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WO2014137420A1 - Procédés de dépistage de récepteur et de modulateur de kinase - Google Patents

Procédés de dépistage de récepteur et de modulateur de kinase Download PDF

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WO2014137420A1
WO2014137420A1 PCT/US2013/070754 US2013070754W WO2014137420A1 WO 2014137420 A1 WO2014137420 A1 WO 2014137420A1 US 2013070754 W US2013070754 W US 2013070754W WO 2014137420 A1 WO2014137420 A1 WO 2014137420A1
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erk
protein
signaling
phosphorylation
cells
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Angelo J. Castellino
Steven K. White
Christopher L. Reading
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Harbor Therapeutics Inc
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Harbor Therapeutics Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation

Definitions

  • the invention relates to methods to identify modulators of mitogen activated protein kinases of low toxicity.
  • the invention further relates to methods to identify modulators of phosphatidylinositide kinases and cell-surface receptors upstream of the protein and phosphatidylinositide kinases.
  • the modulators identified also include dual modulators of extracellular signal-regulated and phosphatidylinositide kinases.
  • Such modulators are useful to treat diseases related to hyperproliferation, including androgen- associated cancers such as prostate cancer, breast cancer, ovarian cancer, lung cancer, liver cancer, bladder cancer, lymphoma, melanoma, thyroid cancer and colon cancer, or unwanted inflammation, which supports initiation or progression of such cancers.
  • the mitogen-activated protein kinases include the extracellular signal- regulated kinase (ERK) isoforms Erk-1 (also referred to as MAPK-3 or p44 kinase) and Erk-2 (also referred to as MAPK-1 or p42 kinase), c-Jun amino-terminal kinase (JNK) and p38 isoforms.
  • ERK extracellular signal- regulated kinase
  • Erk-1 also referred to as MAPK-3 or p44 kinase
  • Erk-2 also referred to as MAPK-1 or p42 kinase
  • JNK c-Jun amino-terminal kinase
  • MAPKs respond to a wide array of stimuli and are involved in a wide range of functions, including phosphorylation of phospho-lipids, transcription factors, cytoskeletal protein and other protein kinase termed MAPK-activated protein kinases (MKs or MAPKAPKs), MAPKs contain similar structural binding domains. Those domains include the ATP binding site, a catalytic active site that transfers a phospho- group from bound ATP to a specific serine or threonine of a MAPK substrate, and protein- protein interaction domains, including the CD (common docking) and FD domains, for recognition of substrates and protein binding partners.
  • CD common docking
  • FD domains for recognition of substrates and protein binding partners.
  • the MAPKs are regulated in part through phosphorylation cascades. Activation of MAPK requires the phosphorylation of conserved tyrosine and serine or threonine in the subdomain VIII activation loop by an upstream protein kinase referred to as a MAPKK or MEK. Various isoforms of this upstream kinase exhibits differing levels of selectivity for their MAPK substrates.
  • the MAPKK in turn are regulated by phosphorylation by upstream kinases referred to as MAPKKK, MEKK or MAP3K, which in turn can be activated through interaction with a protein that becomes activated as a consequence of ligand interaction with its cognate membrane bound receptor.
  • Those membrane receptors typically include receptor tyrosine kinases (RTKs).
  • RTKs receptor tyrosine kinases
  • GPCRs G protein-coupled receptors
  • the phosphorylation cascade in MAPK signaling involve multiple protein kinases that amplifies the initial signal entering into the cascade and appears to be a common feature to the diversity of MAPKs signaling effects. That common cascade allows for multiple unique points of regulation and integration of signaling events originating at the cell membrane or within the cytoplasm that flow through each MAPK signaling node to their diverse array of downstream effectors. That signaling cross-talk sometimes becomes aberrant thereby resulting in excessive signaling through one or more of those nodes, which is often responsible for initiating or propagating neoplasms.
  • scaffold proteins that pre-assemble some of the components of the protein kinase cascade into sub-cellular compartments in order that the incoming signal into the cascade is properly directed to the appropriate downstream effector proteins or integrated with signaling from other signal transduction pathways.
  • the scaffolding protein may in turn be regulated by proteins that affect its phosphorylation state. Additional regulation is provided by phosphatases, which are also regulated by their own phosphorylation states and interactions with scaffolding proteins. Therefore, a tightly regulated network of proteins is required to properly respond to the signaling input and output through each MAPK component so that signaling coming into this network results in the appropriate outcome.
  • Erk-1 and Erk-2 which seem to have the same substrate selectivity in vitro and are often lumped together and described as Erk-1/2, are reported to have over 160 substrates [Yoon, S. and Seger, R. (2006)].
  • Erk-1 and Erk-2 show different substrate selectivities because these isoforms are presumed not to be identically localized due to their different preferences for binding partners.
  • the two Erk isoforms are not influenced identically by the same signaling event.
  • the antiproliferative effect of cytosolic sequestered Erk-1 may be due in part to its improved competition over Erk-2 for the upstream activator MEK as a result of this sequesterization [Vantaggiato, C. et al. (2006)]. That competition between the two Erk isoforms is thought due to differential regulation of Erk-1 , Erk-2 and MEK isoforms by scaffolding proteins.
  • Erk-1 and Erk-2 differ in their nucleo-cytoplasmic transport properties with Erk-1 shuttling more slowly through the nuclear membrane than Erk-2 [Marchi, M. et al. (2008)]. Additionally, the dimerization state of the activated Erk isoform will also influence the isoform's relative contribution between phosphorylating cytoplasmic effector protein and nuclear transcription factors. For example, pErk-2 may access the nucleus either as a monomer (passive transport) or a homodimer (facilitative transport), whereas Docket No. 354. PATENT its phosphorylation of cytoplasmic proteins seems to require the dimerized state, the formation of which is enhanced by scaffolding proteins [Casar, B. et al. (2009)].
  • Inhibitors of MAPKs that have been studied typically target the ATP-binding site (i.e., ATP binding site-dependent inhibitors).
  • Compounds that exert their activity through MAPK signaling through the ATP binding site are generally considered to be toxic, particularly to the liver in view of human toxicity that is observed for p38 MAPK inhibitors [Morel, C. et al. (2005); Laufer, S.A. et al. (2006); Kumar, S. et al. (2003)].
  • Substances that interact at binding domains other than the ATP binding site are sometimes referred to as non-ATP site-dependent inhibitors.
  • phosphatidylinositide kinases having an important role in some hyperproliferation conditions are the phosphatidylinositide kinases.
  • Those kinases include the Class IA phosphatidylinositide-3-kinases (PI3Ks), which are heterodimers composed of a p1 10 catalytic subunit and a p85 regulatory subunit.
  • PI3Ks phosphatidylinositide-3-kinases
  • the Class IA PI3Ks are responsible for production of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), which stimulates the serine kinase Akt.
  • PTEN phosphatase and tensin homolog
  • non-ATP site-dependent MAPK and/or PI3K modulators that suppress (i.e., negatively modulate) excessive pro-proliferative signaling in cancer cells without adversely effecting Ras-Erk and/or PI3K-Akt signaling in normal cells would have improved safety profiles relative to approved standard of care therapies for treating androgen-associated cancers.
  • the present invention relates to screening methods to identify Erk MAPK or Erk MAPK and PI3K modulators having anti-proliferative properties. Moreover, the invention is directed to identifying modulators of those kinase activities that are less toxic to humans than inhibitors presently known and to identifying compounds that oppose or negatively modulate adverse biological activities of DHT and its metabolite, 5a-androstane-3a, 17 ⁇ - diol (3a-diol).
  • the present invention provides for a method to identify a candidate compound, comprising (a) contacting a test compound with a suitable test system; (b) determining phosphorylation states of Erk-1 and Erk-2 resulting from step (a); and (c) selecting a test compound that positively modulated phosphorylation state of Erk-1 activation loop relative to Erk-2 or negatively modulated the phosphorylation state of Erk-2 activation loop relative to Erk-1 of step (b), wherein the test compound selected from step (c) is identified as a candidate compound.
  • the present invention provides screening methods to identify a candidate compound, the method further comprising in step (b) of determining phosphorylation state of a Class 1 PI3K proteins and further comprising in step (c) of selecting a test compound that negatively modulated the tyrosine phosphorylation state of p85a or ⁇ 85 ⁇ regulatory subunit of the Class 1 PI3K of step (b) (i.e., in addition to positively modulating the phosphorylation state of Erk-1 activation loop relative to Erk-2 or negatively modulating the phosphorylation state of Erk-2 activation loop relative to Erk-1 ).
  • the present invention also provides screening methods for identifying those candidate compounds having anti-proliferative properties the method comprising steps Docket No. 354.
  • step (a)-(c) and further comprising the steps of (e) contacting a selected test compound from step (c) with prostate or breast cancer cells of a suitable in vivo test system; (f) determining cancer cell proliferation in the suitable in vivo test system resulting from step (e); and (g) selecting a test compound from step (f) that inhibits cancer cell proliferation statistically significant to cancer cells contacted with vehicle alone.
  • the present invention also relates to screening methods for identifying those candidate compounds having lower toxicities in comparison to known ATP-site dependent inhibitors of PI3K and/or Erk the method comprising steps (a)-(c) and (e)-(g) and further comprising the step of (h) determining a minimum effective amount of a compound selected from step (g) for treating breast or prostate cancer in a mammal; (i) administering the minimum effective amount to healthy mammals so as to provide treated mammals; (j) determining liver enzymes levels for alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ glutamyl transpeptidase of the treated mammals; and (k) selecting a compound from step (i) that did not increase liver enzymes levels for any one of the liver enzymes selected from the group consisting of alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ -glutamyl transpeptidas
  • FIG. 1 E-3a-diol (HE3235) Activity on Prostate Cancer Cells in Suitable In Vitro Test Systems.
  • FIG. 2 E-3a-diol Inhibition of Cell Cycle and Induction of Apoptosis of LP LNCaP Cancer Cells in a Suitable In Vitro Test System.
  • FIG. 3 E-3a-diol Activation of Mutant Androgen Receptor in LP LNCaP cells of a Suitable In Vitro Test System.
  • FIG. 4 Dose Effect of E-3a-diol on Prostate Cancer Tumor Incidence and Tumor Volume in a Suitable In Vivo Test System.
  • FIG. 5 Time Course Effect of E-3a-diol on Established Prostate Cancer Tumors in a Suitable In Vivo Test System.
  • FIG. 8 Effects of E-3a-diol on Phospho-p85a PI3K Levels Induced by DHT
  • FIG. 9 Effects of PI3K Inhibitor (LY295002) and Ras-Erk Signaling Inhibitor Docket No. 354.
  • FIG. 10 Effect of on Erk Phosphorylation of LNCaP Cells in a Suitable In Vitro Test System When Contacted by E-3a-diol (HE3235) and Co-contacted with Kinase Inhibitor.
  • FIG. 11 Protein Phosphorylation in HP LNCaP Cells Determined by KinexTM Antibody Microarray from Contact with E-3a-diol.
  • FIG. 12 Tumor Volume in Response to Contacting E-3a-diol to Breast Cancer Cells of a Suitable In Vivo Test System.
  • FIG. 13 Tumor Incidence Resulting From Contacting E-3a-diol to Breast Cancer Cells of a Suitable In Vivo Test System (average number of tumors per animal).
  • FIG. 14 Tumor Incidence Resulting From Contacting E-3a-diol to Breast Cancer Cells of a Suitable In Vivo Test System (percentage of rats in each group without palpable tumors).
  • FIG. 15 % Tumor Cells Positive for ERa and PARP after Contacting E-3a-diol to Breast Cancer Cells of a Suitable In Vivo Test System.
  • FIG. 16 Expression of Proapoptosis and Differentiation Genes in Breast Cancer
  • FIG. 17 i-AR Phosphorylation Induced by E-3odiol, DHT or in combination after their Contact to Prostate Cancer Cells of a Suitable In Vivo Test System.
  • FIG. 18 Mannose-6-phosphate effect on Proliferation of Prostate Cancer Cells of a Suitable In Vitro Test System When Proliferation is Induced by DHT.
  • FIG. 19 E-3a-diol Anti-Proliferative Effect on Prostate Cancer Cells of a Suitable In Vitro Test System Stimulated by High Dose DHT and Effects of Mannose-6-Phosphate Thereon.
  • Phosphorylation status or "phosphorylation state” as used herein interchangeably refers to the number or pattern of phosphate groups covalently bound to a phospho- protein, such as a phosphorylated protein kinase, which may be membrane bound or in a protein complex.
  • phosphorylation status refers to the overall extent of phosphorylation of a collection of proteins for a specified protein kinase or to the extent to which specified amino acid residue(s) of a specified protein kinase in collection of such proteins that are capable of being phosphorylated in a suitable test system are actually Docket No. 354. PATENT phosphorylated.
  • the phosphorylation status of an Erk isoform may refer to the extent of phosphorylation of the activation loop of that isoform or the extent to which the serine or tyrosine residues in the activation loop are phosphorylated before or after contacting a test compound to a suitable test system.
  • An Erk isoform phosphorylated in the activation loop is represented by pErk-1 or pErk-2.
  • pErk-1 or pErk-2 refers to the isoform that is di-phosphorylated in the activation loop (sometimes identified as ppErk-1 , ppErk-2 or actErk), but also may indicate mono-phosphorylated protein (e.g., pTyr-Erk-1 or pThr-Erk- 1 ) as specified explicitly or implicitly by context.
  • the phosphorylation state of intracellular AR may refer to the extent of tyrosine phosphorylation or to the extent or pattern of serine phosphorylation of the nuclear hormone receptor (NHR).
  • NHR nuclear hormone receptor
  • the phosphorylation state of i-AR refers to specific tyrosine or serine residues as specified explicitly or implicitly by context.
  • i-AR as used herein means any intracellular androgen receptor capable of binding androgen, whether or not that binding events results in its translocation to the nucleus or gene transactivation. Therefore i-AR may refer to functional wild-type AR or a mutant form of the receptor (mt-AR), which may or may not be functional. For suitable test systems, the cells comprising such systems contain either functional wild-type AR or functional mutant AR unless specified otherwise.
  • Modulation of an activity or physical state of a protein means increasing or decreasing an activity of that protein or a property of the protein's physical state resulting from contacting a test or candidate compound to a suitable test system.
  • the modulation may be relative to another activity or property of a different protein, to the same protein in the basal state or subsequent to external stimulation, including contacting a mitogen to the test system prior to contacting of the test compound, or relative to the change in activity or property from contacting the test system with vehicle or reference compound.
  • modulation of an activity includes, for example, Docket No. 354.
  • PATENT increasing or decreasing the capacity of a kinase in a suitable test system to phosphorylate one or more of its downstream effector proteins or substrates of that protein kinase or to increase or decrease signaling through that signal transduction node, cascade or pathway upon contacting a test or candidate compound with any suitable test system relative to one or more other kinases or signal transduction nodes, cascades or pathways within the same test system, e.g., including increasing the phosphorylation capacity of Erk-1 while unaffecting or decreasing the phosphorylation capacity of Erk-2.
  • modulation of an activity includes increasing or decreasing the potential of one Erk isoform to phosphorylate a shared downstream effector protein or substrate relative to the other Erk isoform in a suitable test system.
  • Erk-1/2 activity modulation also includes increasing or decreasing the relative amounts of the two isoforms that are capable of phosphorylation of their downstream effector proteins, molecules or substrates that existed prior to application or administration of a test compound to a suitable test system.
  • test compound may modulate the phosphorylation states of Erk-1 or Erk-2 by, for example, increasing phosphorylation in the activation loop of Erk-1 in comparison to that of Erk-2 or increasing the amount of one phosphorylated isoform in comparison to the other isoform by, for example, increasing the level of phosphorylated Erk-1 (pErk-1 , ppErk-1 or total activation loop phosphorylated Erk-1 protein) in comparison to that of phosphorylated Erk-2 (pErk-2, ppErk-2 or total activation loop phosphorylated Erk-2 protein).
  • a test compound that increases the amount of pErk-1 and/or ppErk-1 without providing an observable ppErk-2 increase or an observable increase in phosphorylated Erk-2 from basal level(s) when both isoforms are present in the same suitable test system is a negative modulation of phosphorylation status of Erk-2, since no phosphorylation of this isoform took place in the presence of test compound, but would have taken place in the absence of test compound as evidenced by positive modulation of phosphorylation states of both isoforms.
  • the phosphorylation status or state of Erk-1 has been positively modulated relative to Erk-2 due to its contact with the test compound (i.e., there has been a change in the relative phosphorylation status between the two isoforms in favor of Erk-1 ).
  • Modulation of phosphorylation status or “modulation of phosphorylation activity” or like terms as used herein means an effected change in activity or phosphorylation state of a specified protein or collection of such proteins in a suitable test system that is capable of being phosphorylated upon contacting a test compound to the test system.
  • PATENT of phosphorylation status or state may mean increasing or decreasing the number of covalently bound phosphate groups in a protein, changing the phosphorylation pattern within a protein, which may or may not be accompanied by an increase or decrease in the number of covalently bound phosphate groups, or increasing the amount of a phosphorylated protein resulting from contacting a test or candidate compound to a suitable test system.
  • Modulation of phosphorylation status or state may also mean changing the number or pattern of covalently bound phosphate groups in a protein or the amount of a phosphorylated protein in comparison to an effect a test compound has on a reference protein that is present in the same suitable test system resulting from contacting a test compound to the same suitable test system. Modulation of phosphorylation status or activity may be described relative to an isoform of the same protein in the same test system or to the same protein in a control test system to which is contacted the same test or candidate compound or from contact of the same test system with a test compound that is a reference compound (e.g., vehicle or positive or negative control compound). Relative modulation of activity or phosphorylation status or state is stated explicitly by describing the comparator protein or by describing the suitable test and control systems, otherwise relative phosphorylation status is implicitly understood by context.
  • modulation of phosphorylation states are exemplified for Erk.
  • An Erk isoform requires activation of tyrosine and threonine residues in the activation loop of the kinase for maximal activity towards its substrates, administration or application of a test compound to a suitable test system will affect one or both of these residue phosphorylations. Therefore, modulation of Erk-1/2 phosphorylation includes decreasing or increasing the phosphorylation state of one Erk isoform relative to the other isoform and includes increasing or decreasing the amount of phosphorylation in the activation loop of one isoform relative to the other isoform.
  • an increase or decrease in the percent total phosphorylation (i.e., phosphorylation extent) of a collection of proteins for one Erk isoform protein relative to another protein collection of the other Erk isoform within the same suitable test system represents a relative change in phosphorylation status between the two Erk isoforms.
  • This change in phosphorylation status may be accompanied by or associated with decreased or increased amounts of protein.
  • increased activation loop Tyr/Thr phosphorylation of Erk-1 may occur with decreased Erk-2 protein level or overall decreased Erk-1/2 protein level favoring Erk-1 (i.e., increased formation of pErk-1 relative to pErk-2 may occur along with increase gene transcription for one or both proteins or with overall decreases in gene transcription that favors protein levels of Erk-1 in comparison to Erk-2).
  • An Erk isoform is typically activated within in vitro cell-based and in vivo systems suitable for determining relative changes in Erk phosphorylation status and usually occurs by sequential phosphorylation of tyrosine and threonine in the activation loop of an Erk isoform protein, wherein the tyrosine is usually first phosphorylated. Therefore, another relative change in phosphorylation status is increasing or decreasing the amount of tyrosine residue phosphorylation in the activation loop for a collection of proteins for one Erk isoform relative to a collection of proteins for the other Erk isoform within the same suitable test system.
  • Another relative change in phosphorylation status of Erk-1/2 is increasing or decreasing the relative amount of di-phosphorylation in the activation loop of a collection of proteins for one Erk isoform protein relative to the other Erk isoform in the same suitable test system, wherein typically one or both isoforms are initial unphosphorylated (i.e., prior to stimulation with mitogen or contact with a test or candidate compound) or have basal levels (i.e., unstimulated) levels of phosphorylation.
  • modulation of phosphorylation status may mean, depending on context, an increase or decrease in the number of phosphate groups covalently bound to a protein, phospho-protein, protein kinase or protein kinase substrate, effector protein other molecule so modulated (i.e., increase or decrease in overall phosphorylation status of the modulated molecule), an increase or decrease in phosphorylation of specified amino acid residues for a collection of specified molecules (e.g., for a specified isoform or a defined set of isoforms), or to an alteration of the phosphorylation pattern of a phospho-protein, which may or may not be accompanied by an increase or decease in the number of covalently bonded phosphate groups.
  • a test or candidate compound that modulates the phosphorylation state of a protein kinase or transcription factor such that an activity of the specified kinase (e.g., phosphorylation of a downstream effector substrate) or transcription factor (e.g., transactivation activity of a nuclear hormone receptor towards a hormone-inducible gene) is increased is referred to as a positive modulator of that activity
  • a test compound that modulates the phosphorylation state of a protein kinase or transcription factor such that the kinase or specified transcriptional activity is decreased is referred to as a negative modulator of that activity.
  • a test or candidate compound that increases overall phosphorylation of a protein is referred to as an overall positive phosphorylation state modulator of that protein.
  • a test or candidate compound that decreases phosphorylation of a protein is referred to as an overall negative phosphorylation state modulator of that protein. This positive or negative modulation of the protein's overall Docket No. 354. PATENT phosphorylation state may consequently modulate the activity of that phospho-protein positively or negatively.
  • test compound when contacted to a suitable test system, diminishes or abrogates phosphorylation of a protein such that the protein appears to remain at its basal phosphorylation state under conditions in which its phosphorylation would occur is also referred to as an overall negative phosphorylation state modulator of that protein.
  • a test or candidate compound that changes the phosphorylation pattern of a protein, whether or not there has been a change in the number of covalently bonded phosphate groups, (i.e., total number of phosphate groups may be increased, decreased or unchanged) is also referred to as a positive phosphorylation state modulator.
  • This positive modulation based upon a change in phosphorylation pattern may be accompanied by positive or negative phosphorylation state modulation of specified residues and by positive or negative modulation of an activity of the phospho-protein whose phosphorylation pattern has been affected.
  • a phosphoprotein containing pSer/pThr and/or pTyr e.g.
  • pErk-1 and pErk-2) that undergoes a change in phosphorylation pattern without a change in the total number of phosphate groups such that the number of phosphorylated tyrosines increases concommitant with decreased number of phosphorylated serine/threonines has its phosphorylation state positively modulated due to the change in phosphorylation pattern, but its overall Ser/Thr phosphorylation state has been negatively modulated.
  • test or candidate compound that results in removal of one or more phosphate groups from a phosphoprotein without addition of at least one phosphate group elsewhere in the phosphoprotein is not considered a change in phosphorylation pattern and thus is not a positive modulator of that phosphoprotein's phosphorylation state. Rather, it is a negative modulator of overall phosphorylation state.
  • test or candidate compound that results in addition of one or more phosphate groups to a protein or phosphoprotein without removal of at least one phosphate group is not considered a change in phosphorylation pattern and thus is not a positive modulator of that phosphoprotein's phosphorylation state. Rather, it is a positive modulator of overall phosphorylation state.
  • Modulation of a phosphorylation pattern is sometimes described functionally such that a modulation by a test or candidate compound of a phospho-protein's phosphorylation pattern that results in negative modulation of pro-survival or pro- Docket No. 354.
  • PATENT proliferative activity or positive modulation of apoptotic or anti-proliferative activity is referred to as a positive modulator of the protein's phosphorylation pattern.
  • modulation of the phosphorylation pattern in a transcription factor that results in negative modulation of nucleo-cytoplasmic transport of that transcription factor negative modulation of its transactivation of anti-apoptotic or pro-proliferative genes or positive modulation of its transactivation of pro-apoptotic or anti-proliferative genes is referred to as a positive modulator of the transcription factor's phosphorylation pattern.
  • a test or candidate compound that decreases phosphorylation of a protein is referred to as a negative phosphorylation state modulator.
  • This positive or negative modulation of a protein's phosphorylation status is sometimes determined relative to a comparator protein present in the same suitable test system (i.e., positive or negative relative phosphorylation status).
  • test compound that increases the amount of a phosphorylated protein due to increased upstream phosphorylation activity acting upon the protein within a kinase cascade is also referred to as a positive modulator of that protein's phosphorylation state
  • a test compound that decreases the amount of phosphorylated protein is referred to as a negative modulator of that protein's phosphorylation status.
  • a positive phosphorylation status modulator of a protein kinase will also be a positive catalytic kinase activity modulator of that kinase and a negative phosphorylation status modulator of a protein kinase will also be a negative catalytic kinase activity modulator for that kinase.
  • modulated phosphorylation states and catalytic kinase activities that are affected by a test or candidate compound may be inversely related.
  • a negative modulator of an activity, phosphorylation state or pattern results in suppression, or lack of an expected qualitative or quantitative increase, of that activity, state or pattern that would otherwise take place in a suitable test system in the absence of test compound.
  • Effective protein or like term means a protein, polypeptide or other molecule that is downstream of a protein kinase, inositol kinase, lipid kinase, lipase or other enzyme (i.e., an upstream enzyme) or is terminal to a kinase cascade or signal transduction pathway whose phosphorylation status or activity is modulated by the upstream enzyme, kinase cascade or signal transduction pathway. This modulation is due to stimulation of a signal transduction pathway that results in modulation of the catalytic activity of the upstream enzyme and results in effect(s) on cellular program(s) not localized to upstream Docket No. 354. PATENT components of the signal transduction pathway. Effector proteins typically refer to those proteins that are direct substrates of an upstream protein kinase or proteins terminal to a kinase cascade or signal transduction pathway within a viable normal or cancerous cell or a cell undergoing apoptosis.
  • Erk effector proteins include certain apoptotic proteins, transcription factors, MAPKAPKs (MAP kinase activated protein kinases), MSKs (mitogen and stress activated kinases) and other cytosolic and nuclear localized proteins as disclosed herein.
  • MAPKAPKs MAP kinase activated protein kinases
  • MSKs mitogen and stress activated kinases
  • Other Erk effector proteins include plasma membrane, endomembrane, mitochondrial and cytoskeletal proteins as disclosed herein.
  • Kinase Substrate or like term means a protein, polypeptide or other molecule that is capable of phosphorylation by a kinase.
  • a kinase substrate includes an effector protein that is directly acted upon by a protein kinase, inositol kinase, lipid kinase or other kinase within a viable normal or cancerous cell or a cell undergoing apoptosis.
  • a kinase substrate also includes a molecule directly acted upon by a protein kinase in a suitable cell-free test system or a suitable test system consisting essentially of cell membrane-disrupted cells. Therefore, a kinase substrate need not be associated with a kinase acting within a viable normal or cancerous cell or a cell undergoing apoptosis.
  • Suitable test system as used herein means an in vitro or in vivo system to which can be contacted with a test compound in order to elicit effect(s) on one or more signal transduction pathways, nodes, complexes, kinase proteins or cascades or effects sequesterization, subcellular localization or nucleo-cytoplasmic transport of one or more protein kinases or signal transduction complexes.
  • the suitable test systems can constitute cells or tissue in vivo (e.g., in xenograft test systems) or cells in tissue culture (i.e., in vitro cell-based test system) or constitute cell extracts in cell-free tests systems, wherein signaling through target molecules of interest, e.g., Erk-1 , Erk-2, PI3K proteins, GPR-C6a and/or AR, is(are) functional and phosphorylation changes or other biological responses as described herein, as for example for E-3a-diol and 3a-diol, in response to the test (or control) compound can be measured.
  • target molecules of interest e.g., Erk-1 , Erk-2, PI3K proteins, GPR-C6a and/or AR
  • cell-free test systems for measuring phosphorylation changes may be artificial in nature by reconstituting a signal transduction pathway comprising one or more proteins of that pathway in a suitable buffer with a downstream substrate that is capable of phosphorylation by at least one of the signal transduction pathway proteins.
  • the compound to be contacted with a suitable test system will usually be in a Docket No. 354.
  • PATENT vehicle, composition or formulation that is compatible with the test system e.g., the test compound can be in a solution or suspension or it can be administered as a solid or liquid formulation to an animal such as a rodent (e.g., mouse or rat), or, for clinical assessment of the test compound, it can be administered to a human patient.
  • Said contact is followed by measurement of target molecule phosphorylation states (e.g., Erk-1 , Erk-2) in cells or tissue samples taken from the animal or patient after administration of the test compound to the animal or patient.
  • target molecule phosphorylation states e.g., Erk-1 , Erk-2
  • test systems will typically be contacted with a positive, negative, placebo and/or vehicle control or reference compound to confirm proper functioning of the test system in vitro or in vivo.
  • the test compound and any control or reference compound or composition will be contacted with the test system (i) under conditions where the test system is functional, e.g., cells in tissue culture are maintained under standard growth conditions, (ii) for one or more sufficient periods of time, e.g., for about 5 seconds-120 minutes for cells in tissue culture or about 0.1 hour to about 48 hours for cells or tissue in vivo, and (iii) in one or more amounts or concentrations suitable to assess biological responses, e.g., dose- responses to the test compound and/or vehicle, placebo or control compound effects, if any.
  • type of biological response is measured typically from within 5 sec to 30 min after contacting the test system with test compound.
  • type of biological response is measured typically from within 60-120 min. after said contact.
  • Typical test compound final concentrations with the test system will be in a range, e.g., about 0.01 nM to about 20 mM or usually about 1 nM to about 10 mM, which can include one or more of about 0.05 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 200 nM, 1 mM, 2 mM and 10 mM. Concentrations of other compounds, e.g., components or excipients in the formulation that contains the test compound will typically be tested at the same or nearly the same concentrations they are at when the test compound is contacted with the test system.
  • Suitable test systems are capable of responding to a test compound to be selected as a candidate compound that modulates an effect as described herein in a qualitatively or quantitatively similar manner when contacted with 17a-ethynyl-5a-androstane-3a, 17 ⁇ - diol (E-3a-diol) or 5a-androstane-3a, 17p-diol (3a-diol) or another positive or negative control.
  • test systems comprise cells in vitro maintained or incubated in growth or culture media, in androgen- and growth-depleted media or under serum-starved conditions or are comprised of mammalian cells in vivo (e.g., as xenograft implanted cells in an animal or in situ tumor cells from carcinogen-induced cancer). Docket No. 354.
  • human mammalian cells having endogenous functional human i-AR protein or transfected to contain functional i-AR gene or genetically engineered to express or overexpress a gene encoding functional i-AR protein are exemplified for some suitable test systems, the invention also contemplates suitable test systems comprising human cells transfected to contain functional rodent i-AR (e.g., murine or rat i-AR) or other mammalian i-AR gene and rodent cells transfected to contain functional rodent, human or other mammalian i-AR gene.
  • functional rodent i-AR e.g., murine or rat i-AR
  • a mutated rodent or other mammalian mt-AR protein in mammalian cells transfected to contain functional mt-AR gene having analogous mutations described herein for the human mt-AR gene are also included, although preferred test systems, such as those in the claims or other embodiments described herein, contain functional human wild-type i-AR protein or the T877A mutant AR.
  • the invention further contemplates suitable test systems comprising mammalian cells other than human cells having endogenous human i-AR protein or transfected to contain functional or i-AR gene or genetically engineered to express or overexpress a functional gene encoding functional i-AR protein.
  • Those human or non-human mammalian cells may contain or be transfected to contain one or more genes encoding other functional nuclear hormone receptors proteins in place of or in addition to i-AR, including estrogen receptor (ER) or ERa and/or ERp.
  • the mammalian cells may also or additionally contain, endogenously or by genetic engineering, one or more surface membrane receptors such as EFGR, ErbB2 or other ErbB receptor or GPR-C6a, an alpha-1 adrenergic receptor or other GPRC coupled to or capable of being coupled to Ga/q. All of the aforementioned human or non-human mammalian cells may also contain an inducible- reporter gene construct that has an upstream promoter for a functional nuclear hormone receptor whose gene is expressed endogenously or is expressed or over-expressed in the cell through genetic engineering.
  • a non-human mammal is transplanted with transformed or cancerous mammalian cells to provide a suitable test system (e.g., from cells that comprise the aforementioned suitable in vitro test systems), sometimes referred to as a xenograft tests system, wherein the cells have or are genetically engineered to have a gene encoding for expression or overexpression of functional i-AR protein with or without additional genetic engineering as previously described.
  • a suitable test system e.g., from cells that comprise the aforementioned suitable in vitro test systems
  • a xenograft tests system wherein the cells have or are genetically engineered to have a gene encoding for expression or overexpression of functional i-AR protein with or without additional genetic engineering as previously described.
  • suitable test system e.g., from cells that comprise the aforementioned suitable in vitro test systems
  • Such in vivo test systems typically are xenograft models that have been used for evaluating anti-cancer treatments. Mammals of those xenograft models are Docket No. 354.
  • PATENT usually immuno-compromised to allow for proliferation of the implanted cells, which may be supported by androgen administration, and include CD-1 nude mouse, nu/nu nude mouse, BALB/c nude mouse and RNU nude rat (T-cell deficient), NIH III nude mouse, SCID hairless outbreed mouse, SCID hairless congenic mouse, CB17 SCID mouse, SCID Beige mouse and NOD SCID mouse.
  • Control test system refers to a suitable test system that is to be sham treated with compound, contacted with vehicle or contacted with a reference compound or composition that, depending on context, may serve as a positive or negative control test compound.
  • the cells of the control test system are genetically the same as the cells comprising the test system to which test or candidate compound is contacted.
  • Control test systems may also be derived from the suitable test system to which a test or candidate compound is to be contacted by genetic alterations to or by external stimulus of signal transductions pathways of the cells comprising the suitable test system. In this context the same test compound may be applied to both suitable test systems (i.e., the control test system and the original test system). Cells within a control test system used in screening of test compounds are sometimes referred to as control test cells.
  • Test compound as used herein means a compound, or a composition comprising the compound, to be evaluated in a suitable test system for the presence of one or more of the activities for E-3a-diol described herein or for the ability to oppose one or more actions of 3a-diol described herein.
  • Test compounds also include reference compounds whose effect on a suitable test system is known and which is to be compared to an effect (or lack thereof) provided by contact of another test compound to the same test system (i.e., a test or reference compound is contacted with a control test system).
  • the reference compound may be a positive or negative control compound.
  • a positive control compound when assessing Galpha/q-mediated signal transduction a positive control compound may be E- 3a-diol and a negative control compound may be 3a-diol.
  • Positive control compounds may also include androgen conjugates as described herein that are cell-impermeable and are useful for evaluation of test compounds for a subset of activities described herein for E-3a-diol.
  • One subset of activities are non-genomic signaling that results from interaction of cell-impermeable conjugates with a membrane androgen receptor, and includes longer term non-genomic signal transduction effects subsequent to initial nongenomic signaling. Another subset of activities includes those associated with secondary genomic effects from nongenomic signaling.
  • initial nongenomic signaling occurs within seconds Docket No. 354.
  • Longer term nongenomic signaling occurs subsequent to the peak of the initial nongenomic signaling and may overlap with the decay phase of the initial nongenomic signaling.
  • the longer term nongenomic signaling is characterized as a plateau and persists after the rapid and transient phase has decayed to baseline.
  • the longer term nongenomic signaling may occur prior to, concurrent with or after induction of secondary genomic effects affected by the initial nongenomic signaling.
  • Test compounds additionally include test compounds shown to have one or more activities qualitatively or quantitatively similar to E-3a-diol, which are required for consideration or selection as a candidate compound, and which may also serve as a positive control compound for that activity. Test compounds also include candidate compounds to be evaluated for identification as low toxicity Erk inhibitors.
  • Test compounds typically have a molecular weight of 200-1 ,000 amu or 200-800 amu, and are non-peptidic, but may be peptidic or have higher molecular weight as, for example, when a test compound is used as reference compounds that selectively elicits extracellular effect(s) by binding to a plasma membrane receptor (e.g. a GPCR) in comparison to intracellular effects (e.g., those resulting from binding to protein complexes or scaffold proteins in the cytoplasm or nucleus).
  • a plasma membrane receptor e.g. a GPCR
  • Such reference compounds are typically cell-impermeable and include androgen-protein conjugates (e.g., testosterone-bovine serum albumin (T-BSA) or DHT-BSA conjugate), optionally coupled to a fluorophore, as described herein.
  • androgen-protein conjugates e.g., testosterone-bovine serum albumin (T-BSA) or DHT-BSA conjugate
  • T-BSA testosterone-bovine serum albumin
  • DHT-BSA conjugate DHT-BSA conjugate
  • Other peptidic substrates recognizes the CD or FD protein-protein interaction domain of a MAPK and may be derived from the amino acid sequence of the corresponding protein-protein interaction domain of a substrate or effector protein for that MAPK.
  • Candidate compound as used herein is a test compound that exhibits one or more of the activities of E-3a-diol or opposes one or more activities of 3a-diol in an in vitro or in vivo model(s) predictive or indicative of efficacy for treating a hyperproliferation condition described herein in a mammal or is expected to have that condition, or is shown to have low toxicity in comparison to clinically studied MAPK inhibitors that are directed to the ATP binding site.
  • candidate compounds positively modulate Erk-1 activation loop phosphorylation or catalytic kinase activity selectively in comparison to Erk- 2, negatively modulate Erk-2 activation loop phosphorylation state or catalytic kinase activity in comparison to Erk-1 or negatively modulate PI3K catalytic kinase activity or Docket No. 354.
  • PATENT negatively modulate the PI3K regulatory subunit tyrosine phosphorylation state. More typically test compounds selected as candidate compounds elicit those Erk-1 and/or Erk-2 and PI3K effects.
  • Reference compound or "control compound” as used herein is test compound that has one or more of the activities as described herein for E-3a-diol or 3a-diol for which comparison is to be made in a suitable test system to another test compound to be screened for that activity (i.e., a positive control).
  • Other reference or control compounds lack one or more of these activities for which comparison is to be made in a suitable test system to another test compound to be screened for that activity (i.e., negative control).
  • Reference or control compounds include E-3a-diol, 3a-diol, 17a-ethynyl-17p-hydroxy-5a- androstan-3-one (E-DHT), 3 ⁇ , 17p-di-hydroxyandrost-5-ene ( ⁇ ), 3 ⁇ , 7 ⁇ 17 ⁇ - ⁇ - hydroxyandrost-5-ene ( ⁇ ), 3p,17p-di-hydroxy-5a-androstane (3p-diol), a hormone i- AR agonist, such as testosterone (T) or 17p-hydroxy-5a-androstan-3-one (DHT) for Galpha/i and/or Galpha/q-mediated signaling, or a cell-impermeable GPR-C6a agonist (e.g., T-BSA and DHT-BSA conjugates) for Galpha/i-mediated signal transduction as described herein.
  • E-DHT E-3a-diol, 3a-diol, 17a-ethynyl-17p
  • Liver toxicity means an increase of one or more liver enzyme (alanine transaminase, aspartate transaminases, alkaline phosphatase, ⁇ -glutamyl transpeptidase) levels when a test or candidate compound is evaluated in a suitable animal model to an extent that is inconsistent with approvable treatment of an intended hyperproliferation condition in humans.
  • a test or candidate compound having low liver toxicity has about 50% or more of the treated animals, e.g., in about 70% to about 90% of the treated animals with liver enzyme levels that are not increased more than about 3-fold compared to normal values.
  • a low liver toxicity compound exhibits liver enzyme level increases less than about 2-fold, typically less than about 1 .5-fold, in about 70% or more of treated humans, more typically in about 70% to about 95% of the treated humans.
  • Indications of low liver toxicity of a test or candidate compound for approvable treatment of an intended hyperproliferation condition in humans are provided by suitable animal models showing liver enzyme levels that are not increased more than about 2.5- fold compared to normal values.
  • Initial assessments of liver toxicity are usually conducted with animal models including those with rodents, such as a mouse or rat.
  • a test or candidate compound selected as a candidate low toxicity Erk modulator for further evaluation for liver toxicity exhibits more than about 2.5-fold increase in a liver enzyme level in no more than 70% of treated rodents, more typically about 70% to about 95% of the treated rodents do not have more than about 2.5-fold increase in a Docket No. 354.
  • PATENT liver enzyme level when the test or candidate compound is administered to animals comprising the animal model at mg/Kg dose levels of 2X to 10X or more than is expected to elicit a desired therapeutic effect (i.e., has a therapeutic index of at least 2-10).
  • a test or candidate compound selected for further evaluation of liver toxicity does not have more than about 2.5-fold increase in level for any of the aforementioned liver enzymes in 95% or more of the treated rodents.
  • Protein-protein interaction domain as used herein means a region in the tertiary structure, which may be comprised of continuous or discontinuous amino acid sequences, of a protein wherein the domain is capable of non-covalently binding to another tertiary structural region of the same or different protein.
  • These interaction domains include the Src homology domains SH2 and SH3.
  • Such protein-protein interaction domain are often found in components of protein kinase signal transduction pathways, e.g., in scaffolding proteins, adaptor proteins, membrane-bound receptors, internalized GPCR or RTK receptors and protein kinases and phosphatases as described herein.
  • Protein-protein interaction domains also include CD and FD domains of MAPKs that recognize complementary regions in their binding partners or substrates such as MAPK activator or inhibitor (i.e., regulatory) proteins, adaptor or scaffold proteins, effector proteins, protein phosphatases or another protein kinase.
  • MAPK activator or inhibitor i.e., regulatory
  • adaptor or scaffold proteins i.e., effector proteins
  • protein phosphatases or another protein kinase.
  • Protein complex refers to a collection of two or more different proteins wherein at least one protein that is a scaffold or adaptor protein and another protein that affects or modulates the phosphorylation (e.g., a protein, lipid or small molecule kinase or phosphatase) or GTP-GDP binding status of a third protein (i.e. a GTP-binding protein) that is typically activated in the GTP bound state.
  • Those GTP- binding proteins include, e.g., Ga-subunits of heterotrimeric G proteins, such as Ga/q family members, and are described in Takai, Y. et al. (2001 ) and Morris, A.J. and Malbon, C.C.
  • a protein complex may also refer to a collection of different proteins wherein at least one protein is a protein, lipid or small molecule kinase or a protein that effects or modulates the phosphorylation or GTP-GDP binding status of a GTP-binding protein.
  • the protein complex typically contains proteins that are components of one or more protein kinase signal transduction pathways or pathways for GPCR or RTK signaling, which often include a scaffolding or adaptor protein such as Ras, SOS, She, ⁇ - arrestin, Tfg, protein 14-3-3 or a kinase or nuclear hormone receptor with several protein- protein interaction domains, including Raf, PI3K, AR and ER.
  • the scaffold or adaptor proteins within these complexes are bound non-covalently to one or more effector or Docket No. 354.
  • PATENT modulator proteins including but not limited to a protein kinase, a protein phosphatase, a G protein monomer or Ga/ ⁇ heterodimer, a membrane bound or internalized GPCR or RTK or a protein kinase effector protein such as a transcription factor, apoptosis-related protein or cytoskeletal protein.
  • the protein complexes may reside preassembled in subcellular locations or domains including the cytoplasm, nucleus, nucleolus, mitochondria, lipid rafts, caveolae and clathrin pits or at membranes including the cytosolic plasma membrane or membranes of subcellular structures including late and early endosomes, the endoplasmic reticulum and Golgi apparatus or may reside bound to cytoskeletal structures.
  • this pre-assembly increases the efficiency of signal transduction by permitting proper recruitment of other signal transduction components upon activation of a protein complex member, allows appropriate crosstalk with parallel signal transduction pathways for signal integration and properly directs the integrated signal to downstream effector proteins.
  • Signal transduction node is a component of a signal transduction pathway capable of having catalytic activity for incoming signal amplification, or is a protein complex containing as a signal transduction component the protein capable of this catalytic activity and one or more other signal transduction components, that signals to multiple downstream effector proteins and/or upstream regulator proteins.
  • the effects of these components on downstream or upstream signaling is dependent on the phosphorylation states of multiple protein kinases that act upon them or on the activities of protein complexes from other signal transduction pathways. Other times the dependency results from sharing of scaffolding protein(s) or other components within a protein complex involved in one signal transduction pathway with a protein complex of another signal transduction pathway.
  • Signal transduction pathway refers to a sequence of biochemical events or the proteins and relay molecules involved in these events that transfer the consequence of a ligand binding event originating externally or internally to a cell to an effector protein or receptor.
  • the external binding events may Docket No. 354.
  • PATENT result, for example, from binding of a cytokine or mitogen to a receptor tyrosine kinase (RTK) or from an agonist binding to a membrane-bound G protein-coupled receptor (GPCR).
  • RTK receptor tyrosine kinase
  • GPCR membrane-bound G protein-coupled receptor
  • Internal binding events that initiate signal transduction may result, for example, from a hormone ligand binding to its nuclear hormone receptor (NHR).
  • the consequence (or signal) from these initial binding events are then transferred to another protein whose catalytic action or its effect on the catalytic action of another downstream protein amplifies the signal, which then may be passed along to yet another protein for further amplification to eventually modulate the activity or phosphorylation state of an effector protein or substrate terminal to the signal transduction cascade.
  • the binding event that initiates signal transduction is superfluous, since the receptor has become constitutively active due to mutation and is thus able to initiate signaling on its own (i.e., in absence of agonist ligand binding).
  • the sequence of biochemical events for signal transduction typically employs a kinase cascade, as defined herein, which is supported by various accessory proteins, including scaffolding and adapter proteins, to guide each biochemical event to the appropriate downstream target.
  • the kinase cascade is engaged by activation of a tyrosine kinase receptor or cytoplasmic protein (e.g., EFGR, PDGFR, Her2/neu, Src) the catalytic kinase activity of which may reside within the binding receptor or a protein kinase recruited to the activated receptor.
  • a tyrosine kinase receptor or cytoplasmic protein e.g., EFGR, PDGFR, Her2/neu, Src
  • the kinase cascade is initiated by G protein activation through a ligand-bound GPCR (e.g., "inverse" agonist activation of a Ga/q coupled GPCR with respect to Galpha/i activation), whereby an activated G protein monomer through scaffolding interacts with a protein kinase, inositol lipid kinase or lipase.
  • a ligand-bound GPCR e.g., "inverse" agonist activation of a Ga/q coupled GPCR with respect to Galpha/i activation
  • engagement of the protein kinase cascade is more indirect as for example when a small molecule second messenger is produced by action of an enzyme (e.g., PI3K, ⁇ _0- ⁇ 1 2 , ⁇ _0- ⁇ 1/2 ) that has been activated by G proteins (e.g., Ga/q monomer or ⁇ / ⁇ heterodimer) such that subsequent binding of the messenger to a downstream protein kinase results in its activation (PIP3 from PI3K to activate Akt or DAG from PLC to activate certain PKC isoforms).
  • an enzyme e.g., PI3K, ⁇ _0- ⁇ 1 2 , ⁇ _0- ⁇ 1/2
  • G proteins e.g., Ga/q monomer or ⁇ / ⁇ heterodimer
  • Kinase cascade refers to a collection of kinases that engage in sequential transfer of ATP ⁇ -phosphate groups catalyzed by one kinase to form another activated protein kinase or to catalyze formation of a kinase or other enzymatic product (i.e., PIP3, DAG, cAMP) that activates a downstream protein kinase within the cascade.
  • a kinase cascade in normal cells typically amplifies a signaling event and directs the Docket No. 354. PATENT outcome to appropriate effector proteins or substrates.
  • a kinase cascade may respond to other signaling events and thus be activated or inhibited by a kinase involved in a different kinase cascade either directly or indirectly through effects on shared scaffolding proteins thereby resulting in signal transduction cross-talk.
  • the signaling events that enter a kinase cascade may originate from a membrane-bound or internalized receptor or from cellular stress response proteins.
  • the signaling event may be triggered by agonist binding to a receptor or spontaneous activity of the receptor. In hyperproliferating cells this spontaneous activity may become constitutively amplified due to mutations of the gene encoding the receptor.
  • Cross-talk refers to the result of one signal transduction pathway influencing the output from another signal transduction pathway wherein the pathways are normally distinguished by their unique sets of effector substrates (some members of which may be shared).
  • aberrant crosstalk typically occurs between the Ras-Erk and PI3K signal transduction pathways.
  • aberrant cross-talk between those two pathways typically results in dysregulated i-AR nuclear transactivation that supports proliferation.
  • the aberrant cross- talk or i-AR dysregulation may be mediated or enhanced by excessive Src kinase activity on or within protein complexes involving one or more scaffolding and/or adaptor proteins that are shared between Ras-Erk and PI3K signal transduction pathways. That excessive activity may result from RTK activation, which itself is able to inappropriately increase pErk-1/2 levels in an isoform indiscriminate manner. That indiscriminate isoform activation also occurs through protein complexes that includes one or more scaffolding and/or adaptor proteins as defined herein. Docket No. 354.
  • a test or candidate compound will typically be capable of disrupting mitogenic-promoting protein complexes so as to re-regulate crosstalk between PI3K-Akt and Ras-Erk signal transduction. That outcome will typically re-regulate i-AR nuclear transactivation such that catastrophic cell differentiation (i.e., differentiation that induces apoptosis) occurs.
  • a test or candidate compounds that re-regulates crosstalk between PI3K-Akt and Ras-Erk signal transduction will do so by redirection of Src activity such that excessive signaling through the Erk and/or PI3K signal transduction node(s) is(are) negatively modulated.
  • Translocation refers to the movement of signal transduction components between the cytoplasm and the nucleus. This translocation or trafficking may result from passive diffusion or facilitative transport. For example, upon activation of a MAPK, its phosphorylated form is transported to the nucleus, which results in phosphorylation of transcription factors responsible for expression of early response genes such as c-fos and myc. Residence time within the nucleus will determine the phosphorylation state of the MAPK when it is translocated back into the cytoplasm, since many of the deactivating phosphatases are found in the nucleus.
  • a test compound that decreases the rate of pErk-1 or pErk-2 transport from the cytosol to the nucleus or increases the fraction of total pErk in the cytoplasm relative to the nucleus is defined as a negative modulator of nucleo-cytoplasmic translocation.
  • a test compound that increases rate of pErk-1 or pErk-2 transport from the cytosol to the nucleus or increases the fraction of total pErk in the nucleus relative to the cytoplasm is referred to as a positive modulator of nucleo-cytoplasmic translocation.
  • Sequester refers to preferential localization of a signal transduction component or a protein complex containing that component to a membrane, cytoplasm, nucleolus, mitochondria or other subcellular compartment.
  • sequesterization provides proper direction of an incoming signal to a signal transduction pathway (or additional pathways by appropriate signal transduction cross-talk) that results in activation of intended effector protein(s) or substrate(s).
  • induced sequesterization by a test or candidate compound of a component of that cascade would negatively modulate the aberrant signaling or additionally redirect this activity away from supporting proliferation or survival.
  • Hyperproliferation condition means a condition or disease state, e.g., a cancer that is characterized by an abnormally high rate or a persistent state of cell Docket No. 354. PATENT division is uncoordinated with that of the surrounding normal tissues, and persists after, e.g., cessation of the stimulus that may have initially evoked the change in cell division. The rate of proliferation by the abnormal cells may be accelerated or similar to that of normal surrounding tissue. This uncontrolled and progressive state of cell proliferation will result in a tumor that is benign, potentially malignant (premalignant) or virtually malignant. Hyperproliferation conditions include those characterized as a hyperplasia, dysplasia, adenoma, sarcoma, blastoma, carcinoma, lymphoma, leukemia or papilloma or other conditions described herein.
  • Hormone-associated cancer precancer or benign hyperplasia as used herein refers to a hyperproliferation condition that responds negatively (i.e., promotes cell cycle progression) or positively in a therapeutic sense (i.e., retards cell cycle progression or promotes apoptosis), to hormone manipulation or is a condition whose genesis, persistence, invasiveness, refractivity, severity in symptoms or responsiveness to cancer chemotherapy are attributable or related, in part or in whole, to one or more hormone levels.
  • Hormone-associated cancers include prostate cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial carcinoma, adenocarcinoma, malignant melanoma or other conditions as described herein.
  • Hyperproliferation conditions in which the hyperproliferating cells contain functional i-AR or which respond to androgen(s) are referred to as androgen-associated conditions.
  • “Scaffold protein” or “adaptor protein” as used herein is a protein capable of bringing together two or more other proteins to facilitate creation of larger signaling complexes and typically contain two or more protein-protein interaction domains for this purpose. By linking specific proteins together, cellular signals can be propagated to elicit an appropriate response from the cell initiated from a membrane-bound receptor or an intracellular receptor.
  • Adaptor proteins include for example 14- 3-3 protein, CrkL, MyD88, Grb2, Nek and She.
  • a scaffold protein typically refers to a protein that recruits or anchors other proteins to a subcellular location. Scaffolding may therefore result in subcellular localization of the components to direct recruitment of other components to the protein complex formed from that scaffolding or may direct signal transduction to kinase effector proteins or substrates localized to a subcellular Docket No. 354. PATENT compartment.
  • a scaffold protein may be devoid of intrinsic activity but may include those proteins having transcriptional activity or protein or inositol lipid kinase activity, lipase activity or other catalytic activity.
  • Non-limiting examples of these dual role proteins include Raf-1 , Tpl-2, Src, PI3K, ⁇ _0- ⁇ /2, PLC-Y ! 2 or are other kinases or lipases associated with components of GPCR, RTK, intracellular AR or ER, Ras-Erk or TNFa-NF- ⁇ signal transduction pathways or other signaling pathways disclosed herein.
  • Scaffold- and adaptor-mediated complexes assemble through protein-protein interactions and engage in signaling resulting from or mediated by a kinase cascade within a signal transduction pathway.
  • Examples of scaffolding and adaptor proteins in MAPK signaling are described in Kolch, W. (2000), Kolch, W. (2005) and Brown, M.D. and Sacks, D.B. (2009). Further examples of scaffolding and adapter proteins for these and other signal transduction pathways are provided in Good, M.C. et al. (2011 ), Ritter, S. L. and Hall, R. A. (2009), Kristiansen, K. (2004) and Hall, R.A. and Lefkowitz, R.J. (2002).
  • Scaffold and adaptor proteins previously found associated with Erk include isoforms of KSR, Sef, 14-3-3, IQGAP1 and ⁇ -arrestin.
  • KSR-1 acts as a positive regulator of Ras-Erk signaling by binding Raf-1 and MEK-1/2 to activate Erk-1/2 in response to growth factor stimulation.
  • a second isoform KSR-2 is implicated in the activation of MEK-3 and Erk by the MAP3Ks MEKK-3 and Tpl-2.
  • Tpl-2 in addition to its protein kinase activity, is also considered to be a scaffold protein and is involved in pro-inflammatory signaling. Undesired inflammation from this signaling may support initiation or progression of hyperproliferation conditions disclosed herein through downstream activation of NF-KB.
  • Protein scaffolding also supports other protein-protein interactions that result in additional cross-talk between Ras-Erk and other kinase signaling cascades.
  • Raf-1 associates with Tpl-2, which therefore permits cross-talk between the p38 MAPK pathway (via Tpl-2 activation of MEKK-3), the TNFCC-NFKB pathway (via Tpl-2 activation of IKK) and the Ras-Erk pathway (via Raf-1 activation of MEK-1 or MEK-2).
  • Tpl- 2 (Cot) is a MAP3K involved in NFKB activation.
  • PI3K may also serve in scaffolding of protein complexes in addition to having kinase activity.
  • the p85 regulatory subunit of PI3K directly interacts with intracellular AR or ERa, which is enhanced by i-AR interaction with androgen.
  • Src positively modulates the interaction of PI3K with AR.
  • PI3K-AK signaling may also cross-talk with AR and Src signaling.
  • Tfg is also considered a scaffold protein as disclosed herein that lacks intrinsic kinase activity due to the presence of various protein-protein interaction domains that may facilitate cross-talk between the Ras-Erk and PI3K-Akt signal transduction pathways.
  • the phosphoserine adapter protein 14-3-3 is involved in regulation of cell-cycle check points, proliferation, differentiation and apoptosis and acts primarily by effecting the subcellular localization of its binding partners.
  • the isoform 14-3-3 ⁇ inhibits Ras-Erk signal transduction by acting as a negative regulator of Raf-1 by binding to phoshoserine- 259 of Raf and sequestering this MAP3K away from the cytoplasmic membrane to the cytosol.
  • the subcellular localization of 14-3-3 ⁇ also affects the pro-apoptotic activity of Bad.
  • Bad promotes apoptosis by binding Bcl-2 and Bcl-x L at the mitochondrial membrane.
  • phosphorylation of Bad generates a binding site to 14-3-3 to cause Bad to disengage from Bcl-2 and Bcl-x L leading to its sequesterization in the cytosol.
  • Those proteins freed from Bad in turn associate with Bax and Bak to prevent their aggregation on mitochondria, and subsequent cytochrome c release, thereby additionally inhibiting apoptosis.
  • dysregulated Erk can indirectly inhibit apoptosis though its phosphorylation of p90 RSK 1 , which then phosphorylates Bad to deactivate this protein's pro-apoptotic activity and whose release from Bcl-2 and Bcl-x L further contributes to inhibition of pro-apoptotic signaling.
  • dysregulated Erk may also directly inhibit apoptosis by its phosphorylation of pro-apoptotic proteins, including caspase-9 or Bim.
  • MP-1 MEK-partner 1
  • MP-1 links Erk-1 with MEK-1 , thus favoring Erk-1 activation over Erk-2.
  • MP-1 requires its interacting protein p14 as for example in EFG-mediated activation of Erk-1 . Therefore, compounds that improve binding interactions between one or more or more of Erk, MP-1 or p14 or can substitute for MP-1 or p14 will typically favor cytosolic signaling through Erk-1 in comparison to nuclear signaling through Erk-2.
  • Raf kinase inhibitor protein is a member of the phosphatidylethanolamine- binding protein family and behaves as an adaptor protein in Ras-Erk and TNFa-NF-kB Docket No. 354. PATENT signal transduction pathway that disrupts rather than facilitates signaling.
  • RKIP has been shown to disrupt the Raf-1 -MEK1/2-ERK1/2 and NF- ⁇ signaling pathways, via physical interaction with Raf-1 -MEK1/2 and the ⁇ (inhibitor of NF-KB)-IKK (IKB kinase- ⁇ / ⁇ ) complexes, respectively, thereby abrogating the pro-survival and anti- apoptotic properties of these signaling pathways.
  • RKIP functions as a metastatic tumor suppressor in prostate and breast cancers and sensitizes human prostate and breast cancer cells to drug-induced apoptosis.
  • IQGAP1 also regulates many signaling pathways and is a scaffold protein that binds directly to B-Raf, MEK-1/2 and Erk-1/2. Analogous to KSR binding to Raf-1 , IQGAP1 may regulate B-Raf signaling. B-Raf is found mutated in various cancers including melanomas, thyroid carcinomas and colorectal cancer.
  • stabilization of the protein complex IQGAP1 -B-Raf-MEK-1 -Erk-2 should compete with Erk-2 binding to MAP-2 or slow nuclear entry (i.e., negatively modulate nucleo-cytoplasmic transport) of activated Erk-2 since MEK-1 is known to preferentially retain unactivated Erk-2 in the cytosol pending its activation upon stimulation of the Ras-Erk signal transduction pathway. That effect on scaffolding on MEK-1 -Erk-2 should thus retard aberrant cytoskeletal rearrangements associated with neurodegenerative diseases without adversely effecting basal levels of Ras-mediated Erk activity (i.e., cytoplasmic activity of pErk-1 ).
  • a steroid binding site is present in the NH 2 -terminal region of MAP-2C, which is a region specific to this isoform that recognizes C19 steroids such as DHEA.
  • Sef or similar expression to FGF is a scaffolding protein first identified as an inhibitor of MEK/Erk-dependent signaling in fibroblast growth. Sef action directs MEK-Erk complexes to the Golgi apparatus to inhibit dissociation of the complex. This in turn results in inhibition of activated Erk translocation to the nucleus and promotes activity at cytoplasmic targets. In aggressive forms of prostate cancer (CaP), Sef is down-regulated. Docket No. 354. PATENT
  • scaffold proteins include Tfg and isoforms of ⁇ -arrestin discussed elsewhere. Scaffold proteins are often devoid of protein kinase or transcriptional activity; however, proteins having such activity, but also serving as scaffolding proteins depending on context as disclosed herein, include Raf-1 , Tpl-2 and i-AR. Scaffolding by i-AR at ErbB receptors may depend upon activation of the intracellular hormone receptor by Src phosphorylation, since AR-mediated non-genomic effects from this scaffolding apparently does not require (but is enhanced by) androgen.
  • a test or candidate compound having anti-proliferative or pro-apoptotic effects prevents or inhibits the formation of protein complexes involved in aberrant cross- talk between the PI3K-Akt and Ras-Erk signal transduction pathways and/or prevents or inhibits the formation mitogenic Src protein complexes.
  • Such protein complexes include those comprising (1 ) Class I PI3K regulatory subunit p85a or ⁇ 85 ⁇ and Ras, (2) p85a or ⁇ 85 ⁇ and intracellular AR or ERa, (3) p85a or ⁇ 85 ⁇ , i-AR and Src, (4) EFGR, i-AR and i- ERa, and (5) i-AR, i-ERa and Src and 6) protein complexes wherein the aforementioned complexes additionally comprise a scaffold or adaptor protein, wherein i-AR and/or i-Era in the aforementioned protein complexes act as scaffold proteins.
  • the negative modulation of aberrant crosstalk between PI3K-Akt and Ras-Erk pathways may result from formation or stabilization of alternative protein complexes that reduces signal transduction through one or both PI3K and Erk signal transduction nodes.
  • alternative complexes include those comprising p85a or ⁇ 85 ⁇ and Galpha/q (GTP) or p-Tyr-Galpha/q.
  • a test or candidate compound having anti-proliferative or pro-apoptotic effects may also stabilize formation of proteins complexes that isoform selectively localizes pErk-1 into a cytoplasmic compartment relative to its nuclear translocation or isoform selectively localizes Erk-2 in comparison to Erk-1 to a cellular compartment that prevents or inhibits its phospho-activation, thereby resulting in preferential phospho- activation of Erk-1 on stimulation of Ras-Erk signaling.
  • Such complexes include those comprising Erk-1 and ⁇ -arrestin, Erk-1 and MAP-2, or Erk-2 and MEK-1 and may additionally comprise an Erk scaffold protein as described herein.
  • Non-genomic effect means an effect or activity from a transcription factor not directly related to transactivation of gene transcription or an effect or activity from an agonist of an intracellular transcription factor that is not initiated by binding of agonist to the transcription factor and does not directly result in transactivation of gene transcription. Those effects are typically due to intracellular nuclear hormone receptors (i-NHRs) acting upon cytosolic proteins or from activation of membrane receptors that respond to extracellular hormones or synthetic analogs thereof. These Docket No. 354. PATENT non-genomic effects are typically mediated by kinase cascades that terminate in phosphorylation of cytosolic proteins.
  • An activity from such a kinase cascade is sometimes initiated by i-NHR agonist binding to a GPCR or from scaffolding of an i-NHR to an activated RTK, an intracellular membrane or a cytoskeletal structure.
  • other non-genomic effects include effects on ion transport (e.g., intracellular Ca 2+ mobilization) or Erk kinase activation, which typically are hallmarks of GPCR activation.
  • a non-genomic effect may result in apoptosis, survival, cell cycle arrest, proliferation or variation in metabolism.
  • gene transactivation may eventually result. Those secondary consequences and activities resulting therefrom are indirect and therefore are not considered effects directly related to gene transcription.
  • Gene effect refers to transactivation of gene transcription directly resulting from an activated transcription factor binding to its cognate promoter upstream of the gene to be transcribed. Gene transcription that results as a secondary consequence of a primary non-genomic effect is excluded as a direct genomic effect.
  • Substantially similar to refers to an activity or property of a test or candidate compound equal or nearly equal to that same activity or property determined in identical suitable test systems for a reference compound, usually a positive control test compound.
  • “Quantitatively similar to” as used herein refers to a numerically determined activity or property of a test or candidate compound or is a numerically determined change in a property or activity of a suitable test system to which a candidate or test compound is contacted that is similar in, or the same as, the magnitude of that same activity or property determined for another test or candidate compound, usually a positive control compound, when contacted to an identical test system or is 50-150%, 60-140%, 80-120% or 90-1 10% of that activity or property numerically determined for a positive control test compound.
  • test or candidate compound that exhibits an activity or property or affects a change in a suitable test system to which the compound is contacted in the same direction as that determined or observed for another test or candidate compound, usually in reference to a positive control test (i.e., both result in positive or negative modulation of the determined activity or property).
  • a positive control test i.e., both result in positive or negative modulation of the determined activity or property.
  • two compounds that negatively modulate the phosphorylation state of protein to observable extents when applied separately to identical suitable test systems containing Docket No. 354.
  • PATENT the protein to be affected exhibit qualitatively similar activities.
  • a test or candidate compound that is absent an observable activity or property in relationship to a negative control compound may also be characterized as qualitatively similar to that negative control compound.
  • Alkyl refers to moieties with contiguously linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof wherein all of the carbon atoms are saturated.
  • An alkyl substituent for a structure is comprised of an alkyl moiety that is single bonded to that structure through a saturated (i.e., sp 3 ) carbon atom of the alkyl moiety.
  • alkyl moiety that is substituted with one or more moieties as described below for alkenyl, alkynyl, cycloalkyl, aryl and heterocycle (including heteroaryl) provides an alkyl substituent having unsaturated carbons.
  • the number of carbon atoms in an alkyl moiety or substituent is typically 1 to about 10.
  • Expressions such as d- 6 alkyl or C1 -6 alkyl mean an alkyl moiety or substituent containing 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • an alkyl substituent of an organic moiety such as an androst-5-ene, androst-4-ene, androst-1 -ene, androst-1 ,5-diene androst-1 ,4-triene, 1 ,3,5(10)-estratriene, 5a-androstane, 5a-androstan-1 -ene, 5 ⁇ - androstane or 5p-androstan-1 -ene moiety is specified, an unsaturated carbon of that substituent is covalently attached by a single bond to that organic moiety.
  • Alkyl species include by way of example and not limitation the fully saturated groups methyl, ethyl, 1 - propyl (n-propyl), 2-propyl (/so-propyl, -CH(CH 3 ) 2 ), 1 -butyl (n-butyl), 2-methyl-1 -propyl (/so-butyl, -CH 2 CH(CH 3 ) 2 ), 2-butyl (sec-butyl, -CH(CH 3 )CH 2 CH 3 ) and 2-methyl-2-propyl (f- butyl, -C(CH 3 ) 3 ).
  • alkyl moieties or substituents are species or genera selected from the group consisting of Ci -8 alkyl, d- 6 alkyl, Ci -4 alkyl moieties, methyl and ethyl, substituted or unsubstituted (i.e., optionally substituted).
  • An alkenyl substituent for a structure is comprised of an ethenyl moiety that is single bonded to that structure through an unsaturated carbon atom of the ethenyl moiety.
  • Alkenyl moieties or substituents may be comprised, in addition to the ethenyl moiety, of contiguously linked normal, secondary, tertiary or cyclic carbon atoms that are fully saturated, i.e., linear, branched, cyclic and/or one or more unsaturated alkyl moieties as described below for alkenyl, alkynyl, and aryl moieties.
  • the number of carbon atoms in an alkenyl moiety typically is 2 to about 10. C 2 .
  • alkenyl or C2-6 alkenyl means an alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms and is inclusive of the carbons of the ethenyl moiety that defines it as an alkenyl substituent. Docket No. 354. PATENT
  • an alkenyl moiety is specified as a substituent of an organic moiety, such as an androst-5-ene, androst-4-ene, androst-1 -ene, androst-1 ,5-diene androst-1 ,4-triene, 1 ,3,5(10)-estratriene, 5a-androstane, 5a-androstan-1 -ene, 5p-androstane or 5 ⁇ - androstan-1 -ene moiety, an sp 2 carbon of that substituent is covalently attached by a single bond to that organic moiety.
  • alkenyl moieties or substituents are species or genera selected from the group consisting of C 2 . 8 alkenyl, C 2 . 6 alkenyl, C 2 - 4 alkenyl, or vinyl, substituted or unsubstituted (i.e., optionally substituted).
  • Alkynyl refers to linked normal, secondary, tertiary or cyclic carbon atoms where one or more triple bonds are present, usually 1 .
  • Alkynyl moieties or substituents may be comprised, in addition to the ethenyl moiety, of contiguously linked normal, secondary, tertiary or cyclic carbon atoms that are fully saturated, i.e., linear, branched, cyclic and/or one or more unsaturated alkyl moieties as described below for alkenyl and aryl moieties.
  • an alkynyl moiety is specified as a substituent of an organic moiety, such as an androst-5-ene, androst-4-ene, androst-1 -ene, androst-1 , 5- diene androst-1 ,4-triene, 1 ,3,5(10)-estratriene, 5a-androstane, 5a-androstan-1 -ene, 5 ⁇ - androstane or 5p-androstan-1 -ene moiety, a non-aromatic sp carbon of that substituent is covalently attached by a single bond to that organic moiety.
  • an alkynyl substituent is comprised of an ethynyl moiety (i.e., -C ⁇ C-) that is single bonded to that structure through an unsaturated carbon atom of the ethynyl moiety.
  • the number of carbon atoms in an alkynyl moiety or substituent is typically 2 to about 10.
  • C 2 . 6 alkynyl or C2-6 alkynyl means an alkynyl moiety containing 2, 3, 4, 5 or 6 carbon atoms and is inclusive of the carbons of the ethynyl moiety that defines it as an alkynyl substituent.
  • alkynyl moieties or substituents include by way of example and not limitation any of the alkyl moieties or substituents described herein that incorporates a triple bond such as -C ⁇ CH, -C ⁇ CCH 3 , -C ⁇ CCH 2 CH 3 , -C ⁇ CC 3 H 7 or -C ⁇ CCH 2 C 3 H 7 .
  • alkynyl moieties or substituents are selected from the group consisting of C 2 . 8 alkynyl, C 2 . 6 alkynyl, C 2 . 4 alkynyl, more typically ethynyl, 1 -propynyl and 1 -butynyl, substituted or unsubstituted (i.e., optionally substituted).
  • Aryl refers to an aromatic ring system or a fused ring system with no ring heteroatoms (i.e., the ring(s) that are composed of only carbon atoms) comprising 1 , 2, 3 or 4 to 6 fused rings, typically 1 to 3 fused rings and is characterized by a cyclically conjugated system of 4n+2 electrons (Huckel rule), typically 6, 10 or 14 electrons some of which may additionally participate in exocyclic conjugation (cross-conjugated).
  • Huckel rule typically 6, 10 or 14 electrons some of which may additionally participate in exocyclic conjugation (cross-conjugated).
  • species include by way of example and not limitation phenyl, naphthyl, phenanthryl and quinone.
  • an aryl moiety or substituent is phenyl, substituted or unsubstituted (i.e., optionally substituted).
  • aryls are unsubstituted phenyl or phenyl substituted with one or more, typically 1 or 2 independently selected halogen, d- 4 alkyl, C 2 - 4 alkenyl, C 2 - 4 alkynyl or monovalent oxygen-bound substituents as defined herein such as -OH, ether or ester.
  • heteroaryls the heteroatom(s) of the heteroaryl participates in a conjugated system either through pi-bonding with an adjacent atom in the ring system or through a lone pair of electrons on the heteroatom.
  • the heteroaryl ring system may be optionally substituted on one or more its carbon atoms or heteroatoms, or a combination of both, in a manner which retains the cyclically conjugated system.
  • mono cyclic carbon-bonded heteroaryls are bonded at position 2, 3 or 4 of a pyridine, position 3, 4, or 5 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2 or 3 of a furan, thiophene or pyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole
  • bicyclic fused carbon-bonded heterocycles are bonded position 2, 3, 4, 5, 6, 7, or 8 of a quinoline, position 1 , 3, 4, 5, 6, 7, or 8 of an isoquinoline, position 2, 3, 4, 5, 6, or 7 of indole, position 2, 3, 4, 5, or 6 of 7-aza-indole, position 2, 4, 5, 6 or 7 of benzimidazole, positions 2,
  • carbon bonded heteroaryls have one ring oxygen or one or more ring nitrogens, typically 1 or 2, and include by way of example and not limitation 2- furanyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyridyl, 4-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrimidinyl,
  • a heteroaryl attached to those organic moieties through a nitrogen atom of the heteroaryl aromatic ring system is referred to as a nitrogen-bonded heteroaryl or N-heteroaryl.
  • Optionally substituted alkyl means an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycle or other group or moiety as defined or disclosed herein that has a substituent(s) that optionally replaces a hydrogen atom(s) in the group or moiety.
  • substituents are as described above.
  • Optionally substituted phenyl moieties include unsubstituted phenyl, Ph-N0 2 , and Ph-(halogen) 1 2 or 3, wherein halogen independently selected is -F, -CI, -Br or -I, typically - F, -CI or -Br, more typically -F or -CI, and Ph-(optionally substituted alkyl) 2 or 3, wherein optionally substituted alkyl independently selected is typically optionally substituted d- 6 alkyl, more typically methyl, ethyl or isopropyl.
  • Optionally substituted heteroaryl moieties include unsubstituted d- 6 heteroaryl (HetAr), HetAr-N0 2 , and HetAr(halogen) 1 2 0 r 3, wherein halogen independently selected is -F, -CI, -Br or -I, typically -F, -CI or -Br, more typically -F or -CI, -Ph-(optionally substituted alkyl) ! 2 or 3, wherein optionally substituted alkyl independently selected is typically optionally substituted d- 6 alkyl, more typically methyl, ethyl or isopropyl.
  • HetAr unsubstituted d- 6 heteroaryl
  • HetAr-N0 2 HetAr(halogen) 1 2 0 r 3
  • halogen independently selected is -F, -CI, -Br or -I, typically -F, -CI or -Br, more typically -F
  • Optionally substituted phenyl and HetAr moieties further include Ph(0-linked) 1 2 0 r 3 and HetAR (0-linked) 1 2 or 3, wherein the O-linked moiety independently selected is monovalent, typically -OH, ester, ether or divalent, typically -0-(optionally substituted d- 6 alkyl)-0-. More typically the monovalent and divalent O-linked substituents are independently selected from the group consisting of -OH, -OC(0)CH 3 , -OC(0)CH 2 CH 3 , - OCH 3 , -OCH 2 CH 3 , -OCH 2 0-, -OCH 2 CH 2 0-.
  • a substituted phenyl or HetAr having the divalent -0-(optionally substituted d- 6 alkyl)-0- substituent is considered disubstituted with the substituents open valent oxygen atoms bonded to two adjacent carbons of the aromatic ring and therefore is included within Ph(0-Iinked) 2 and HetAR(0-linked) 2 .
  • Ph(0-Iinked) 3 and HetAR(0-linked) 3 all three O-linked substituents independently selected Docket No. 354.
  • PATENT may be monovalent or may contain a divalent O-linked substituent and one monovalent O-linked substituent.
  • phenyl and HetAr moieties have multiple substitutions, typically totaling 2 to 4, more typically 2-3 that are combinations of those previously described and therefore include Ph(0-linked) 1 . 2 (halogen), Ph(0-linked)(halogen) 2 , Ph(0- linked)i -2 (Ci-8 alkyl), Ph(0-linked)(C 1 - 6 alkyl) 2 , Ph(halogen) 1-2 (Ci-8 alkyl), Ph(halogen)(d- 6 alkyl) 2 , Ph(0-linked)i -2 (Ci-8 alkyl)(halogen), Ph(0-linked)(C 1 - 6 alkyl) 2 (halogen), Ph(0- linked)(d- 6 alkyl) 2 (halogen), Ph(0-linked)(d- 6 alkyl)(halogen) 2 , HetAriO-linked) !
  • O-linked, d_ 6 alkyl and halogen substituents are independently selected and are typically -OH, -OC(0)CH 3 , - OC(0)CH 2 CH 3 ,-OCH 3 , -OCH 2 CH 3 , methyl, ethyl, -F or -CI.
  • Optionally substituted alkyl includes unsubstituted d_ 6 alkyl, -CH 2 Ph, -CF 3 , - CH 2 OH, -CH 2 -halogen, wherein -halogen is -F, -Br, -CI or -I, typically -I or -Br, and optionally substituted alkynyl includes -C ⁇ CCH 2 OH, -C ⁇ C-halogen, typically C ⁇ C-CI or - C ⁇ C-Si(R 13 ) 3 , with R 13 as previously defined for silyl ether. More typically an optionally substituted alkynyl is -C ⁇ C-Si(CH 3 ) 3 or -C ⁇ C-Si(t-Bu)(CH 3 ) 2 .
  • Oxygen-bonded moiety, O-linked moiety refers to an oxygen-containing moiety that is attached to an organic moiety, such as steroid moiety including by way of example and not limitation an androst-5,16-diene, androst-1 ,5,16- triene, androst-4,16-diene, androst-1 ,4,16-triene 5a-androstan-16-ene, 5p-androstan-16- ene, 5a-androstan-1 ,16-diene, ⁇ -androstan-l ,16-diene, androst-5-en-17-one, androst- 1 ,5-dien-17-one, androst-1 , 4-dien-17-one, 5a-androstan-17-one, 5p-androstan-17-one, 5a-androstan-1 -en-17-one or an 5p-androstan-1 -ene-17-one moiety, directly though an oxygen atom of the
  • an oxygen-bonded moiety may be a monovalent O-linked moiety and include by way of example and not limitation moieties such as -OH, -OP PR , wherein PR is a protecting group as defined herein, an ester, such as acetoxy, i.e., -0-C(0)-CH 3 , acyloxy, i.e., -0-C(0)-R 12 , wherein R 12 is -H (i.e., formyl ester), optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl or optionally substituted heteroaryl (including optionally substituted heterocycle).
  • moieties such as -OH, -OP PR , wherein PR is a protecting group as defined herein, an ester, such as acetoxy, i.e., -0-C(0)-CH 3 , acyloxy, i.e., -0-C(0)-R 12 , wherein R
  • Monovalent oxygen-bonded moieties further include ether and silyl ether moieties such as Docket No. 354.
  • PATENT alkyloxy Alkyl-O-
  • aryloxy Aryl-O-
  • phenoxy Ph-O-
  • benzyloxy Bn-O-
  • heteroaryloxy Het-O-
  • silyloxy i.e., R 11 0-
  • R 11 is optionally substituted alkyl, aryl, phenyl, benzyl (i.e., -CH 2 Ph), heteroaryl or silyl, i.e., (R 13 ) 3 Si-, wherein R 13 independently are alkyl, aryl or heteroaryl, optionally substituted.
  • monovalent O-bonded moieties are -OH, esters that have the structure - 0-C(0)-R 12 or silyl ethers that have the structure (R 13 ) 3 SiO-.
  • R 12 is d- 6 alkyl or is -CH 3 (i.e., acetate), -CH 2 CH 3 (i.e., propionate), -Ph (i.e., benzoate), -CH 2 Ph (phenylacetate) and 4-nitrophenyl (i.e., p-nitrobenzoate) with -CH 3 especially preferred.
  • R 13 independently are d- 6 alkyl or aryl including -CH 3 , -CH 2 CH 3i f-butyl or -Ph. More typical oxygen-bonded silyl ethers are trimethylsilyloxy and f-butyldimethylsilyl-oxy moieties.
  • Carbon-bonded moiety refers to a moiety or substituent that is attached to another organic moiety, such a steroid moiety, including by way of example and not limitation an androst-5,16-diene, androst-1 ,5,16- triene, androst-4,16-diene, androst-1 ,4,16-triene 5a-androstan-16-ene, 5p-androstan-16- ene, 5a-androstan-1 ,16-diene, ⁇ -androstan-l ,16-diene, androst-5-en-17-one, androst- 1 ,5-dien-17-one, androst-1 , 4-dien-17-one, 5a-androstan-17-one, 5p-androstan-17-one, 5a-androstan-1 -en-17-one or an 5p-androstan-1 -ene-17-one moiety, directly though a carbon
  • An C-bonded moiety include groups such as acyl, i.e., -C(0)-R 12 , wherein R is -H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted C- heterocycle or carboxylate, i.e., -C(0)-OR 12 , wherein R 12 is -H or its corresponding salt, - C(0)-0 " , or is as previously defined for ester as for non-limiting examples where R 12 is alkyl, aryl, a C-bonded heteroaryl or a C-bonded heterocycle.
  • Protecting group as used here means a moiety that prevents or inhibits the atom or functional group to which it is linked from participating in unwanted reactions.
  • Preferred R 11 for oximes moieties are -H, alkyl or -Si(R 13 ) 3 , with R 13 as defined for silyl ether.
  • Ketals include cyclic ketals that contain structures such as -0-C(R 16 ) 2 - C(R 16 ) 2 0-, wherein R 16 independently selected are -H or alkyl.
  • R PR is a carbonyloxy protecting group
  • R PR is a protecting group for sulfur in thiols, for instance, and for -NHR PR or -N(R PR ) 2 -
  • R PR independently selected is a nitrogen atom protecting group for primary or secondary amines.
  • the protecting groups for sulfur or nitrogen are usually used to avoid unwanted reactions with electrophilic compounds.
  • the protecting groups for oxygen are used to avoid unwanted reactions with electrophiles, and are typically esters (e.g.
  • organometallic reagents or other highly basic reagents are typically ethers, optionally substituted, including alkyl ethers, (e.g., methyl or tetrahydropyranyl ethers) alkoxymethyl ethers (e.g., methoxymethyl or ethoxy-methyl esters), optionally substituted aryl ethers and silyl ethers (e.g.
  • TMS trimethylsilyl
  • TES triethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • TIPS triisopropylsilyl
  • SEM [2-(trimethylsilyl)ethoxy]methylsilyl
  • Halogen means fluorine, chlorine, bromine or iodine.
  • Ras-Erk signaling Erk-1 and Erk-2 have opposing roles in mediating signaling through Ras referred herein as Ras-Erk signaling.
  • Ras is a small G protein with intrinsic GTPase activity that associates with the cell membrane.
  • Raf When in the activated GTP-bound state Ras recruits Raf, which is a MAP3K, by its interaction with the GDP/GTP exchange factor son-of sevenless (SOS). That exchange factor is brought to the cell membrane by growth-factor-receptor-bound protein 2 (Grb-2) adapter protein, which recognizes phosphorylated receptor tyrosine kinase (pRTKs) docking sites.
  • Grb-2 growth-factor-receptor-bound protein 2
  • Erk phosphorylates SOS to disrupt the cell membrane-based complex that had led to its activation through Ras signaling.
  • Examples of opposing isoform roles include overexpression of ERK1 by not ERK2 gene that was found to inhibit Ras-dependent cell growth, which is an effect independent of its phosphorylation status of the MAPK.
  • Erk-1 deficiency results in the enhanced stimulus-dependent activation of Erk-2 without a compensatory increase in Erk- 2 protein levels, with an opposite effect on proliferation observed with ERK2 knockdown.
  • Erk-1 and Erk-2 when inactive are retained in the cytoplasm in heterodimeric complexes with MEK-1 or MEK-2. Phosphorylation of the MEK isoforms by upstream kinases triggers phosphorylation of its Erk partner, which then dissociates from the heterodimer complex.
  • a serine residue Ser-298
  • PAK-1 or Rac which are protein kinases activated upon integrin-mediated adhesion
  • the signaling output from Ras-Erk pathway may be dependent on the relative concentrations or activities of Erk-1 and Erk-2. Test or candidate compounds that increase signaling activity through Erk-1 in comparison to Erk-2 are therefore expected, based upon the insights provided by the invention disclosed herein, to have anti- proliferative properties.
  • Erk-1 associates with both MEK isoforms and activated Erk-1 typically acts upon cytoplasmic targets, and therefore does not require dissociation as a predicate for activity as does activated Erk-2 for activity in the nucleus
  • compounds that stabilize the interaction of MEK-1 or MEK-2 with Erk-1 would increase retention of pErk-1 in the cytoplasm.
  • compounds that preferentially stabilize the interaction of MEK-1 with Erk-1 would allow Erk-1 to more effectively compete with Erk-2 for this MAPKK isoform, thus reducing the ability of MEK- 1 to properly sequester inactive Erk-2 in the cytoplasm for activation and subsequent translocation of pErk-2 to the nucleus upon growth factor stimulation. Therefore, test compounds that preferentially stabilize the interaction of MEK-1 with Erk-1 are expected to be useful in the treating hyperproliferation conditions described herein.
  • Erk is dephosphorylated in the nucleus it is rapidly exported out by a mechanism that is mediated in part by re-association with MEK that had entered the nucleus independently.
  • PEA-15 also known as PED, which is a death effector domain (DED)-containing protein, also associates with Erk in the nucleus and due to it nuclear export sequence also mediates relocation of this MAPK to the cytosol. Therefore, some preferred test or candidate compounds modulate the interaction of Erk with PED, for example, by stabilizing the interaction of Erk-2 with PED, thus truncating long-term signaling through Erk-2 due to its more rapid nuclear export. Such test compounds having that characteristic for selection as a candidate compound are thus expected to be useful in the treating hyperproliferation conditions described herein. Docket No. 354.
  • MAPKs are also known to associate with MAPK phosphatases (MKPs), also known as dual-substrate phosphatases (DUSPs), since they hydrolyze tyrosine and Ser/Thr phosphate residues, and are mediated by at least two protein-protein interaction sites.
  • MKPs MAPK phosphatases
  • DUSPs dual-substrate phosphatases
  • the nine known MKP isoforms are unrelated to the Ser/Thr phosphatases but belong to the superfamily of protein tyrosine phosphatases (PTPase).
  • PTPase protein tyrosine phosphatases
  • One interaction site on MAPKs recognizing MKPs is the common docking (CD) domain, previously discussed, and another site is a domain referred to as the FXFP or F-domain (FD), so named for the conserved amino acid sequence in this MAPK binding domain.
  • CD and FD domains among other potential protein-protein interaction sites are thought to confer selectivity
  • MKP-3 is predominately located in the cytoplasm due to the presence of a putative nuclear export signal, which may, along with CD, FD and other potential protein- protein binding domains, contribute to Erk binding, and are highly specific for deactivating Erk1/2.
  • a putative nuclear export signal which may, along with CD, FD and other potential protein- protein binding domains, contribute to Erk binding, and are highly specific for deactivating Erk1/2.
  • CD binding is thought more important to binding affinity compared to the other domains it is postulated to be not essential to the Erk- induced activation of MKP-3.
  • productive interaction of activated Erk with MPK-3 is believed to occur. This interaction is expected, based upon the insights provided by the invention disclosed herein, to activate MKP-3 phosphatase activity to efficiently deactivate Erk.
  • phosphatases of appropriate specificity to be physiologically relevant regulators of Erk include the tyrosine phosphatase HePTP and the Ser/Thr phosphatase PP2A. Therefore, the strength and duration of the Erk-2 signal from MEK-1 activation is postulated to be balanced by the opposing action of MEK-1 and multiple protein phosphatases that includes single specificity and dual specificity phosphatase and are expected, based upon the insights provided by the invention disclosed herein, to affect the time course and threshold for Erk-2 reactivation. Therefore, test compounds that preferentially stabilizes the interaction of Erk-2 with MKP-3 to preferential retain that isoform in an unphosphorylated or deactivated state MEK-1 are expected to be useful in the treating hyperproliferation conditions described herein.
  • E-3a-diol 17a-ethynyl-5a-androstane-3a,17p-diol, referred herein as E-3a-diol, primarily affects Erk MAPK signaling in comparison to p38 or JNK MAPK signaling. Additionally, it has been unexpectedly found that E-3a-diol selectively affects the phosphorylation status of Erk-1 in comparison to the isoform Erk-2. E-3a-diol also unexpectedly exerts different localization effects on the two isoforms that are believed to be mediated by scaffold-adaptor proteins. Thus, preferred candidate Docket No. 354.
  • PATENT compounds positively modulate the phosphorylation state of Erk-1 or negatively modulate the phosphorylation state of Erk-2 in comparison to the other isoform.
  • Other preferred compounds are negative modulators of nucleo-cytoplasmic translocation.
  • phosphorylation states of the Erk isoforms or nucleo-cytoplasmic translocation of total pErk or for an isoform thereof i.e., pErk-1 or pErk-2
  • a suitable cell-based test system preferably an in vitro cell-based test system, with a test compound as compared to a sham contacted cells in an identical test system.
  • Erk phosphorylation states or pErk nucleo-cytoplasmic transport may also be evaluated relative to that observed when test cells are contacted with a positive control for negative modulation of nucleo-cytoplasmic transport (e.g., E-3a-diol) or when test cells are contacted with a positive control for negative modulation of nucleo-cytoplasmic transport (e.g., 3a-diol) or positive modulation of Erk-2 phosphorylation state.
  • a positive control for negative modulation of nucleo-cytoplasmic transport e.g., E-3a-diol
  • a positive control for negative modulation of nucleo-cytoplasmic transport e.g., 3a-diol
  • positive modulation of Erk-2 phosphorylation state e.g., Erk phosphorylation states or pErk nucleo-cytoplasmic transport
  • the invention provides methods to identify candidate compounds having desirable biological activities, e.g., as anti-cancer drugs or anti-inflammation drugs.
  • Characteristics of preferred candidate compounds include properties such as (i) a capacity of differentially regulate Erk-1 compared to Erk-2, (ii) a capacity to promote or increase apoptosis, e.g., by re- regulating dysregulated AR signaling, (iii) a relatively low toxicity in a mammal, e.g., having a therapeutic index of at least two, more preferably at least about five, most preferably at least about 10 and/or (iv) other biological properties or activities described herein.
  • test compounds have a molecular weight of 200-1 ,000 amu or 200-800 amu, and are non-peptidic, but may be peptidic or have higher molecular weight as, for example, when a test compound is used as a reference compound that selectively elicits extracellular effect(s) by binding to a plasma membrane receptor (e.g., a GPCR) in comparison to intracellular effects (e.g., those resulting from binding to protein complexes or scaffold proteins in the cytoplasm or nucleus).
  • a plasma membrane receptor e.g., a GPCR
  • Such reference compounds are typically cell-impermeable and include androgen-protein conjugates (e.g., testosterone-bovine serum albumin (T-BSA) or DHT-BSA conjugate), optionally coupled to a fluorophore, as described herein.
  • androgen-protein conjugates e.g., testosterone-bovine serum albumin (T-BSA) or DHT-BSA conjugate
  • T-BSA testosterone-bovine serum albumin
  • DHT-BSA conjugate DHT-BSA conjugate
  • Other peptidic substrates recognizes the CD or FD protein-protein interaction domain of a MAPK and may be derived from the amino acid sequence of the corresponding protein-protein interaction domain of a substrate or effector protein for that MAPK.
  • a test compound is initially screened for a biological response (e.g., modulation of a phospho-protein's activity or phosphorylation state as described herein) Docket No. 354.
  • PATENT by contacting the test compound with a suitable test system in a concentration range of about 100 ⁇ to 0.001 ⁇ , preferably in a range including, e.g., about 25 ⁇ , 10 ⁇ or 1 ⁇ concentration (final concentration within the test system).
  • Preferred initial suitable test systems are in vitro test systems that screen test compounds for one or more biological responses qualitatively similar to an activity elicited by E-3a-diol when that reference compound is contacted with a control test system.
  • test compounds that oppose one or more biological responses elicited by 3a-diol when that reference compound is contacted to those same test systems.
  • a test compound for consideration as a candidate compound that provides a desired biological response within the concentration range of the initial screen provides an EC 50 of 0.1 ⁇ or less, more preferably between about 0.1 ⁇ to 0.001 ⁇ (i.e., 100 nM to 1 nM) or less or about 0.005 ⁇ (i.e., 5 nM) or less in a subsequent screen for that biological response (e.g., negative modulation of PI3K p85 regulatory subunit tyrosine phosphorylation state or positive modulation of Erk-1 activation loop phosphorylation state in comparison to that of Erk-2).
  • Other preferred candidate compounds are test compounds that have the aforementioned Erk-1 and/or Erk-2 effects and additionally have negligible effect on one or more isoforms of p38 or JNK MAPK. Such compounds have a negligible effect on the phosphorylation capacity or rate of pErk-1 or pErk-2 towards an Erk-1/2 substrate or effector protein in a suitable cell-free in vitro test system as described herein. More preferred candidate compounds have negligible binding to the ATP binding site of Erk-1 or Erk-2. Other more preferred candidate compounds are test compounds that exhibit efficacy in treating chemically induced or xenograft implanted tumors and have a therapeutic index of 2 or more when tested in relevant animal model(s).
  • Particularly preferred candidate compounds include test compounds that exhibit efficacy against cancer or hyperproliferating cells and have low liver toxicity or are characterized by a therapeutic index of 10 or more as when contacted with cancer or hyperproliferating cells in a suitable animal model. More preferred are candidate compounds that are efficacious (e.g., demonstrates statistically significant reduction in tumor burden, progression or incidence) when administered at about 0.1 mg/kg to 350 mg/Kg to an animal suffering from a hyperproliferation condition or about 0.01 mg/Kg to 25 mg/Kg in a human. The preferred candidate compounds may be further evaluated for their ability to induce one or more other effects observed E-3a-diol (i.e.,. in addition to effects of E-3a-diol on Erk-1/2 and PI3K phosphorylation states) as described herein.
  • E-3a-diol i.e.,. in addition to effects of E-3a-diol on Erk-1/2 and PI3K phosphorylation states
  • Other preferred candidate or test compounds will mediate sequesterization of Erk Docket No. 354. PATENT or pErk in the cytoplasm so as to positively modulate Erk-1 phosphorylation or favor cytosolic retention of pErk-1 (i.e., negatively modulate pErk-1 nucleo-cytoplasmic translocation) . More preferred compounds will mediate subcellular localization of Erk-2 so as to negatively modulate this isoform's phosphorylation state.
  • cytoplasmic Erk-1 or pErk-1 will mediate subcellular localization of cytoplasmic Erk-1 or pErk-1 so to positively modulate this isoform's cytosolic activity (e.g., by promoting Erk-1 phosphorylation or inhibiting pErk-1 nucleo-cytoplasmic translocation) with minimal or no activation of anti-apoptotic proteins.
  • Other more preferred compounds will mediate subcellular localization of cytoplasmic Erk-1 or pErk-1 so to positively modulate this isoform's cytosolic activity (i.e., by promoting Erk-1 phosphorylation or inhibiting p-Erk-1 dephosphorylation) with minimal or no activation of anti-apoptotic proteins.
  • a preferred candidate or test compound will localize pErk-1 to an endomembrane other than that of mitochondria, with localization to endosomes more preferred.
  • Other preferred candidate or test compounds modulate spatial or temporal regulation of signaling from the signal transduction nodes Erk-1 and/or Erk-2 that are expected, based upon the insights provided by the invention disclosed herein, to have anti-proliferative or anti-inflammatory effects and may result from candidate or test compounds that (i) promote stabilization of Erk-2 containing protein complexes through sequesterization of one or more of its component(s) in the cytoplasm to prevent Erk-2 signal transmission, (ii) prevent activated Erk-2 from translocating to the nucleus (i.e., negatively modulating pErk-2 nucleo-cytoplasmic translocation), (iii) retaining deactivated Erk-2 in the nucleus (i.e., positively modulating Erk-2 nucleo-cytoplasmic translocation), (iv) promote stabilization or retention of protein complexes containing unphosphorylated Erk-2 in a subcellular compartment less accessible to its activating MAPKK, (v) promote stabilization of Erk-1 containing protein complexes
  • Additional preferred candidate or test compounds mediate cytosolic Docket No. 354.
  • Downstream outcomes from this negative nucleo-cytoplasmic transport of actAR includes re-regulation of AR signaling whereby differentiation (and hence apoptosis) is promoted, anti-apoptotic gene transactivation is negatively modulated, pro-apoptotic gene transactivation is positively modulated or some combination of these effects.
  • Scaffold or adaptor proteins effect the spatial regulation of Erk1/2 activation by directing the activity of Erk-1 and Erk-2 to the appropriate subcellular compartments. Furthermore, the effect of scaffold or adaptor proteins are temporally-dependent with respect to concurrent signaling through mitogenic and pro-inflammatory signaling cascades due to assembly of protein complexes with one or more components in common with complexes that are assembled during Ras-Erk signaling. Thus, the assembly of a multi-component complex will sequester the components within it away from other signaling pathways. Therefore, scaffold proteins will participate in specifically activating some signaling cascade while inhibiting others.
  • Some preferred candidate compounds affect protein complex assembly and localization of these complexes by modulating KSR- mediated scaffolding such that Erk-2 activity or phosphorylation state is negatively modulated in comparison to that of Erk-1 or by modulating the behavior of another or one or more other Erk- interacting scaffolding and adaptor proteins in the manner indicated below.
  • RKIP interferes with assembly of a competent Ras-Raf-MEK-Erk protein complex by acting as a competitor of MEK binding to Erk.
  • RKIP negatively modulates Erk activity through negative phosphorylation state modulation of MEK. Therefore, based upon the insights of the present invention, a candidate or test compound that recapitulates or enhances the competitive binding behavior of RKIP may temper long-term mitogenic signaling through RTKs mediated by Erk to avoid triggering an undesired gene transcription program while interfering with pro-inflammatory cascades mediated through Raf-1 and NF-kB.
  • Such compounds identified by the screening methods described herein should be useful in treating cancers such as breast and prostate cancers and the underlying inflammation that supports their metastatic potential. Docket No. 354.
  • a candidate compound that affects IQGAP1 scaffolding of Erk will disrupt cytoskeletal dynamics, which will adversely affect proliferating cells in comparison to quiescent cells. Such compounds will be useful in treating certain cancers, including those dependent on Erb-signaling, and screening methods described herein should be useful in their identification. Therefore, some preferred candidate compounds affect IQGAP1 scaffolding involving Erk to inhibit B-Raf signaling, which should be useful in treating hyperproliferation conditions disclosed herein.
  • 14-3-3 protein is involved in cross-talk between the proinflammatory pathway TNFCC-NFKB and the mitogen-activated Ras-Erk pathway and 14-3-3 ⁇ also affects the pro-apoptotic activity of Bad, then some preferred candidate compounds negatively modulate the adaptor function of 14-3-3 protein. Such compounds should exert antiproliferative effects by re-regulating aberrant or excessive cross-talk between these two signal transduction pathways in cancer cells.
  • chaperone proteins may affect particular protein complexes involving Erk, e.g., by stabilizing Hsp-90-Erk-1 or Hsp-90-Erk-2 complexes, thereby negatively modulate nucleo-cytoplasmic transport of activated Erk (e.g., pErk-1 ) or negatively modulate the phosphorylation state of Erk-2, some preferred test or candidate compounds will positively modulate these interactions in order to direct pErk activity to cytosolic targets required for differentiation. Stabilization of Hsp-90-Erk complexes should also inhibit other signaling cascades that aberrantly or excessively cross-talk with the Ras-Erk pathway (e.g., TNFa- NFKB pathway). Such compounds should then exert concurrent pro-apoptotic and antiinflammatory effects useful for treating the hyperproliferation conditions disclosed herein.
  • Test or candidate compounds that bind to the NH 2 -terminal region of MAP-2C may positively modulate binding of Erk-2 to MAP-2C to form a non-productive complex that retards this isoform's phosphorylation and negatively modulates nucleo-cytoplasmic Docket No. 354. PATENT transport of pErk-2 which does form. The resulting cytoplasmic localization of inactive Erk-2 should therefore negatively modulate Erk-mediated deactivation of pro-apoptotic proteins and transactivation of pro-survival genes. Therefore, such compounds are useful in treating one or more of the hyperproliferation conditions described herein.
  • E-3a-diol is a biased GPCR ligand due to its engagement of an alternative signal transduction pathway.
  • T-BSA cell conjugates contacted with LNCaP cells can induce Erk phosphorylation [Wang, Z. et al. (2008)], which is an effect sensitive to the MEK-1 inhibitor PD098059, inhibit cell proliferation [Hatzoglou, A. et al. (2005)] with an IC 50 of 5 nM and induce PSA secretion Docket No. 354.
  • 3a-diol increases Akt signal transduction [(Nunlist, E.A. et al. (2004); Zimmerman, R.A. et al. (2004); Yang, Q. et al. (2008); Dozmorov, M.G. et al. (2008)] and T-BSA increases PI3K activity in LNCaP [Kampa, M. et al. (2003); Papakonstanti, E.A. et al. (2003)) , MCF-7 (Kallergi, G. et al.
  • T-BSA has been shown to increase total pErk in vascular smooth muscle and skeletal muscle cells whereas in such normal cells DHT selectively increases pErk-2, but not pErk-1
  • DHT selectively increases pErk-2, but not pErk-1
  • E-3a-diol selectively permits Erk-1 phosphorylation in LNCaP to the exclusion of Erk-2 without increasing gene expression of either isoform.
  • T-BSA also increases total pErk levels, but no isoform specific effects were described.
  • T-BSA is reported to have no effect on Erk phosphorylation in hippocampal and i-AR transfected PC12 cells [Nguyen, T.-V. V. (2005)] whereas DHT induces rapid (within 5 min.) preferential Erk-2 phosphorylation over Erk-1 in hippocampal cells with no effect on isoform protein levels and is neuroprotective (i.e., anti-apoptotic).
  • E-3a-diol and T-BSA have opposite effects on phosphorylation of PI3K with E-3a-diol decreasing tyrosine phosphorylation of the p85 regulatory subunit whereas T-BSA increases that phosphorylation in LNCaP and MCF-7 [Papakonstanti, E.A. et al. (2003); Kampa, M. et al. (2004); Kallergi, G. et al. (2007)].
  • DU-145 it is only on longer exposure to T-BSA (2h) is decreased p85 tyrosine phosphorylation observed Docket No. 354.
  • E-3a-diol which is structurally unrelated to the aforementioned conjugates by virtue of its free C-3 hydroxy substituent, would promote apoptosis and thus be considered an anti-proliferative m-AR agonist and would do so by engaging a different G-protein (i.e., Galpha/i for T-BSA and Galpha/q for E-3a-diol).
  • G-protein i.e., Galpha/i for T-BSA and Galpha/q for E-3a-diol
  • E-3a-diol pro-apoptotic effects of E-3a-diol are mediated by reduced PI3K activity through decreasing tyrosine phosphorylation of it p85 regulatory subunit and by isoform selective increases in p-Erk-1 levels without Erk-2 activation, which are unexpected given the opposite effects observed for cell-impermeable androgen-protein conjugates.
  • Natural androgens such as T and DHT at physiological concentrations and the synthetic androgen R1881 also interact at m-AR in addition to i-AR. The combined effects from interaction at the two receptors are to promote proliferation.
  • E-3a-diol will also oppose pro-proliferative effects of T, DHT and R1881 by direct competition of these androgens at m-AR thereby disrupting those combined effects and further to induce nongenomic signaling at m-AR contrary to that induce at m-AR by the aforementioned androgens.
  • contrary nongenomic signaling by E-3a-diol is due to that compound acting as a biased GPCR ligand at m-AR.
  • Ga Protein-dependent Non-qenomic Activity The m-AR is now identified as the G protein-coupled receptor GPR-C6a [Pi, M. et al. (2010)], which previously had been identified as an amino acid sensor [Wellendorph, P. et al. (2005)].
  • GPR-C6a which shares homology with the calcium sensor receptor CaSR, is coupled to the G proteins Ga/i and Ga/q. Unlike CaSR, Ca 2+ binding to GPR-C6a is not required for receptor activation although it does positively modulate that activity.
  • GPCR signaling that is dependent on activation of Ga/i, which inhibits adenylate cyclase activity, can enter the Ras-Erk signaling cascade through cross-talk with other signaling pathways that involve, for example, PI3K (phosphoinositide-3-kinase), Akt (a member of the non-specific serine/threonine kinase family, which is a downstream effector protein of PI3K), Src (a non-receptor tyrosine kinase) or transactivation of RTKs (receptor tyrosine kinases).
  • PI3K phosphoinositide-3-kinase
  • Akt a member of the non-specific serine/threonine kinase family, which is a downstream effector protein of PI3K
  • Src a non-receptor tyrosine kinase
  • transactivation of RTKs receptor tyrosine kina
  • Signaling to the Ras-Erk pathway may also be effected by Ga/q activation of certain isoforms of phospholipase C (PLC), which generate the second messengers inositol triphosphate (IP3) and diacyl glycerol (DAG), to trigger intracellular mobilization of Ca 2+ and to activate phosphokinase C (PKC).
  • PKC phospholipase C
  • IP3 second messengers inositol triphosphate
  • DAG diacyl glycerol
  • PKC phosphokinase C
  • a G protein-independent pathway dependent on ⁇ -arrestin scaffolding is responsible for slower activation of Ras-Erk signaling through recruitment of Src, which is also dependent on ⁇ -arrestin scaffolding. Docket No. 354.
  • PLC- ⁇ ! is also known to be effected through direct interaction with Ga/q in its activated GTP-bound state, Ga/q (GTP).
  • Deactivation of Ga/q (GTP) occurs in a feedback inhibitory manner by the GTPase activity of activated PLC- ⁇ ! .
  • Ga/q is associated with a sub-population of PLC- ⁇ ! as Ga/q (GDP) at the plasma membrane, and the affinity for this association is increased on Ga/q activation. That pre- association of PLC- ⁇ ! and Ga/q provides for a rapid cellular response mediated by PLC- ⁇ ! upon GPCR-Ga/q activation that does not depend on Ga/q (GTP) release from the receptor.
  • Ga/q(GTP) inhibits p1 10a PI3K [Ballou, L.M. et al. (2003)] by binding directly to p100a/p85 and displacing Ras(GTP) [Ballou, L.M. et al. (2006); Jimenez, C. et al. (2002); Carpenter, C.L. et al. (1993)], whereas receptors coupled to the Gi/o family of G proteins increase PI3K activity through stimulation of the isoforms p1 10 ⁇ - and p1 10 ⁇ - ⁇ 3 ⁇ .
  • Phosphorylation of p85 Ser-608 on p85a also is inhibitory and may be effected through feedback inhibition by the intrinsic serine kinase activity of PI3K.
  • Ga/q and PLC- ⁇ ! Ga/q and PI3K may also exist in pre-associated complexes, with the Ga/q-PLC- ⁇ and Ga/q-PI3K protein complexes probably localized in different subcellular compartments [Golebiewska, U. et al. (2008)].
  • the p85 regulatory subunit has an SH3 domain, a BcR homology domain (BH) flanked by proline-rich sequences and two SH2 domains separated by a domain that binds to the catalytic domain p1 10a (the inter-SH2 domain). Due to these binding domains, p85 also mediates interaction of PI3K with the deactivating phosphatase SHP- 1.
  • the p100a domain contains a p85-interacting region and a Ras-binding domain.
  • the p85 domain mediates translocation to activated RTKs, which induces an activating conformational change in p1 10a, and phosphorylation of p85 Tyr-688 by Src increases PI3K activity by releasing p1 10a from p85 inhibition [Kodaki, T. et al. (1994)].
  • Ras (GTP) binding to the p1 10a subunit further increases catalytic activity [Rodriquez-Viciana, P. et al. (1994)].
  • tyrosine kinase stimulation releases inhibition of p85, which permits Docket No. 354.
  • PATENT further enhancement of PI3K catalytic kinase activity through interaction of p1 10a subunit with Ras(GTP).
  • Those various interactions between the p85 and p100 subunits with components of other signal transduction pathways provide for cross-talk between PI3K-Akt signaling with that of Ras-Erk, Src and RTK.
  • Galpha/q disrupts the p85-Src interaction that otherwise would lead to stimulation of the PI3K/Akt pathway through Src tyrosine phosphorylation of the p85 regulatory subunit.
  • tyrosine phosphorylation of p85 is negatively modulated by Galpha/q activation.
  • E- 3a-diol restores the activity of the pro-apoptotic protein thereby promoting apoptosis.
  • the screening methods disclosed and claimed herein are useful to identify compounds having low toxicity and/or capacity to elicit that pro-apoptotic activity in hyperproliferation conditions such as AR signaling-dependent or androgen-associated cancers.
  • a test or candidate compound that has an activity qualitatively or quantitatively similar to E-3a-diol at m-AR will activate Ga/q to negatively modulate the activity of a Class I A PI3K such as ⁇ 1 10 ⁇ / ⁇ 85 ⁇ and ⁇ 1 10 ⁇ / ⁇ 85 ⁇ .
  • This negative regulation is believed to occur by negative modulation of tyrosine phosphorylation status of the PI3K regulatory subunit p85 (as is observed for E- 3a-diol) and possibly by interfering with the binding of activated Ras to the catalytic Docket No. 354.
  • PATENT subunit p100a is believed to occur by negative modulation of tyrosine phosphorylation status of the PI3K regulatory subunit p85 (as is observed for E- 3a-diol) and possibly by interfering with the binding of activated Ras to the catalytic Docket No. 354.
  • GPR-C6a signaling through Ga/q counteracts PI3K signaling that would be due to that receptor's alternative engagement of Ga/i.
  • the prosurvival effect from GPRC signaling through Ga/i that would result from Akt activation by PI3K activity through the intermediacy of phosphatidyl inositol-(3,4,5)-triphosphate (PIP3), would be reversed or opposed by GPR-C6a-Ga/q signaling.
  • PLC- ⁇ ! by Ga/q activation leads to consumption of PI3K's substrate phosphatidyl inositol (4,5)-bisphosphate (PIP2) to further limit Akt activation.
  • G protein class switching by inverse agonist binding to GPR-C6a of a test or candidate compound to release Ga/q with qualitatively or quantitatively similar activity in this regard to E-3a-diol will elicit an anti-proliferative effect by preferentially engaging PI3K in comparison to PLC- ⁇ ! , or if additionally engaging PLC- ⁇ ! doing so such that activation of PKC or nuclear accumulation of pErk associated with that activation does not occur to an extent that obviates the antiproliferative effect from PI3K inhibition.
  • Those dual effects on PI3K and PLC- ⁇ ! activities may provide an additional selection criterion for identifying candidate compounds.
  • E-3a- diol is attributable to biological effects arising from cytoplasmic scaffolding of Erk-1 to retain pErk-1 in a cytosolic subcellular domain.
  • This localization effect may be enhanced by negatively modulating nuclear translocation of pErk-1 that may be formed as a result of PLC- ⁇ ! activation (i.e., E-3a-diol redirects pErk-1 activity due to PLC- ⁇ ! activation to desired cytosylic effector proteins).
  • a test or candidate compound having qualitatively or quantitatively similar effect to E-3a-diol on PLC- ⁇ ! stimulation of Ras-Erk signaling or PI3K activity should also be expected based upon the insights of the present invention to be anti-proliferative. Docket No. 354.
  • Erk-1/2 activity is involved in several cellular processes other than inhibiting apoptosis by phosphorylated deactivation of pro-apoptotic proteins in hyperproliferating cells, and total Erk activity will influence an observed biological outcome [Lefloch, R. et al. (2008)].
  • Ras-Erk signal transduction is important for cellular differentiation [Yashuda, T. (201 1 ); Robitaille, H. (2010); Gaest, C. et al. (2009); Jirmanova, L. et al. (2002)].
  • Ras-Erk signal transduction is important for apoptosis in normally proliferating cells [Ishihara, Y. et al. (201 1 )] and for chemotherapeutic induction of apoptosis in cancer cells [Pal, P. and Kanaujiya, J.K. (201 1 ); Kim, M.J. et al. (2010)]. Additionally, in some malignant cells, particularly those which are PTEN-deficient, Ras-Erk signal transduction is actually inhibited due to Raf phosphorylation by high levels of activated Akt.
  • reactivation of Ras-Erk signaling may have an anti-proliferative effect [McCubrey, J. A. et al. (2007)], and based upon the insights of the invention that anti-proliferative effect would most likely occur with a test or candidate compound that selectively phospho-activates Erk-1 vs. Erk- 2.
  • Raf-1 is inactive when Ser-43, Ser-259 and Ser-621 are phosphorylated, which allows binding of 14-3-3 to induce an inactive configuration.
  • Phosphatases dephosphorylate pSer-259, whereupon 14-3-3 disassociates to allow Raf to be activated by phosphorylation of Ser-338, Tyr-430 and Tyr-431 .
  • a test or candidate compound that reactivates Ras-Erk signal transduction in PTEN-deficient cancer cells to have an anti-proliferative effect may do so by negative modulation of the phosphorylation states of one or more of Ser-43, Ser-259 and Ser-621 , positive modulation of the Ser phosphorylate state of inactive Raf-1 (i.e., by altering the serine phosphorylation pattern) or negatively modulate binding of the scaffold protein 14-3-3 to Raf-1 .
  • Such effects on Raf-1 Ser phosphorylation state or 14-3-3 binding are alternative criteria for identifying a candidate compound.
  • this engagement of the apoptotic program may result from phosphorylated Elk-1 (a pErk substrate) preferentially retained in the cytoplasm and/or stimulation of p53 signaling in cancer cells retaining functional p53. It is believed those non-genomic effects from reactivation of Ras-Erk signal transduction (i.e., cytosolic p-Elk-1 or p53 activation) stem from negative modulation of pErk-1 nucleo-cytoplasmic transport and is due to scaffolding effects on this MAPK isoform induced by E-3a-diol activity.
  • CaP cell lines such as LNCaP and PC-3 cells have PTEN mutations resulting in high levels of activated Akt and low levels of activated Raf, MEK and Erk.
  • CaP cell lines have varying p53 states, e.g., LNCaP cells (bearing wild-type p53), DU145 cells (bearing de-activated functional p53) and PC3 cells (lacking functional p53). Therefore, some preferred test systems are comprised of hyperproliferating cells having inactive or deleted PTEN, a high level of activated Akt or a preponderance of Raf-1 in its inactive state.
  • test systems have wild-type or de-activated functional p53 (i.e., mutated p53 that binds more tightly to MDM2 than wt-p53, thereby repressing p53 pro-apoptotic activity) with inactive or deleted PTEN, a high level of activated Akt or preponderance of Raf-1 in its inactive state. More preferred are test systems comprising cancer cells with inactive or deleted PTEN, wild-type or inactivated functional p53 and no observable pErk-1/2 when incubated under serum-starved conditions or in androgen- and growth factor-depleted serum.
  • test systems comprise cancer cells with inactive or deleted PTEN that harbor gain of function mutant p53 (p53(GOF)) including LNCaP-R273H cells [Carroll, A.G. (1993)) and those described in Tepper, C.G. et al. (2005) and Nesslinger, N.J. et al. (2003).
  • Preferred test or candidate compounds will positively modulate activity of one or more pro-apoptotic proteins or negatively modulate activity of one or more anti- apoptotic proteins in cells of such test systems despite dysregulated p53 activity from the presence of p35(GOF).
  • a preferred test or candidate compound will reactivate Ras-Erk signal transduction, preferably with negative modulation of nucleo-cytoplasmic translocation of pErk-1 or pElk-1 .
  • the test compound exerts an anti-proliferative effect or sensitizes the cancer cell to apoptosis from co-contact with a cancer chemotherapeutic compound.
  • chemotherapeutic compounds include tubulin disrupting agents, DNA damaging agents, topoisomerase I and II inhibiting agents, disrupters of p53-MDM2 binding, inhibitors of RTKs, PI3K, Akt, NF- ⁇ , AR and ERa, anti-androgens and ERp agonists.
  • Preferred cancer chemotherapeutic compounds are tubulin disruptors, including taxane-based compounds such as paclitaxel, docetaxel, Docket No. 354. PATENT cabazitaxel and other tubulin disruptors described in Jordan, A. et al. (1998) and Perez, E.A. (2009).
  • E-3a-diol-induced localization of pErk-1 in the cytosol results from scaffolding of pErk-1 within a protein complex onto a subcellular cytoplasmic endomembrane. That endomembrane is believed to be those of endosomes and localization of pErk-1 to this structure results from scaffolding, which may result from G protein-independent signaling that is likely initiated by inverse agonist action of E-3a-diol at GPR-C6a (i.e., results from Ga/q release from this GPCR).
  • GPR-C6a-Ga/q signaling may also modulate aberrant or excessive cross-talk between Src and Ras-Erk or Src and PI3K-Akt signal transduction pathways.
  • Src may tyrosine phosphorylate Ga/q at Tyr-356 (henceforth referred to as pGa/q) to increase the amount of bound GTP [Simon, M.I. et al. (1991 ); Umemori, H. et al. (1997)].
  • G protein-dependent signaling through Ga/q in effect converts pro-proliferative signaling from aberrantly activated Src to anti-proliferative signaling.
  • additional selection criteria for identifying a candidate compound can include an intracellular effect on Src activity that redirects PLC- ⁇ ! activity from PKC activation (pro-proliferative) to that of stimulating Ga/q phosphorylation (anti-proliferative).
  • Ga monomer subunits including Ga/i
  • PATENT is positive modulation of Ga/q Tyr-365 phosphorylation in comparison to the phosphorylation state of the corresponding residue in Ga/i, preferably with concommitant negative modulation of the phosphorylation state of that Ga/i tyrosine.
  • This selective engagement of Ga/q can be another selection criterion for identifying a candidate compound.
  • GPR-C6a-Ga/q G protein-dependent signaling is also believed to stimulate Ras- Erk signaling in the absence of RTK activation, within the context of re-regulating signal transduction cross-talk. That occurs, through Ga/q activation of PLC- ⁇ ! (a G protein- dependent effect) believed to occur at the cytosolic plasma membrane and by G protein- independent effects mediated by ⁇ -arrestin scaffolding of a cytosolic Src-containing protein complex or by ⁇ -arrestin scaffolding of Erk at early endosomes, particularly phosphorylated Erk-1 .
  • test or candidate compounds will increase cytosolic Erk-1 within 15 min. without a corresponding increase in nuclear Erk- 1/2. It is further believed that endosomal localization of pErk-1 is responsible for the cytoplasmic subcellular sequesterization described herein that permits cellular functions that support differentiation without deactivation of mitochondrial associated pro-apoptotic proteins by phosphorylation.
  • Ga/q effector protein i.e., between negative modulation of the tyrosine phosphorylation state of PI3K p85 regulatory subunit (inhibition of activity) and positive PLC- ⁇ ! phosphorylation modulation (stimulation of activity), which is presumed to take place in different subcellular compartments, will also affect the strength of anti-proliferative activity resulting from engagement of Ga/q signaling.
  • Ga/q downstream effector protein is also expected to be ligand dependent.
  • Ga/q engagement of PLC- ⁇ ! may lead to activation of protein kinase C, which is associated with nuclear accumulation of pErk and is thus pro-proliferative, while engagement of Ga/q with PI3K decreases its kinase activity and is thus anti-proliferative.
  • test compounds that preferentially engage PI3K, or additionally engage PLC- ⁇ ! without positive modulation or with minimal increase in PKC activity are preferred when considering selection of test or candidate compounds for further studies as described herein.
  • test or candidate compounds will positively modulate intracellular Ca 2+ levels from mobilization of intracellular stores when contacted with cells having or engineered to have functional GPR-C6a. That would occur without significant accumulation of pErk in the nucleus that would otherwise obviate the antiproliferative effect of negatively modulating the phosphorylation state of p85 PI3K subunit.
  • Non- genomic effects from i-AR recruitment to activated RTKs are discussed elsewhere in respect to other desirable selection criteria.
  • Membrane AR Ga/q signaling may also cross-talk with TNFCC-NFKB signaling since TNF-R1 (tumor necrosis factor receptor-1 ) is localized with various GPCRs and many other signaling components in the same subcellular compartments that include lipid rafts and caveolae. Furthermore, it has been reported that the TNF-R1 -TRAF2 (TNF-receptor- associated factor 2) receptor complex associates with Ga/q through ⁇ -arrestin-l scaffolding [Kawamata, Y. et al.
  • scaffolding of Src by a particular ⁇ -arrestin isoform may result from Ga/q-dependent or G protein-independent signaling and is also determined by the Ga/q coupled GPCR and the activating ligand at this receptor.
  • test or candidate compounds to be identified as a candidate compound simulates G protein-dependent signaling through diversion of Ga/q from ⁇ - arrestin-1 to p-arrestin-2 scaffolding are sometimes preferred since such compounds are expected based upon the insights of the present invention to diminish or prevent potential cross-talk between Ga/q and NF- ⁇ . That redirection of Ga/q scaffolding provides an anti-inflammatory effect and avoids confounding signaling from Ga/q activation. Since chronic non-productive inflammation is pro-proliferative and promotes metastasis, the antiinflammatory effect from preferential scaffolding should augment an antiproliferative effect resulting from Ga/q inhibition of PI3K instead of this kinase participating in NF- ⁇ activation.
  • cytosolic retained Erk-1 in preference to Erk-2 activation negatively modulates Erk-2 activation of nuclear transcription factors, thereby negatively modulating the phosphorylation states of cytosol-residing transcription factors (i.e., inhibits their nucleo-cytoplasmic transport) and pro-apoptotic proteins (i.e., inhibits their deactivating phosphorylation). That would occur in part by Erk-1 outcompeting Erk-2 for the upstream activator MEK.
  • the disruption of Erk-2 activation will therefore re-regulate or normalize apoptotic signaling (i.e., will provide an anti-proliferative effect).
  • test or candidate compound that exhibits one or more of the effects associated with Ga/q activation or "inverse" agonist binding to GPR-C6a when contacted with cells in a suitable test system that have or are genetically engineered to have a GPCR coupled to Ga/q provides selection criteria that can be used in identifying a test compound.
  • G protein-dependent effects associated with Ga/q activation or inverse agonist binding by a test or candidate to GPR-C6a include negative modulation of one or more of (i) PI3K regulator subunit tyrosine phosphorylation state, (ii) ⁇ activity, (iii) PIP2 phosphorylation, (iv) PIP2 level without conversion to PIP3 or pErk nuclear accumulation, (v) Ras-PI3K interaction and (vi) Akt activity or phosphorylation state.
  • Other effects include positive modulation of one or more of (vii) GTP-bound Ga/q level, (viii) PLC- ⁇ !
  • Particular preferred test or candidate compounds are those exhibiting one or more of those aforementioned GPR-C6a or Ga/q effects and that positively modulates Docket No. 354.
  • Other particularly preferred compounds will affect scaffolding of pErk-1 through G protein-independent signaling subsequent to Ga/q activation and its release from GPR-C6a (i.e., Ga/q-dependent and -independent signaling acting in parallel) such that pErk-1 is preferentially localized to endosomes or to a subcellular compartment that does not deactivate mitochondrial-associated pro-apoptotic proteins.
  • G Protein-independent Non-qenomic Activity After ligand stimulation, GPCRs are phosphorylated by GPCR kinases (GRKs) to recruit ⁇ -arrestin whose role was thought restricted to agonist-dependent desensitization by internalizing and translocating the receptor to endocytic compartments. In what is known as classical G protein- independent signaling, scaffolding by ⁇ -arrestin then promotes recruitment and activation of Src and also recruits Raf, which is then activated by Src.
  • GPCR kinases GPCR kinases
  • GPCR ligands have dual efficacy effects by signaling through G protein-dependent and -independent pathways with the latter occurring through the intermediacy of the scaffolding protein ⁇ -arrestin. Therefore, in some situations a ligand for the same GPCR exhibits an "inverse agonist" effect by primarily signaling through ⁇ -arrestin scaffolding.
  • a different signaling pathway e.g., Ga/i to Ga/q
  • ⁇ -Arrestin scaffolding of Src in G protein-independent signaling is required for receptor internalization to endosomes and transactivation of RTKs such as EGFR. Since ⁇ -arrestin will also scaffold with components of the Ras-Erk signal transduction pathway it is believed that endosomal targeting of Erk that is mediated by ⁇ -arrestin-Src protein complexes will result in sequestering of pErk to the cytosol whereas ⁇ -arrestin scaffolding of Src at the plasma membrane (i.e., from Src recruitment to membrane- Docket No. 354. PATENT bound GPRC-p-arrestin protein complexes) will transactivate RTKs.
  • both Ga/q and the G protein-independent pathway using p-arrestin-2 scaffolding from GPCR-Ga/q signaling will usually provide the necessary Erk- 1 activation.
  • p-arrestin-2 scaffolding is usually associated with cytoplasmic retention of pErk and is believed to be important for restoring apoptotic signaling while scaffolding by ⁇ -arrestin-l provides cross-talk to pro-inflammatory or pro-proliferative TNFa-NF- ⁇ or MAPK signaling.
  • ⁇ -arrestin-l mediate TNFa-induced Erk activation. Since cross-talk between Ras-Erk signaling and PI3K-Akt signaling may occur, ⁇ -arrestin-l scaffolding of cytosolic Erk may potentiate growth factor induced phosphorylation of Akt by positively modulating Src-mediated tyrosine phosphorylation state of PI3K regulatory subunit. That positive modulation of PI3K phosphorylation state is mediated in part by Ras acting as a scaffold protein.
  • G protein-independent signaling downstream from GPR-C6a-Ga/q activation most likely requires p-arrestin-2 scaffolding in preference to that its isoform ⁇ -arrestin-l in order for a test or candidate compound to negatively modulate the tyrosine phosphorylation state of the p85 regulatory subunit and to exhibit antiproliferative effects that are enhanced by an anti-inflammatory activity.
  • a test or candidate compound that induces p-arrestin-2-mediated G protein-independent signaling emanating from GPR-C6a activation in preference to that mediated by ⁇ -arrestin-l is one selection criterion for identifying a candidate compound.
  • a test or candidate compound that results in positive modulation p-arrestin-2 interaction with Erk- 1 whereby cytoplasmic localization of pEk-1 is promoted or translocation of pErk to the nucleus is inhibited, is a preferred characteristic for identifying a candidate compound in some embodiments.
  • Preferred test systems for determining a test compound's activation of Ga/q and ⁇ -arrestin pathways include ⁇ -arrestin-l , -2 (single) and -1/2 (double) KO and Gq (double) KO mice as described in Kohout, T.A. et al. (2001 ) and Zywietz, A. et al. (2001 ).
  • cancer cells have mutations or amplifications of genes encoding Rel/NF- KB transcription factors or mutations in genes encoding NF- ⁇ signaling regulatory proteins that lead to constitutive activation of NF- ⁇ . It is thought that continuous nuclear Rel/NF- ⁇ activity protects cancer cells from apoptosis (i.e., is pro-survival) and in some cases is pro-proliferative.
  • Rel/NF- ⁇ proteins include the NF- ⁇ proteins p105 and p100.
  • the second class of Rel/NF- ⁇ proteins includes cRel, RelB and RelA (p65), referred to collectively as Rel proteins.
  • E-3a-diol negatively modulates cRel expression
  • a test or candidate compound that inhibits NF-KB transcriptional activity by modulating or normalizing Ras-Erk and/or PI3K-Akt signal transduction in the manner described herein for E-3a-diol would be useful as an anti- proliferative compound. That anti-proliferative effect may be observed in single agent therapy using the test compound or may be observed by way of tumor cells sensitized to other cancer chemotherapeutic compounds, including tubulin disrupting agents.
  • That sensitization may be mediated in part by negatively modulating the phosphorylation state of one or more NF- ⁇ signaling components that results in negative modulation of activity(ies) of the component(s). That sensitization also may be mediated in part by or negatively modulating gene expression for those components.
  • a test or candidate compound that affects transcription for a Rel protein in qualitatively similar manner to E-3a-diol would negatively modulate NF-KB transactivation activity and thus would have an anti-inflammatory effect. That anti- inflammatory effect should prevent initiation or retard progression of a hyperproliferation Docket No. 354. PATENT condition. Negative modulation of one or more of cRel expression, Rel protein level or NF-KB transcription of a ⁇ -inducible promoter genetically engineered into cell of a suitable test system are other selection criteria for identifying a candidate compound.
  • selection criteria related to effects of E-3a-diol on NF- ⁇ activity for indentifying a candidate compound include negative modulation of (i) Rel/NF- ⁇ activity in the nucleus, (ii) NF-kB dimerization, (iii) NF-kB dimer nuclear transport, (iii) phosphorylation state of p100 COOH-terminal region or some combination of the aforementioned effects attributable to decreased cRel expression.
  • the IKK protein complex includes catalytic IKKa and ⁇ subunits and the regulatory NF- ⁇ essential modulator (NEMO), which is also required for NF- ⁇ activation.
  • NEMO regulatory NF- ⁇ essential modulator
  • TANK TRAF family member-associated ⁇ - ⁇ activator
  • Tfg TRK fusion gene
  • Tfg has no known inherent activity, but contains numerous protein-protein interaction domains. Therefore, Tfg exhibits the hallmarks of a scaffolding protein due to its numerous protein-protein interaction domains (which are also present in other scaffolding proteins, including Ras, ⁇ -arrestin and others disclosed herein) and potential binding partners. Tfg is thus capable of bringing together signaling components that are common to several signal transduction pathways. As a further example, Tfg is a direct substrate of c-Src in vitro [Amanchy, R. et al. (2008)] and interacts with TANK and NEMO, which are components of the TNFa- ⁇ - ⁇ signaling pathway.
  • Tfg binds to Src
  • this interaction (mediated by SH3 binding domains) is expected, based upon the insights provided by the invention disclosed herein, to be responsible for additional cross-talk between TNFa-NF- ⁇ and Ras-Erk signaling pathways. Those pathways often become aberrant in various hyperproliferation conditions and chronic inflammatory states that support initiation or progression of such conditions.
  • interaction between Src and Tfg provides another intersection for NF- ⁇ and Erk signal transduction cross-talk with G protein-independent GPCR signaling, which is mediated by ⁇ -arrestin and PI3K-Akt signaling.
  • Tfg associates with phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor that modulates PI3K-Akt signal transduction, which is an activity often lost in caner.
  • PTEN phosphatase and tensin homolog deleted on chromosome 10
  • E-3a-diol through Docket No. 354.
  • PATENT selective activation of Erk-1 in contrast to Erk-2 contributes to restoring normal PI3K activity or reduces aberrant PI3K-Akt signaling in hyperproliferating cells having pro- proliferative RTK or PTEN mutations through its effect on Tfg scaffolding.
  • a test or candidate compound that exhibits one or more of the effects associated with E-3a-diol on Tfg scaffolding when contacted with a suitable cell-based or cell-free test system can satisfy a selection criterion for identifying a candidate compound.
  • Selection criteria include one or more of negative modulation of (i) Tfg with SHP-1 , which will negatively modulate phosphorylation state of PI3K regulatory subunit thereby inhibiting PI3K kinase activity, (ii) Tfg interaction with NEMO or TANK, both of with will negatively modulate NF-kB activity, (iii) Tfg interaction with Src, which will disrupt aberrant Src signaling or redirect Src activity to promote anti-proliferative Ga/q signaling, or (iv) some combination of such effects.
  • E-3a-diol exhibits favorable biologically active in patients with castrate-resistant prostate cancer (CRPC), in addition to patients with androgen-dependent CaP, without eliciting liver toxicity. It is believed that favorable biological activity is mediated through extracellular interaction of E-3a-diol at GPR-C6a and intracellular interaction(s) of this compound with component(s) of the Ras-Erk signaling pathway, including physical interaction with Erk-2, which is believed to be in association with one or more scaffolding proteins. Those favorable activities are applicable to other hyperproliferation conditions where AR signaling can occur due to the presence of functional i-AR (wild-type or mutant). That assertion is supported by anti-tumor effects in breast cancer models, the tumor cells of which are known to contain i-AR, which were observed or are observable in suitable in vivo test systems as described in the examples.
  • E-3a-diol inhibits PI3K activity, the effect of which is to reduce downstream activation of Akt, and possibly that of NFK-B, all of which would otherwise lead to pro-proliferative signaling. Furthermore, it has been unexpectedly found that induced phosphorylation of the MAPK isoform Erk-1 by E-3a-diol occurs in the absence of external growth factors, which is normally required for Erk activation, with or without androgen stimulation (e.g., in intracellular AR signaling- dependent cancer cells incubated in growth factor and androgen-depleted media, with or without support from externally added androgen) or in the absence of observable constitutive RTK activation.
  • G protein-independent signaling through p-arrestin-2 is accompanied by G protein-dependent signaling that releases Ga/q (GTP) from GPR-C6a such that tyrosine phosphorylation of PI3K p85 regulatory subunit is negatively modulated.
  • GTP Ga/q
  • Those effects on GPR-C6a signaling modulation are believed to exert an anti-proliferative effect without significant activation of PKC from PLC- ⁇ (i.e., there is insufficient activation of PKC to exert a pro-proliferative effect that negates the effect from inhibiting PI3K activity).
  • Erk-2 scaffolding is believed to be directly modulated by E-3a-diol acting upon an Erk-2 containing intracellular protein complex, whereas Erk-1 scaffolding is indirectly affected, through the intermediacy of Ga/q and p-arrestin-2, by extracellular action of E-3a-diol on GPR-C6a.
  • E-3a-diol or compounds identified as candidate compounds by methods disclosed herein redirects improper signaling through sequesterization of signal transduction components to restore proper activation of effector substrates.
  • Erk-2 is sequestered in a state resistant to activation in a Docket No. 354. PATENT cellular compartment within a protein complex.
  • That protein complex is supported by one or more scaffold proteins that prevent phosphorylation of Erk-2 and transport of any pErk- 2 that may be formed to the nucleus (or alternatively or additionally prevents transport of deactivated Erk-2 from the nucleus for cytosolic reactivation) thereby inhibiting activation of transcription factors that would otherwise support proliferation or survival. It is further believed that sequesterization of Erk-2 allows for activation of Erk-1 so that preferential phosphorylation of cytosolic effector substrates that support differentiation occurs without activating anti-apoptotic signaling. That selectivity for cytosolic effector proteins by pErk-1 is believed to result from pErk-1 (or Erk-1 prior to activation loop phosphorylation) sequesterization to a subcellular cytoplasmic domain.
  • E-3a-diol and similar test or candidate compounds modulate the phosphorylation states of the Erk isoforms so as to redirect proliferative and survival signaling to pro-apoptotic signaling or differentiation (which induces apoptosis).
  • aforementioned effects on Erk isoform scaffolding results in alteration in the phosphorylation status of i-AR as described herein such that signaling characteristic of differentiation, which is supported from i-AR activation in normal cells, is restored sufficiently in AR-dependent cancer cells to result in cell death.
  • pro-proliferative signaling of mt-AR may be redirected by E-3a-diol to support differentiation.
  • Modulators of PI3K and/or Erk identified by the methods disclosed herein are expected to have anti-proliferative effects while having lower toxicity in comparison to ATP-site dependent inhibitors of these proteins due to the conditional nature of their effects.
  • basal signaling in normal proliferating cells is not adversely effected in comparison to aberrantly proliferating cells since the result of the intercellular interaction(s) at GPR-C6a and/or on the Erk isoforms is to disrupt formation of inappropriately formed protein complexes that occur primarily in the abnormal cells.
  • the non-ATP site dependent modulators identified by the methods disclosed herein effect specific protein interactions that are formed under specified conditions as described herein, their modulating activities are not always "on”. That is in contrast to ATP-site inhibitors that offer no distinction between protein complexes in which the kinase participates and hence these inhibitors can exhibit no isoform selectivity.
  • E-3a- diol contributes to the observed anti-proliferative effects of this compound. That activity is now expected, based upon the insights of the invention, to occur when tumor cells expressing GPR-C6a are contacted with E-3a-diol. Since release and phosphorylation of Ga/q (GTP) resulting from activation of a GPCR coupled to this G protein monomer decreases PI3K regulatory subunit phosphorylation, it is believed that E-3a-diol acts as an agonist for GPRC-6a-Ga/q signaling (and thus is an inverse agonist for Ga/i signaling from this same receptor).
  • GTP Ga/q
  • E-3a-diol to inhibit the pro-proliferative effect of androst-5-ene-3p, 17 ⁇ -diol (PAED) on low passage (LP) LNCaP is not only opposed by competitive binding of E-3a-diol to GPR-C6a, but is actually reversed due to activation of a counteracting signal transduction cascade due to Ga/q release that inhibits PI3K.
  • Unopposed Ga/i signal transduction is postulated to occur by pAED agonist engagement of intracellular mutant AR (m-AR) and possibly by agonist engagement GPR-C6a such that signaling occurs through activation of Ga/i to increase PI3K activity.
  • E-3a-diol reverses this signaling and results in re-regulated cross-talk between the Ras-Erk and PI3K-Akt pathways such that signaling through i-AR to promote differentiation occurs instead of supporting survival or proliferation when Ras-Erk signal transduction is aberrantly activated. That alternative engagement of i-AR in i-AR signaling-dependent or androgen- associated cancer cells is thus in opposition to pro-proliferative signaling from this nuclear hormone receptor in these cancer cells and contributes to the observed anti-proliferative effects of E-3a-diol.
  • the anti-proliferative effect of E-3a-diol results in part from subcellular sequesterization of Erk-2 and/or, or alternatively from binding Erk-2 in a non-productive protein complex, that prevents its phosphorylation and subsequent translocation of pErk-2 to the nucleus and/or translocation of deactivated Erk-2 in the nucleus to the cytosol.
  • the effect(s) on Erk-2 sequesterization limits cell proliferation or survival by reducing phosphorylation of certain transcription factors in the nucleus, such as Elk1 or CREB.
  • Suitable test systems can thus comprise androgen-associated tumor cells with lost PTEN activity.
  • Ga/q- Src scaffolding not only results in Ga/q (GTP) phosphorylation that leads to negative modulation of PI3K phosphorylation status, but also redirects excess Src activation from Docket No. 354. PATENT pro-proliferative to anti-proliferative effects mediated by Ga/q.
  • Ga/q activation is responsible at least in part to the observed inhibition of PI3K that occurs as a result of "inverse" agonist action of E-3a-diol at GPR-C6a to activate Ga/q in preference to Ga/i that would otherwise result from agonist action of 3a-diol at this same receptor.
  • Scaffolding of Ga/q from GPR-C6a activation by E-3a-diol with Src is probably enhanced by either intracellular action of E-3a-diol on this scaffolding or is indirectly due to selective recruitment of 3-arrestin-2 (vide infra) to phosphorylated GPRC-6a occurring subsequent to Ga/q release from the receptor.
  • 3-arrestin-2 vide infra
  • Those effects on Src scaffolding diverts activated Src, which represents a significant signal transduction node often aberrantly stimulated in cancer cells, away from PI3K activation toward its inactivation.
  • Modulation by E-3a-diol of G protein-independent signaling through GPR-C6a may further contribute to the redirection of aberrant Src activity away from PI3K activation through Src subcellular sequesterization away from pro-proliferative effector proteins. That sequesterization of Src may also be mediated by intracellular action of E-3a-diol on protein scaffolding and may involve disruption of protein complexes involving ⁇ -arrrestin-l . Scaffolding of Erk-2 that results in its sequesterization to inhibit its activation from stimulation of the Ras-Erk signal transduction pathway may occur through scaffolded protein complexes. Those scaffolded protein complexes contain one or more scaffolding proteins described herein and likely includes the putative scaffolding protein Tfg.
  • That re-regulation or normalization may occur by E-3a-diol favoring the scaffolding of Src with p-arrrestin-2 instead of ⁇ -arrestin-l , which is believed to occur when aberrant Src signaling is present.
  • E-3a-diol physically interacts (i.e., directly binds) with Tfg to contribute to the observed antiproliferative and antiinflammatory properties of E-3a-diol.
  • E-3a-diol mediates cross-talk between one or more of GPCR, Ras-Erk, TNFa-NF- ⁇ , PI3k-Akt and c-Src signaling pathways, and is believed due to the commonality of scaffold proteins used in these signal transduction Docket No. 354.
  • PI3K scaffolding with Tfg which is postulated to be enhanced by E-3a-diol, may promote PI3K deactivation due to the aforementioned redirection of Src activity to tyrosine phosphorylation of Ga/q.
  • E-3a-diol binding to Tfg may dislodge SHP-1 thereby restoring that protein's phosphatase activity to ameliorate excessive signal transduction by PI3K resulting from aberrantly activated tyrosine kinases.
  • E-3a-diol modulates cross-talk between one or more of GPCR, Ras-Erk, TNFa-NF- ⁇ , PI3k-Akt and native Src (or its v- Src mutant) signaling pathways initiated through RTK, GPCR G protein-dependent or GPCR G protein-independent signaling by affecting scaffolding of protein complexes that involve one or more of ⁇ -arrestin, Ras/Raf, Tfg and other scaffolding proteins (including kinases and phosphatases acting as scaffolding proteins) disclosed herein.
  • E-3a-diol or preferred test or candidate compounds which exhibits one or more of the intracellular effects of E- 3a-diol as described herein, affects component scaffolding within an Erk signal transduction node. It is further believed this scaffolding effect inhibits or compensates for the abnormal signal transduction cross-talk such that pro-proliferative signaling is attenuated or reversed (i.e., promotes differentiation-induced apoptosis) within cancer cells. It is further believed such test or candidate compounds will influence protein-protein interactions with activated i-AR so as to reverse pro-proliferative signaling to drive back tumor cells to the differentiated state thereby inducing apoptosis.
  • PATENT a powerful cytosolic AR activator (which may also stimulate GPR-C6a Ga/i signaling), to prevent its accumulation in prostatic tissue while counteracting the activity of 3a-diol and DHT at GPR-C6a. That counteraction may occur not only by E-3a-diol acting as a competitive inhibitor for 3a-diol or residual DHT binding to the m-AR, but also by E-3a-diol differentially engaging (i.e., Ga/q vs. Ga/i) the Ras-Erk signaling pathway that is stimulated by GPR-C6a signaling so as to promote apoptosis rather than inhibition of this process by 3a-diol. Stated differently, E-3a-diol opposes Ga/i signaling from GPR-C6a by acting as an "inverse agonist" of that activity.
  • That disruption of cross-talk between i-Ar and Src signaling is postulated to occur through re-direction of Src activity either by scaffolding with Ga/q or by scaffolding of Src by p-arrestin-2 (or inhibiting scaffolding of Src with ⁇ - arrestin-1 ). Scaffolding by p-arrestin-2 during G protein-independent signaling, or through Tfg scaffolding of PI3K, will also influence Src signaling through these protein complexes such that re-regulation of i-AR tyrosine phosphorylation occurs.
  • Such negative modulators of 17HSD10 activity are not only useful candidate compounds for treating cancer, they are also candidate compounds for treating other conditions or diseases having an unwanted inflammatory component, which also can promote these hyperproliferating conditions.
  • a test compound contacted with a suitable in vivo test system that decreases the serum or intraprostatic concentration of DHT in comparison to placebo or decreases the serum or intraprostatic concentration of DHT in qualitatively similar manner to a positive control inhibitor of 17HSD10 or to that of E-3a-diol exhibits an activity that Docket No. 354.
  • PATENT can be used in selecting a candidate compound.
  • a test compound found to bind to Tfg or to selectively bind Erk-2 in comparison to Erk-1 in intact cells or cell-lysates provides an activity that may be used in identifying a candidate compound.
  • LNCaP clone FGC was originally derived from a lymph node biopsy of a patient with confirmed diagnosis of metastatic CaP. These cells are responsive to DHT (growth modulation and acid phosphatase production) and are androgen receptor, positive; estrogen receptor, positive. Through titration experiments, a concentration of 10 nM ⁇ was found to provide maximal stimulation of LP LNCaP, while no proliferation of LP LNCaP cells was observed without the addition of ⁇ . High-passage (HP) LNCaP cells are castration-resistant, but they still express AR and require the presence of functional i-AR to proliferate. HP and LP LNCaP cell both contain the T877A mt-AR. Other cell lines for evaluating test compound activity in CaP include PC-3 and DU-145
  • PC-3 cell line was originally derived from advanced androgen-independent, bone- metastasized prostate cancer.
  • DU-145 cell line was originally derived from a brain lesion in a patient with metastatic CaP and history of lymphocytic leukemia.
  • DU-145 and PC-3 cells are reported to be AR-negative and thus are not detectably hormone sensitive.
  • other studies have provided evidence that the DU-145 and PC-3 cell lines contain AR mRNA and it has been subsequently found that that DU-145 and PC-3 cell lines produce AR protein, but the relative levels of the AR mRNA and protein were lower than in LNCaP, an AR-positive cell line.
  • treatment of those cell with DHT resulted in measurable increases in the AR protein levels and considerable nuclear accumulation.
  • E-3a- diol may have an effect on the phosphorylation state of this mutant or affect crosstalk between AR, Ras-Erk or TNFa-JNK signaling, but is not sufficient in of itself to promote apoptosis (through inducement of differentiation). That may occur if E-3a-diol is an inadequate agonist (i.e. partial agonist) at this mt-AR for eliciting the required AR signaling that needs to be redirected for cell differentiation.
  • E- 3a-diol may be sufficient to sensitize PC-3 and DU-145 to apoptosis from co-contacting cancer cells with a tubulin disrupting agent or other cytotoxic cancer chemotherapeutic compound.
  • the anti-proliferative effect of E-3a-diol can be observed in a suitable in vitro or in vivo test system wherein the cells comprising the test system express or overexpress GPR-C6a and the antiproliferative effect is due in part to re-regulation or normalization of cross-talk between signal transduction through Src and GPCR signaling through Ga/q activation.
  • Cross-talk of Src and GPR-C6a Ga/q signaling is believed to occur through scaffolding of the G protein and the tyrosine kinase or through Src scaffolding by ⁇ - arrestin-2 in a G protein-independent manner, and may occur in combination with other additional scaffolding proteins.
  • Src signaling is redirected away from activation of i-AR that occurs through the "outlaw pathway", so named for ligand independent activation of i-AR through its tyrosine phosphorylation by Src.
  • AR activation by this "outlaw pathway” is postulated to become dominant during the transition to CRPC (castrate-resistant prostate cancer).
  • Cross-talk between Ga/q signaling or G protein-independent signaling with Src signaling, both of which are believed mediated by protein complexes with scaffolding of Src, will divert Src activity away from PI3K activation (passive inhibition) while providing Ga/q-mediated PI3K inhibition (active inhibition).
  • Prostate cancer cells known to endogenously have m-AR include LNCaP and DU145 [Kampa, M. et al., (2002); Papadopoulou, N. et al. (2008)].
  • Other cancer cells known to endogenously have m-AR include breast cancer cells T47D and MCF-7 [Kampa, M. et al. (2005); Kallergi, G. et al. (2007)] and colon cancer cells including Caco2 and HCT1 16 [Gu, S. et al. (201 1 ); Gu, S. et al. (2009)].
  • m-AR is also found in carcinogen- induced colon tumors.
  • suitable test systems for screening test compounds for E-3a-diol activity associated with its extracellular interaction at m-AR thus include a suitable cell-based in vitro or in vivo test system comprising LNCaP, MCF-7 or T47D cells, which are AR signaling-dependent, or DU145, and probably PC-3 cells, that are AR _/" but are GPR-C6a + + (i.e., are androgen-associated).
  • Other suitable tests systems for that purpose are HCT1 16 or transformed Caco2 cells or colon tumor cells that are carcinogen- induced in Balb/c mice [Wang, J.G. (2004)].
  • E-3a-diol contacted with cancer cells in a suitable test system with proliferating cancer cells that are AR _ " , incubated in growth factor- and androgen-depleted media, and found to have endogenous m-AR may exhibit anti-proliferative effects when the cells are co-contacted with a cancer chemotherapeutic agent.
  • Other suitable test systems are GPR-C6a +/+ AR' ' cancer cells that are engineered to be AR /+ and which are proliferating in growth factor- and androgen-depleted media.
  • E-3a-diol is expected to exert an antiproliferative effect that is enhanced or synergistic Docket No. 354.
  • PATENT with a cytotoxic chemotherapeutic compound such as a tubulin disrupting agent.
  • Still other suitable tests are transformed normal cell not endogenously expressing either gene, but are genetically engineered to express GPR-C6a and AR.
  • a test or candidate compound with qualitatively or quantitatively similar activity to E-3a-diol with respect to modulation of PI3K activity or its phosphorylation state may result in inhibition of survival or proliferation of the cancer cells through negative modulation of activity(ies) of downstream effector protein(s) in these cells, including that of Akt and/or PKC, when the cells are (1 ) AR positive, with or without GPR-C6a over- expression and dependent on context (i.e., in growth and androgen-depleted media vs. external mitogen or i-AR stimulation) or in (2) AR negative cells with GPR-C6a expression and co-contacted with a cancer chemotherapeutic compound.
  • test systems include cancer or transformed cells that have or are engineered to have i-AR with GPR- C6a present, either endogenously or through genetic engineering, incubated in growth factor and androgen-depleted media with or without co-contact of test compound with an externally added androgen or cancer chemotherapeutic compound.
  • a test or candidate compound contacted with such test systems that negatively modulates PKC or Akt activity or negatively modulates phosphorylation states of one or more downstream effector proteins that include adaptor proteins (Bcl-2, BAD, MDM2, PRAS40), other kinases (glycogen synthase kinase-3 [GSK-3], ⁇ kinase- ⁇ [ ⁇ - ⁇ ], mTOR), GTPases (Rac/cdc42, Rho), caspases (CASP9), metabolic enzymes (endothelial nitric oxide synthase [eNOS], 6-phosphofructo-2-kinase), cell cycle regulators (MDM2, p21 Cip1 , p27 Kip1 ), transcription factors (FOXO/Forkhead), and cancer susceptibility genes (BRCA1 ) can be used as additional criteria for identifying a candidate compound.
  • adaptor proteins Bcl-2, BAD, MDM2, PRAS40
  • other kinases glyc
  • Modulation of AR transactivation In /-AR, there are at least 1 1 serine residues that may be phosphorylated, depending on the cell culture system and the hormonal or growth factor stimulation employed. Although, distributed throughout the length of i-AR, a majority of these serine residues are located in the NH 2 -terminal domain (NTD) with many of these having an adjacent proline residue COOH-terminal to the phosphorylated serine, which can be recognized by the peptidyl prolyl isomerase Pin-1 .
  • NTD which has little intrinsic tertiary structure, contains the hormone-independent coactivator interface AF-1 .
  • NTD serines may result in a conformational change to a more ordered structure to promote cofactor and DNA binding.
  • the NTD contains several tyrosines that may become phosphorylated by Src, which have importance for nuclear translocation and Cdc42 associated kinase (Ack)-induced activation that promotes CaP progression.
  • Src nuclear translocation
  • Cdc42 associated kinase (Ack)-induced activation that promotes CaP progression.
  • serine residues that become specifically phosphorylated upon stress kinase Docket No. 354.
  • PATENT activation, EFG stimulation and by the PI3K-Akt signaling pathway Therefore, different phosphorylation patterns may occur in a context-specific manner to modulate the nucleo- cytoplasmic trafficking of i-AR and recruitment of various co-regulators that effect transcription.
  • co-activator stimulation of i-AR may also be dependent on the phosphorylation status of the co-activator.
  • the anti-proliferative effect of E-3a-diol may also be due in part to modulation of the phosphorylation status of one or more p160 co-activators, including that of Ser-738 in TIF2.
  • Protein levels of this co-activator are increased in various recurrent CaP cell lines, which in some embodiments are included in certain suitable test system as described herein, concomitant with increased phosphorylation status of Ser-738.
  • the increased TIF2 phosphorylation which increases the interaction between this coactivator and i-AR is presumably due to activation of Ras-Erk signaling resulting from aberrant or excessive cross-talk with RTK signaling, including signaling from EGF stimulation.
  • ErbB2 receptors may be important modulators of i-AR activity, since these receptors associate with TGF-R that participates in the "outlaw pathway" through activation of Src, and may mediate Erk activation by EFG signaling. Additionally, EGF- induced transactivation activity of i-AR results in increased levels of TIF2 as well as increased circulating levels of EFG to establish an autocrine loop. Therefore, without being bound by theory, it is believed that Erk-2 sequesterization in activation-resistant form by E-3a-diol disrupts this autocrine loop through the resulting decrease in the phosphorylation status of TIF2 Ser737 thereby contributing to the anti-proliferative effects of this compound.
  • E-3a-diol exhibits agonist activity for transactivation of an androgen responsive element (ARE) promoter-reporter gene construct. That contrary activity indicates the importance of a functional membrane androgen receptor in order to recapitulate some of the observed anti-proliferative effects for E-3a-diol seen in LNCaP cells.
  • ARE androgen responsive element
  • E-3a-diol therefore is postulated to result in part from establishment of cross-talk with Ras-Erk signaling resulting from m-AR activation to differentially engage mutant i-AR (mt-AR) signaling from pro-survival to pro-apoptotic gene expression. Those effects are further supported by reduction of Erk-2 stimulation of anti-apoptotic gene expression. Therefore, the action of E-3a-diol at membrane AR is consistent with its role as an mutant-AR agonist that supports an anti-proliferative gene transcription program resulting from its differential engagement of mt-AR.
  • DHT is expected, based upon the insights provided by the invention disclosed herein, to interact at GPR-C6a in similar manner to 3a-diol, i.e., through release of Ga/i. Therefore, it is believed that differences in the resulting cross-talk with Src and Ras-Erk signaling affected by E-3a-diol in comparison to 3a-diol should translate into differences in mt-AR phosphorylation patterns in the cytosol that are reflected in differences in subsequent gene transcription programs in the nucleus.
  • E-3a-diol when E-3a-diol is combined with pAED potent inhibitory activity on LP LNCaP tumor cells in vitro is observed and this activity translates to anti-tumor activity in vivo).
  • the action of pAED in the presence of E-3a-diol indicates the role of pAED as an AR activator that supports pro-apoptotic gene transcription resulting from differential engagement of this nuclear hormone receptor Docket No. 354.
  • PATENT in the presence of pAED alone. Since pAED weakly induces i-AR transactivation that activation may in part be due to direct interaction with i-AR, whose activity is sufficiently enhanced to support proliferation by interaction of pAED at m-AR.
  • That enhancement results from PI3K activation and aberrant crosstalk of the PI3K-Akt and Ras-Erk signaling pathways with i-AR.
  • the support by PAED for pro-apoptotic gene transcription induced by E-3a-diol in LP LNCaP instead of proliferation is due to selective activation of the Ras-Erk pathway affected by that compound's alternative engagement of m-AR (due to Ga/q-mediated signaling).
  • the pre-requisite for cytosolic AR activity in HP LNCaP is provided by E-3a-diol acting at mt-AR sensitized by Src phosphorylation (and is in conjunction with its activity at m-AR).
  • i-AR signaling In order to maintain the differentiated state in normal cells, i-AR signaling is required, which apparently becomes subverted so as to promote proliferation in cancers dependent on aberrant i-AR signaling. Without being bound by theory it is believed that E-3a-diol re-establishes i-AR signaling for differentiation and is thus catastrophic for cancerous cells, and is concommitant with the switch from target activation by pErk-2 to that of pErk-1 localized to a cytosolic subcellular compartment.
  • PSA prostate specific antigen
  • mt-AR expressing C4-2B cells are able to utilize E-3a-diol as a partial agonist to provide the required AR signaling, which is subsequently redirected towards inducing apoptosis. That belief is based upon the capacity of the T877A mutant to accommodate steroids having a C-linked substituent at position 17 as found in some C21 steroid mt-AR activators.
  • anti-proliferative effects of E-3a-diol or test compound with qualitatively or quantitatively similar activity may result from redirection of AR signaling.
  • the redirected AR signaling may be elicited by agonist binding of an endogenous or supplemented androgen to wt-AR or in the case of the T877A mt-AR from E-3a-diol partial agonist binding to this mutant AR.
  • Suitable test systems thus include cancer or transformed cells that have or are engineered to have functional mt-AR protein with functional GPR-C6a protein, or wt-AR protein (preferably in the presence of ⁇ ) with functional GPR-C6a protein, in order to recapitulate one or more of the cellular activities that have been observed for E-3a-diol.
  • Those proteins may be present either endogenously or through genetic engineering, and the cancer or transformed cell so obtained may by be incubated in growth factor- and androgen-depleted media with or without co-contact with an externally added androgen.
  • interruption of pAED signaling is an activity of E-3a-diol that is useful when treating prostate cancer, or other cancers that express AR, with this compound, particularly after ADT has failed.
  • ErbB receptor signaling is upregulated to promote AR transactivation through stabilization of AR protein and enhanced AR binding to promoters of androgen-regulated genes.
  • HER2/neu or ErbB2 a member of the ErbB receptor family whose gene is often overexpressed in breast and ovarian cancers
  • MAPK and PI3K pathways which stimulates AR [Traish, A.M. and Morgentaler, A. (2009); Berger, R. et al. (2006)].
  • CWR-R1 a CRPC cell with functional i-AR
  • EGF and heregulin-induced AR transactivation of androgen-inducible reporter was 10 fold greater than in its absence indicating the importance of AR signaling in CRPC that is supportable by residual serum DHT found after androgen ablation [Gregory, C.W. et al. (2005)].
  • the LBD or AR is sufficient to stimulate anti-apoptotic effects through Src/Shc/Erk scaffolding with HER2/neu receptor [Kousteni, S. et al. (2001 )].
  • rapid signaling from AR activation is now thought to occur in MCF-7 and LNCaP cells from physical interaction of phosphorylated EGFR with a AR/ER/Src complex and requires ER tyrosine-537 phosphorylation elicited by EGF [Migliaccio, A. et al. (2005)].
  • ErbB2 or EFGR activation results in AR transactivation by inducing phosphorylation of AR in the NH 2 -terminal domain by Erk-2 and subsequent AR activation of Erk-1/2 in CaP and other AR signaling-dependent cancer cells to stimulate proliferation [Zhu, X., et al. (1999); Peterzeil, H. et al. (1999); Migliaccio, A. et al. (2000)].
  • Those are rapid effects (within 2 min.) distinct from AR genotropic effects (i.e., they are non-genomic effects involving i-AR).
  • Activated Akt is also able to deactivate pro-apoptotic proteins including Bad, capase-3, GSK-3P (through ⁇ -catenin) and forkhead transcription factors (through p27 Kip1 ) through phosphorylation of these effector proteins.
  • cell signaling pathways leading to cell proliferation diverge with C2-4 cells that require phosphorylation of p70 S6 kinase, which is an independent downstream effector of PI3K and promotes cell cycle progression through activation of transcription factors downstream of this protein kinase [Ghosh, P.M. et al. (2005)].
  • PC-3 cells which are AR 1' , also depend on p70 S6 kinase activity for proliferation.
  • reducing PI3K activity should disrupt cross-talk between PI3K-Akt and AR signaling mediated by Src.
  • AR transactivation in intracellular AR signaling-dependent CaP cell lines, or nongenomic AR signaling that supports survival or proliferation is inhibited or is redirected to support differentiation and apoptosis.
  • Negative modulation of PI3K activity also inhibits activation of other pro-proliferative transcription factors stimulated by Akt.
  • suitable test systems further include those comprising GPR-C6a +/+ AR +/+ (endogenously or by genetic engineering ) cancer or transformed normal cells that are EFG responsive.
  • Suitable test systems also include cancer cells or transformed cells that have or are engineered to have functional i-AR and GPR-C6a, with or without expression or engineered overexpression of an Erb receptor gene such as HER2/neu.
  • PATENT test or candidate compound in one or more of these suitable test system that is found to negatively modulate the activity of PI3K or Akt or negatively modulate the phosphorylation state of Akt or the NTD of i-AR would exhibit an effect or activity qualitatively similar to E- 3a-diol and thus may be criteria for identifying candidate compounds.
  • a test or candidate compound that negatively modulates the phosphorylation state of an effector protein downstream of Akt such as that of Bad, capase-3 or GSK-3P, disrupts Src or AR scaffolding at ErbB2
  • negatively modulates the phosphorylation state of an AR co- activator or negatively modulations transactivation activity of a transcription factor activated through PI3K-Akt signal transduction could also exhibit a selection criteria for identifying a candidate compound.
  • Erk MAPK phosphorylation that occurs in the absence of mitogenic signaling, either from external growth factors or from constitutive activity of mutated RKT, will result from GPCR activation form an agonist that releases Ga/i from the receptor, as seen for example with 3a-diol activation of GPR-C6a. Erk MAPK activation oftentimes seen in this type of GPCR agonist activation is due to Gp/ ⁇ heterodimer release after Ga/i activation.
  • a suitable test system will include cancer cells or transformed cells that have or are engineered to have functional i-AR and functional GPR-C6a.
  • a test or candidate compound that positively (i) modulates Ga/q activity, (ii) an activity of a downstream effector protein consistent with this positive modulation of Ga/q activity, or (iii) positively modulates Ga/q phosphorylation state or GTP binding, or negatively modulates (iv) Ga/i activity or (v) activity of a downstream effector protein in a manner consistent with this negative modulation in one or more of these suitable test systems would exhibit an effect or activity qualitatively similar to E-3a-diol and thus may be criteria for identifying a candidate compound.
  • test or candidate compound that (i) exhibits "inverse agonist" activity at GPR-C6a, (ii) competes with 3a-diol binding at this receptor to inhibit Ga/i release or (iii) opposes an activity or modulation of phosphorylation state effected by 3a-diol in one or more of these suitable test systems would exhibit an effect or activity that may become other criteria for identifying candidate compounds.
  • a test compound that re-regulates Src activity on AR or Raf-1 when cells in these suitable test systems are stimulated by external contact with a mitogen may be criteria for identifying a candidate compound.
  • Cross-talk between these two signal transduction pathways is believed to be mediated in part through the negative feedback mechanism by pErk-1 , which is selectively increased in comparison to pErk-2, to disengage Ras from the cytoplasmic membrane through its phosphorylation of SOS.
  • E-3a-diol stemming Another contribution to the anti-proliferative effect of E-3a-diol stemming from selective formation of pErk-1 in comparison to pErk-2 is believed due to redirection of i-AR activity from abnormal activation of Src. In normal cells i-AR signaling is required for maintenance of the differentiated state whereas in cancer cells this activity is subverted to support survival or proliferation. Without being bound by theory, it is believed that E-3a- diol re-establishes differentiation activity of i-AR by affecting the phosphorylation state of i- AR.
  • That modulation in phosphorylation state results in redirection of aberrant i-AR activity in the nucleus towards a transcriptional program that supports differentiation or negative modulation of non-genomic i-AR effects in the cytosol that support apoptosis through re-normalization of signal transduction cross-talk.
  • the scaffolding of i-Ar to activated RTKs e.g., EGFR or HER2/neu
  • RTKs e.g., EGFR or HER2/neu
  • a test compound or candidate compound that (i) negatively modulates AR tyrosine phosphorylation status or (ii) positively modulates AR serine phosphorylation status through alteration of the phosphorylation pattern in the NTD in comparison to the pattern obtained from indiscriminate Erk isoform activation, (iii) re-regulates AR nucleo- cytoplasmic translocation in cancer cells to that of the differentiated normal cell from which the cancer cells arose, (iv) negatively modulates AR co-activator binding or recruitment for transactivation of pro-proliferative genes or (v) negatively modulates transactivation of ARE-inducible genes or (vi) exhibits one or more of these effects may be criteria for identifying a candidate compound.
  • Erk substrates are cytoplasmic proteins involved in apoptotic signaling, including FOX03a (Ser-294, Ser-344, Ser-425), p90 Rsk 1 (Ser-364, Thr-574), Bim (Ser-55, Ser-65, Ser-100), capase-9 (Thr-125) and perhaps Bcl-2 (Ser-70).
  • FOX03a Ser-294, Ser-344, Ser-425
  • p90 Rsk 1 Ser-364, Thr-574
  • Bim Ser-55, Ser-65, Ser-100
  • capase-9 Thr-125
  • Bcl-2 Bcl-2
  • soluble cytoplasmic proteins that are Erk substrates include cPLA 2 (Ser- 505), p70S6 kinase (Thr-421 , Ser-424), phosphodiesterase 4D (Ser-579), MKP-3-DUSP6 (Ser-159, Ser-197), MAP kinase activated protein kinases (MAPKAPKs), which includes p90 , mitogen and stress activated protein kinases (MSKs) and MAP-integrating kinases (MNKs).
  • MAPKAPKs MAP kinase activated protein kinases
  • MSKs mitogen and stress activated protein kinases
  • MNKs MAP-integrating kinases
  • Additional non-nuclear Erk targets include plasma membrane, endomembrane, mitochondrial and cytoskeletal proteins (e.g., CD120a, calnexin, paxillin, nucleoporins, EFGR, cortactin). Erk phosphorylation of one or more of those substrates in suitable in vitro test systems may also be used to asses Erk activity.
  • cytoskeletal proteins e.g., CD120a, calnexin, paxillin, nucleoporins, EFGR, cortactin.
  • Other preferred test or candidate compounds negatively modulate phosphorylation states, protein levels or activities of one or more transcription factors associated with intermediate early gene expression (e.g., c-Myc, c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 , Fra-2), negatively modulates transactivation by AP-1 (dimers of proto-oncogene products encoded by jun and fos families) or negatively modulates phosphorylation state of an Ets transcription factor (e.g., Elk-1 ). Phosphorylation of c-Fos by Erk occurs at Thr-325 and Thr-331 to extend the half-life of this transcription factor and is dependent on the duration Erk signal.
  • c-Myc c-Jun
  • JunB JunD
  • c-Fos FosB
  • Fra-1 Fra-2
  • Ets transcription factor e.g., Elk-1
  • Phosphorylation of c-Fos by Erk occurs at Thr-325 and Thr-3
  • Additional preferred test or candidate compounds increase retention of pErk in the cytosol (i.e., negatively modulates pErk nucleo-cytoplasmic transport) with minimal or no positive modulation of ser/thr phosphorylation status in one or more of the apoptotic proteins described herein at least one of the indicated residues (in parentheses) such that pro-apoptotic signaling is not negatively modulated or anti-apoptotic signaling is not positively modulated to an extent that cell survival is promoted or anti-proliferative effects from negative modulation of PI3K p85 regulatory subunit tyrosine phosphorylation are not abrogated or adversely diminished.
  • E-3a-diol is believed to induce a change in the gene transactivation pattern by i-AR away from proliferation to supporting differentiation- induced apoptosis.
  • An example of that switch in gene expression patterns is the decreases expression of IGFBP-3 (i.e. negative modulation )in LNCaP cells (which express the T877A mutant AR) and in MDA-MD453 breast cancer cells (which express the wild-type AR) by E-3a-diol.
  • IGFBP-3 has been implicated in cell growth inhibition and initiation of apoptosis in various cancer cell types and androgens.
  • genes involved in various phases of the cell cycle including G1 phase and G1/S transition effected by E-3a-diol include CCNE1, CDK4 and CDKN1B as well as cell cycle checkpoint and cell cycle arrest (ATM & CHEK2).
  • CCNE1, CDK4 and CDKN1B as well as cell cycle checkpoint and cell cycle arrest (ATM & CHEK2).
  • ATM & CHEK2 cell cycle checkpoint and cell cycle arrest
  • Bcl-2 and Caspase-9 were the most notable.
  • Also down-regulated were ABCG2 and ABCC5, both members of the ABC transporter family involved in drug resistance to cancer chemotherapy.
  • test or candidate compound that is identified as a further characterized candidate compound affects transcription of one or more genes of Table 2-5 in qualitatively similar manner.
  • the further characterized candidate compound down-regulates Bcl-2 or IGFBP-3 expression; more preferably down-regulates both of these genes.
  • AR the proteins levels from which are often found increased in CRPC, is down- regulated by E-3a-diol.
  • AR i.e., cytosolic androgen receptor
  • AP-1 activated protein-1
  • Erk-2 but not Erk-1 was found to complex with AP- 1 [Kumar, N.V. et al. (2001 )]. That observation is consistent with sequesterization of Erk-2 in inactive form due to intracellular action of E-3a-diol.
  • IRX5 Iroquois homeobox protein 5 inhibits apoptosis, and promotes cell cycle progression in LNCaP prostate cancer cells.
  • JUN v-jun sarcoma virus 17 homolog
  • ETV1 Ets variant gene 1
  • CD44 acts as an adhesion molecule for invasion, and a docking receptor for matrix metalloproteinase-9.
  • CD44 is also a receptor for osteopontin, which is involved in prostate tumor metastasis.
  • CD44 also acts as a scaffold protein for the Sialyl Lewis X binding determinant for endothelial cell E- selectin during metastasis.
  • CD44 expression is high on disseminated breast tumor cells in bone marrow, on prostaspheres and prostate cancer stem cells, and potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells, and invasion and growth.
  • CD44 is associated with the neuroendocrine tumor stem cells.
  • CD44 is more proliferative, clonogenic, tumorigenic and metastatic than the CD44- cells.
  • CD44 is also expressed on colon cancer stem cells, which can reconstitute the original human tumor in vivo, and on pancreatic cancer stem cells.
  • Expression of CD44 variants is correlated with drug resistance during prostate cancer metastasis.
  • Stromal hyaluronan interaction with epithelial CD44 variants promotes prostate cancer invasion by augmenting expression and function of hepatocyte growth factor and androgen receptor [Ghatak, S. et al. (2010)].
  • JAG1 Another gene down-regulated by contacting E-3a-diol with LNCaP cells is JAG1. Jagged 1 [Alagille syndrome] and Notchl ligand (JAG1 ) protein levels are increased in Docket No. 354. PATENT prostatospheres, representing tumor-initiating cells. Inhibition of Notch signaling increases susceptibility to apoptosis and targeted knockdown of Notchl inhibits invasion of human prostate cancer cells. Thus, down-regulation of Notchl and Jaggedl inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF- ⁇ signaling. Both CD44 and NOTCH are present in breast cancer stem cells, as well as prostate cancer stem cells and Notch signaling is important for the osteomimetic properties of prostate cancer bone metastatic cell lines.
  • RIS1 is up-regulated by contacting E-3a-diol with LNCaP cells.
  • Expression of RIS1 (Ras-induced senescence 1 ) can be silenced in prostate cancer, and is decreased in non-small cell lung cancer.
  • RIS1 is mutated in melanoma and colorectal cancers.
  • TIMP2 TIMP2 (TIMP metallopeptidase inhibitor 2) whose overexpression in prostate cancer is associated with longer disease free survival.
  • RUNX1 Another gene up-regulated by E-3a-diol is RUNX1.
  • Runt-related transcription factor 1 AML1 oncogene was specifically bound to and activated the PSA regulatory region in chromatin immunoprecipitation assays.
  • a RUNX1 polymorphism was significantly associated with a higher risk for advanced pathologic stage, a higher risk for lymph node metastasis, and poorer PSA-free survival.
  • RUNX1 induces senescence-like growth arrest in primary murine fibroblasts.
  • CASP10 Another up-regulated gene is CASP10.
  • Capase-10 is involved in prostate cancer cell apoptosis induced by various pro-apoptotic agents.
  • E-3a-diol Another gene up-regulated by E-3a-diol is /.OX (lysyl oxidase), whose polypeptide product has been shown to inhibit Ras signaling and the transformed phenotype of prostate and other cancer cells, and to sensitize pancreatic and breast cancer cells to doxorubicin-induced apoptosis.
  • LOX expression is progressively lost in primary human prostate cancer and associated metastatic lesions.
  • E-3a-diol affects expressed of the aforementioned genes by inhibiting pErk-2 activity in the nucleus through sequesterization of unactivated Erk-2 in the cytoplasm or deactivated Erk in the nucleus (or both) and by modulating the phosphorylation status of i-AR or of an AR co-regulator whereby transcriptional activity of agonist-bound AR is redirected to support differentiation.
  • results of the oligo arrays and real-time PCR results presented in Example 3 show that levels of AR transcript become decreased by treatment with E-3a-diol in cancer cells comprising a xenograft in vivo test system. Those effects correlated with the effects of E-3a-diol on levels of AR protein.
  • a test or candidate compound that effects transcription of one or more genes in qualitatively similar manner observed for E-3a-diol in some embodiments is a property for identifying a candidate compound.
  • expression or more of AR, IRX5, JUN, CD44 and JAG1 (decreased expression), and RIS1, TIMP2, RUNX1, CASP10 and LOX (increased expression) are affected in the manner indicated.
  • cRel is a class II member of the Rel/NF- ⁇ family of transcription factors. All Rel family members contain an NH 2 -terminal Rel Homology Domain (RHD) that is involved in dimerization, DNA and ⁇ binding, and nuclear localization. Activation of cRel from TNF-a signaling is dependent on PI3K and PLC- ⁇ phosphorylation of serine residues in the COOH-terminal domain that individually result in distinct, but overlapping, phosphorylation patterns.
  • RHD Rel Homology Domain
  • cRel contains a PKA recognition site in its RHD and an Erk consensus site in its COOH-terminal domain.
  • Bcl XL is likely to be a direct target for cRel activation.
  • cRel is involved in differentiation and apoptosis during T-cell and B-cell maturation including thymic differentiation of Foxp3 + Treg cells.
  • cRel activity becomes aberrant, due to over-expression of cRel and/or loss of function mutations in the gene encoding ⁇ , such that cRel retention by ⁇ in the cytoplasm is impaired.
  • the phosphorylation status of cRel is affected by activities of several protein kinases that include Erk1/2, PI3K, PKA and PLC- ⁇ .
  • E-3a-diol may affect the phosphorylation pattern (i.e., positively modulates phosphorylation status) in cRel in analogous manner to i-AR thereby switching its aberrant activity back towards supporting differentiation and promoting apoptosis or by inhibiting proliferation by binding cRel translocated to the nucleus to components of the transcriptional machinery in non- productive complexes. Therefore, a test or candidate compound that affects the Docket No. 354. PATENT phosphorylation state of cRel in like manner to E-3a-diol is in some embodiments a property for identification of candidate compound.
  • Rrebl binds and represses expression of the p16(lnk4a) promoter.
  • the p16INK4a (p16) gene is a tumor suppressor involved in regulating cell cycle checkpoints.
  • the p16 protein specifically binds to and inhibits the cyclin-dependent kinases CDK4/6, which regulate cell cycle progression in G1 through phosphorylation of the retinoblastoma protein (pRb) [Goodrich, D.W. et al. (1991 ); Ewen, M.E. et al. (1993); Kato J.-Y. et al. (1993)].
  • E-3a-diol may affect Rrebl binding activity to inhibit cell cycle progression from d phase. Therefore, a test or candidate compound that interacts with one or more transcription factors in qualitatively similar manner observed for E-3a-diol or which arrests tumor cell cycle in Gi is in some embodiments an additional property for identification of a candidate compound.
  • E-3a-diol was found to modulate phosphoprotein recognition by SH2 domains as shown by Example 13. Because of the binding specificity of the SH2 domain to phosphorylated tyrosine residues, a specific pattern or "fingerprint" of tyrosine phosphorylation can be elucidated.
  • SH2 domains for tyrosine phosphorylation profiling focuses on specific pathways and thus avoids binding preference to dominant populations of tyrosine kinases.
  • SH2 profiling arrays for fingerprinting tyrosine kinase activity within cells experiencing aberrant signal transduction by Western blot analysis are further described in Nallou, P. and Mayer, B.J. (2001 ).
  • a candidate compound may be characterized as positively modulating p-Tyr phosphoprotein recognition by the N-SH2 domain of RasGAP optionally with no discernable effect of such recognition by the C-SH2 domain.
  • this effect on N-SH2 phosphoprotein recognition is observable at about 15- min upon contacting the candidate compound to a suitable test system, preferably with a significantly smaller or no observable effect on this recognition with 5 min.
  • a candidate compound may be characterized by positive modulation of RasGAP phosphorylation state, preferably with negative modulation of Ser/Thr phosphorylation state or negative modulation of RhoGAP tyrosine phosphorylation state, more preferably by inducing both of these modulations.
  • E-3a-diol antagonizes the pro-proliferative effects of 3a-diol, which is an androgenic compound in its own right, and perhaps the pro-proliferative effects of the powerful androgen DHT (to which 3a-diol is converted by oxidase activity of a 3a-hydroxysteroid dehydrogenase). Without being bound by theory, it is believed this antagonism results from a combination of intracellular and extracellular interactions initiated or induced by E-3a-diol.
  • That cytoplasmic sequesterization of pErk-1 is believed to occur to a subcellular cytosolic domain that does not allow for phosphorylated- deactivation of pro-apoptotic proteins or activation of anti- apoptotic proteins that would otherwise support anti-apoptotic signaling.
  • the cytoplasmic localization is not considered or required to be absolute, but permits diminished pErk translocation to the nucleus in order that a differentiation program may be engaged as well as permitting non-genomic signaling of cytosolically retained pErk to support this same apoptotic-promoting program.
  • Ras-Erk signaling will selectively reactivate Ras-Erk signal transduction whereby apoptosis is promoted, probably as a result from negatively modulation of Mdm2 binding to p53 (i.e., positive modulation of functional p53 activity).
  • E-3a-diol The intracellular interaction of E-3a-diol is believed to occur through stabilization of a protein complex comprising Erk-2 and a scaffold protein or Erk-2 and a dual-substrate phosphatase (which may act as a scaffolding protein).
  • That protein complex stabilization preferentially sequesters Erk-2 to a subcellular compartment to inhibit its phosphorylation due to activation of the Ras-Erk signal transduction pathway (which may result from activation of GPR-C6a-Ga/q signaling through the aforementioned signal transduction cross-talk), thus negatively modulating Erk-2 phosphorylation state, negatively modulating nucleo-cytoplasmic translocation of pErk-2 that may be formed as a result of this crosstalk or preventing or inhibiting translocation of deactivated Erk-2 out the nucleus for reactivation in the cytosol.
  • Erk isoform redistribution is to positively modulate the phosphorylation state of actAR or a co-regulator thereof such that aberrant or excessive i-AR signaling in androgen-associated cancer cells is re-regulated in a manner that promotes execution of a differentiation gene transcriptional program leading to apoptosis instead of survival or proliferation.
  • E-3a-diol antagonizes the interconversion of 3a-diol and DHT by inhibiting a 3a-hydroxysteroid dehydrogenase believed to be 17HSD10 to negatively modulate prostatic tissue levels of DHT
  • the invention provides screening methods in vitro or in vivo that determines one or more of the aforementioned characteristic effects reported herein for E-3a-diol on kinase signal transduction cascade(s).
  • Such test compounds can be anti-proliferative candidate compounds and thus these screening methods would be useful for identifying compounds having anti-proliferative or anti-inflammatory activity(ies) in vivo.
  • Those compounds will Docket No. 354.
  • PATENT typically have low toxicity in vivo, and thus may be particularly useful in comparison to existing therapies for treating a human experiencing a hyperproliferation conditions as disclosed herein, and may additionally be useful in treating unwanted inflammation that initiates or supports these conditions.
  • a test or candidate compound found to have a characteristic effect or activity reported herein for E-3a-diol (i.e., desired antiproliferative or pro-apoptotic) or 3a-diol (i.e., undesired pro-proliferative or pro-survival) on kinase signal transduction cascade(s) would be useful as a reference compound or positive or negative control for the effect or activity screened.
  • the invention further provides methods to identify compounds that are selective or differential modulators of Erk-2 and Erk-1 activity.
  • Test or candidate compounds so identified will usually exhibit low toxicity, e.g., liver, skin or CNS activity, in contrast to ATP binding site-dependent MAPK inhibitors, when administered in therapeutic effective amounts to humans for treating hyperproliferation conditions disclosed herein.
  • the invention further provides screening methods that identify one or more of the aforementioned characteristic effects on Ras-Erk signaling, as described herein for E-3a- diol resulting from contact of a test compound with a suitable test system that would be useful in selecting candidate compounds or identifying further characterized candidate compounds having such effects.
  • Those screening methods thus would prove useful in identifying compounds having anti-proliferative activity of low liver toxicity in order to treat humans experiencing the hyperproliferation conditions disclosed herein.
  • the test or candidate compounds so identified may also have anti-inflammatory effects that would ameliorate unwanted inflammation that initiates or supports these hyperproliferation conditions in a mammal.
  • the invention additionally provides screening methods that identifies one or more of the aforementioned characteristic effects on signal transduction pathways or signal nodes including (i) Ras-Erk, (ii) TNFa-NF- ⁇ , (iii) PI3K-Akt, (iv) Src or (iv) i-AR, as described herein for E-3a-diol resulting from contact of a test compound with a suitable test system.
  • the characteristic effects so identified may be used to select test or candidate compounds.
  • the invention further provides screening methods that identifies one or more of the aforementioned characteristic effects on GPR-C6a-Ga/q signaling for selecting candidate having such effects and thus would prove useful in identifying compounds having antiproliferative and optionally anti-inflammatory activity(ies) of low liver toxicity in order to treat a mammal experiencing the hyperproliferation conditions as disclosed herein and those compounds additionally treating unwanted inflammation that initiates or supports these conditions.
  • the invention also provides methods to identify agonists of GPR-C6a that oppose the proliferative effect of 3a-diol by this compound's action at this receptor (i.e., inverse agonists of 3a-diol signaling at GPR-C6a).
  • the invention additionally provides for methods to identify non-ATP binding site- dependent ligands selective for Erk-1/2 in comparison to p38 and JNK MAPKs.
  • the invention further provides methods to identify non-ATP binding site-dependent ligands selective for Erk-2 in comparison to Erk-1 .
  • the invention also provides methods to identify differential modulators of Erk-1 and Erk-2 phosphorylation such that activation of Erk-1 occurs in preference to Erk-2.
  • the invention also provides methods to identify differential modulators of Erk-1 and Erk-2 phosphorylation such that inhibition of Erk-2 activity occurs in preference to inhibition of Erk-1 activity.
  • the invention additionally provides methods to identify differential modulators of Erk-1 and Erk-2 phosphorylation such that activation of Erk-1 occurs in preference to activation of Erk-2 activity.
  • the invention additionally provides methods to identify modulators of protein kinase activit(ies) whereby excessive tyrosine phosphorylation of Raf-1 resulting from external mitogen stimulation or constitutive RTK activity is negatively modulated.
  • the invention further provides methods to identify modulators of protein scaffolding mediated by scaffold proteins with one or more other components of kinase signal transduction pathway(s) whereby selective or differential modulation of Erk-1 and Erk-2 occurs to reduce survival or decrease proliferation of cancerous cells, preferably by additionally reducing signal transduction through one or more pro-inflammatory signal transduction nodes.
  • the invention further provides screening methods that identifies modulators of cytosolic AR (wt-AR or mt-AR) phosphorylation to redirect activity of this nuclear hormone Docket No. 354. PATENT receptor away from supporting proliferation towards supporting differentiation and hence apoptosis.
  • cytosolic AR wt-AR or mt-AR
  • the invention additionally provides screening methods that identifies negative modulators of undesired non-genomic activity of the cytosolic androgen nuclear hormone receptor or negatively modulates RTK signal transduction resulting from external mitogen stimulation of one or more mitogen-responsive RTKs or from constitutive activation of the RTK(s)
  • the invention further provides screening methods that identifies modulators of Src whereby activity of this tyrosine kinase is directed away from supporting proliferation or directed towards supporting differentiation.
  • the invention additionally provides methods to identify compounds that interact with the scaffold protein Tfg, or interact with 17pHSD10, or decreases 17pHSD10 conversion of 3a-diol to DHT, or decreases PI3K intracellular activity or downstream effects resulting therefrom, or increases intracellular levels of pErk-1 relative to pErk-2 or a combination of these effects wherein the intracellular activities detected occur in an artificial cell cancer cell line expressing GPR-C6a and wt-AR or mt-AR or artificial transformed cell lines containing gene constructs encoding functional GPR-C6a and wt- AR or mt-AR.
  • the invention also provides screening methods that identifies modulators of i-AR phosphorylation status such that transcription of pro-survival or anti-apoptotic genes in cancer cells is negatively modulated.
  • the invention further provides methods to identify compounds that induce differentiation of cancer or normal transformed cells.
  • the invention further provides methods to identify compounds that induce arrest in proliferating cancer or normal transformed cells.
  • the invention additionally provides methods to identify compounds that modulate PI3K or Akt phosphorylation status such that prosurvival or anti-apoptotic activity in cancer or transformed normal cells is negatively modulated.
  • the phosphorylation status of a protein kinase refers to the extent of phosphorylation of a collection of proteins present in the suitable test system for a particular protein kinase, which leads to activation or deactivation or modulation of the Docket No. 354.
  • PATENT activity of this protein kinase or isoform thereof as specified explicitly or implicitly by context, before or after contacting a test or candidate compound to a suitable test system.
  • phosphorylation status is stated relative to the number of amino acid residues capable of being phosphorylated or the number of phosphate groups covalently attached to a phospho-protein, when the phospho-protein or protein that is to be phosphorylated is present in a suitable test system.
  • phosphorylation status of one protein kinase isoform is stated relative to another isoform of the same protein kinase before or after contacting a test or candidate compound to a suitable test system containing the isoforms.
  • the phosphorylation status of a protein kinase after contact with a test or candidate compound is stated relative to the phosphorylation status of the same protein kinase in the same suitable test system prior to contact to the test or candidate compound or relative to the same protein kinase in a control test system to which is contacted the same test or candidate compound or a reference test compound.
  • phosphorylation status refers to the pattern of phosphorylation of amino acid residues in a phospho-protein.
  • the phosphorylation status of a protein is sometimes stated relative to one or more specified amino acid or amino-acid type (e.g., Ser/Thr or Tyr) residues that are phosphorylated or capable of phosphorylation in a suitable test system before or after contact with a test or candidate compound.
  • the phosphorylation pattern in a receptor or a protein kinase prior to contact of a suitable test system containing the receptor or protein kinase with a test or candidate compound is compared with the phosphorylation pattern of the same protein in the same test system after contact with a test or candidate compound or with the same protein in a control test system contacted with the same test or candidate compound or a reference test compound.
  • differences in phosphorylation patterns may not be accompanied by changes in the total number of phosphate groups covalently bonded to the phospho-protein or the overall extent of phosphorylation of a collection of such proteins.
  • Those differing phosphorylation patterns may have in common one or more phosphorylated amino acid residues or have common un-phosphorylated residue(s) that are capable of phosphorylation in the suitable test system.
  • modulation of phosphorylation status is based upon comparison of a test or candidate compound's effect on a comparator protein present in the same test system.
  • the compared protein kinases may be the same protein as when Docket No. 354. PATENT comparing changes in basal phosphorylation state or activity of a protein kinase upon contacting a test or candidate compound to a suitable test system.
  • modulation of a protein kinase capacity includes increasing or decreasing the rate of phosphorylation of one or more downstream effectors or substrates of the protein kinase or modulating the phosphorylation status of the effectors or substrates upon contacting a test or candidate compound with a suitable test system relative to the basal state of the system.
  • the phosphorylation status of one or more isoforms of PI3K of Class I or their capacity to convert phosphatidyl inositol (4,5)-bisphosphate (PIP2) to phosphatidyl inositol-(3,4,5)-trisphosphate (PIP3), or their capacity to effect activation of the downstream effector protein Akt before and after contacting a test compound to a suitable test system are compared.
  • comparisons with test compound are typically made using 3a-diol or E-3a-diol as control compounds.
  • modulation of phosphorylation status of Erk includes increasing or decreasing the amount of pErk-1 relative to pErk-2 in a suitable test system after contacting the test compound to the suitable system.
  • the modulation in phosphorylation state of one Erk isoform relative to the other isoform is concommitant with or in absence of an effect on the relative protein levels of the two isoforms.
  • phosphorylation status of a protein kinase influences its nucleo-cytoplasmic translocation by effecting dimerization of the protein or its interaction with other components of the signal transduction cascade in which it participates either directly or through cross-talk.
  • E-3a-diol, or test or candidate compounds identified as having one or more of the activities of E-3a-diol by methods disclosed herein modulates nucleo-cytoplasmic translocation of Erk-1/2, such that Erk-2 is preferentially retained in a cellular compartment in its un-phosphorylated state. This retention prevents activation of transcription factors in the nucleus that otherwise would support proliferation or survival.
  • Erk-1/2 nucleo-cytoplasmic translocation is modulated by preferentially retaining some fraction of Erk-1 in its phosphorylated state in the cytoplasm (i.e., negatively modulating pErk nucleo-cytoplasmic transport) in order to activate cytosolic targets for promoting differentiation or re-regulating apoptosis.
  • phosphorylation status of i-AR influences its nucleo- cytoplasmic translocation so that transactivation of genes that support survival or proliferation is inhibited.
  • the induced i-AR phosphorylation additionally influences the transcriptional program of activated i-AR translocated into the Docket No. 354. PATENT nucleus to redirect transactivation away from supporting survival or proliferation to that which promotes differentiation.
  • the differentiation promoting effects of E-3a-diol may involve differential recruitment of co-activators as described herein that result in protein complexes that are more favorable for transactivation of pro-apoptotic genes or are sub-optimal for transactivation of anti- apoptotic genes or is a consequence of differential recruitment of co-repressors that result in protein complexes that are less favorable for transactivation of anti-apoptotic genes.
  • modulating the phosphorylation status of a nuclear hormone receptor such as i-AR to inhibit proliferation may include decreasing tyrosine phosphorylation, particularly in relationship to decreased aberrant Src activity at i-AR or increased phosphorylation of Ser/Thr residues, particularly in the NH 2 -terminal domain of i-Ar attributable to increased pErk-1 formation relative to pErk-2 (and which may be concommitant with decreased protein levels of Erk-2).
  • contacting a suitable test system comprising LNCaP cells with a test or candidate compound may result in positive modulation of the phosphorylation status of i-AR translocated into the nucleus, which may be accompanied by inhibition of Ser-210 or S-213 phosphorylation in the NH 2 -terminal domain that is typically associated with transactivation of pro-proliferative genes.
  • preferred test systems are capable of responding to 3a-diol or E-3a-diol whereupon contact of 3a-diol or E-3a-diol to the test system results in modulation of the aforementioned pathways, nodes, protein complexes, cascades, proteins or cellular trafficking of the complexes or proteins or downstream effects resulting from this modulation, including changes in phosphorylation states or activities of one or Docket No. 354. PATENT more protein kinases, transcription factors or nuclear hormone receptors in qualitatively similar manner to one or more of the effects described herein for 3a-diol or E-3a-diol.
  • test systems are capable to responding to 3a-diol and E-3a-diol in qualitatively similar manner to one or more of the effects described herein such that 3a- diol and E-3a-diol have opposing effects.
  • modulation of a kinase activity includes increasing or decreasing the capacity of a kinase in a suitable test system to phosphorylate one or more of its downstream effectors or substrates of that protein kinase or to increase or decrease signaling through that signal transduction node, cascade or pathway upon contacting a test or candidate compound with a suitable test system relative to the basal state of the system.
  • E-3a-diol or compounds identified as candidate compounds by methods as disclosed herein positively modulates the phosphorylation status of Erk-1 in its activation loop or negatively modulated the phosphorylation status of Erk-2 in its activation loop in comparison to the other isoform.
  • the modulation of Erk isoform phosphorylation state(s) is accompanied by negative modulation of PI3K p85 phosphorylation state.
  • in vitro test systems include a cell-based system wherein the cells comprising the test system express genes encoding functional GPR-C6a, wt-AR or T877A mt-AR or other mutant forms of cytosolic androgen nuclear hormone receptor such as those found in tumor tissue cells or in cancer cell lines.
  • Functional GPR-C6a is capable of signaling through Ga/i, Ga/q or Gp/ ⁇ (concommitant with activation of Ga/i or Ga/q) release upon binding to the GPCR of a known agonist or reference compound in qualitatively similar manner to the full-length m-AR as described herein.
  • Functional GPR- C6a includes mutants that lack the extracellular Venus fly trap (VFT) domain, which is responsible for sensing certain amino acids.
  • functional GPR-C6a contains the human amino acid sequence.
  • functional GPR-C6a contains the amino acid sequence for a rodent homolog such as that for mouse or rat. Docket No. 354.
  • Functional i-AR is capable of stimulation by contact of the cell-based system with a known agonist or reference compound in qualitatively similar manner to full-length wt-AR or mt- AR.
  • agonist stimulation of i-AR in those contexts results from or is enhanced by one or more non-genomic effects initiated at GPRC-6a described herein or results in translation of a gene containing an ARE promoter (i.e., genomic effects).
  • Candidate compounds are selective Erk modulators or selective modulators of Erk isoform phosphorylation status or activity.
  • test compounds that are identified as candidate compounds are potentiators of Erk-2 activity or enhancers of Erk-1 activity.
  • candidate compounds are selective GPR-C6a- Galpha/q agonists, sometime referred to as inverse agonists (with respect to Ga/i activation or release).
  • candidate or further characterized candidate compounds are test compounds that exhibit efficacy or low liver toxicity when evaluated in a suitable animal model.
  • One mutation frequently detected in advanced prostate cancer biopsies is at codon 877 where a switch from threonine to alanine occurs in the ligand-binding domain. This mutation is typically present in LNCaP cells.
  • Another mutation H874Y is found in the recurrent human prostate cancer xenograft cell line CWR-R1 . That mutation retains wild- type sensitivity to androgens, but has increased responsiveness to other steroids. Therefore, some embodiments use a cell line as described herein expressing, or made to express by transfection (stably or transiently), a gene coding one of those particular mutant i-ARs.
  • transformed HEK293 (HEK293T) cells that express a gene encoding functional LNCaP mt-AR are used in a suitable test system.
  • gene mutations incorporated into gene constructs used for producing mt-AR expressing cell lines that provide suitable test systems disclosed herein include one or more of the mutations described in Table 1 [see, Taplin, M.E. et al. (1995)] and are numbered according to the gene sequence provided by Lubahn, D.B. et al. (1988).
  • the mt-AR resulting from transfection may be from expression of a gene having single, double, triple, quadruple or more of the point mutations described herein that do not involve the DNA-binding (DBD) domain in order to provide functional i-AR in a cell line disclosed herein.
  • mt-AR is present in a cell-line that results from a single point mutation in AR.
  • the mt-AR is constitutively active due to partial or completed deletion of the COOH-terminal region that contains the LBD or is responsive to atypical ligands due to gene shuffling that result in an AR with the LBD of another nuclear hormone receptor.
  • NH 2 -terminal activation function-1 (AF-1 ) is constitutively active in truncated AR that lacks the LBD. It is thus believed that in native i-AR the NTD domain competes with co-activators for AF-2 and this competition is lost in the truncated AR or in certain LBD point mutation that allow for greater co-activator recruitment. Therefore, in some embodiments an mt-AR receptor is introduced into a cell-line with an AR transfection vector containing a point mutation in exon 1 (encodes NTD) of the AR gene and is sometimes combined with a point mutation in a LBD exon that will permit mt-AR activation by wt-AR antagonists.
  • an mt-AR receptor is introduced into a cell-line with an AR transfection vector containing a point mutation in exon 1 (encodes NTD) of the AR gene and is sometimes combined with a point mutation in a LBD exon that will permit mt-AR activation by wt-AR antagonists.
  • the Hinge region located between the NH 2 -terminal domain (NTD) and the DNA- binding domain (DBD), is important for heat shock protein (Hsp) binding, which also involves the LBD. Hsp dissociation upon agonist binding is required for AR dimerization and eventual co-activator recruitment and binding of the activated receptor complex to ARE.
  • the hinge region is also important for AR transport since a nuclear localization Docket No. 354. PATENT signal spans the region between DBD and the hinge region. Therefore, in some embodiments the mt-AR will contain one mutation in the NTD that forms the AF-2 region or one mutation in the hinge region, each of with is sometimes combined with one mutation in the LBD that permits agonist activation by wt-AR antagonists.
  • the mutation in the NTD domain permits or disrupts formation of the AF-2 region on agonist activation.
  • the NTD mutation permits formation of the AF-2 region while disrupting the N/C interaction in preference to a co- activator or co-repressor protein AF-2 interaction or selectively inhibits this protein-protein interaction.
  • the mutation in the hinge region stabilizes or disrupts interaction of the mt-AR with an Hsp protein or disrupts translocated of the activated nuclear receptor to the nucleus (i.e., negatively modulates nucleo-cytoplasmic transport.
  • an mt-AR is introduced that is constitutively active due to disruption of the N/C interaction that allows for more facile co-activator recruitment.
  • the mt-AR will contain a Ser to Ala mutation wherein the wild type Ser residue is located in the NTD which is sometimes combined with one mutation in the LBD that permits agonist activation by wt-AR antagonists.
  • the mutant AR results from expression of a gene lacking one or more of the LBD exons that sometimes may be combined with a point mutation in exon 1 .
  • the mutant ARs from the aforementioned mutations are useful for developing suitable test systems for screening of test compounds according to the methods described herein that disrupt the survival or proliferative advantage enjoyed by AR signaling- dependent cancers harboring those mutations (and not to be confused with androgen- dependent vs. androgen-independent CaP, both of are postulated to require AR signaling, but which have differing sensitivity to androgen).
  • the advantages conferred include improved binding to co-activators, inhibition of co-repressor binding, improved NF-kB-Rel binding interactions, disrupted internal AR N/C interaction for greater recruitment of co- activators or increased ligand retention due to stabilization of the H12 lid of the ligand- binding pocket.
  • the cell-based system is comprised of mammalian cells capable of DNA transcription or RNA translation (i.e., are viable cells) and express AR and optionally ERa or GPR-C6a.
  • in vitro test systems comprise viable mammalian cells expressing GPRC6a and wt-AR or mt-AR.
  • the cell-based system is comprised of viable mammalian cells that are derived from a deposited cancer cell line and express AR, which encodes wt or mutant intracellular AR, and are either quiescent or induced to proliferate.
  • PATENT the cell-based system is comprised of viable mammalian cells that are derived from a normal cell line and are induced to proliferate, for example, by mitogen or cytokine stimulation, or through transformation.
  • the suitable cell-based system is comprised of quiescent cancer cells derived from a deposited cell line that expresses mt-AR.
  • the derived cell-based system is comprised of quiescent cancer cells that additionally express GPR-C6a.
  • Mammalian cells are "quiescent" (but not necessarily synchronized), as for example when maintained in charcoal stripped CS or FBS media or in growth factor and androgen-depleted media or in media where steroids are not present at concentrations more than about 100 pM, more than about 10 pM or more than about 1 pM.
  • DHT is present at no more than 10 pM or 100 pM concentration in order to mimic the androgen-depleted state resulting from maximal androgen blockade therapy.
  • an suitable in vitro cell-based test system will comprise cells expressing AR and GPR-C6a that additionally express one or more genes encoding a mitogen-responsive membrane-bound receptor or a functional RTK, including but not limited to EGFR, IGFR, ErbB2 or Her2/neu, either having native or constitutive activity, or a functional membrane hormone receptor (m-NR) other than GPRC-6a, including but not limited to membrane estrogen receptor (m-ER), membrane progesterone receptor (m-PR) or membrane glucocorticoid receptor (m-GR), or a functional cytosolic nuclear hormone receptor (c-NR) other than a i-AR, including but not limited i-ERa, i-ERp, i-PR or i-GR, or a functional cytokine receptor, including but not limited to receptors for TNFa or IL-6.
  • m-NR membrane hormone receptor
  • m-ER membrane estrogen receptor
  • m-PR membrane progesterone receptor
  • m-GR membrane
  • a suitable in vitro cell-based test system will comprise cells expressing AR, GPR-C6a and ERfi.
  • a suitable in vitro cell-based test system will comprise cells additionally expressing one or more genes encoding functional GPR-C6a protein, a functional or constitutively active RTK or one or more of the aforementioned functional hormone membrane or cytosolic receptors wherein the gene encoding the receptor protein is transiently or stably transfected into a normal or cancer cell line.
  • GPRC6a gene is transfected into an androgen- or estrogen-associated cancer cell line in order to express or overexpress GPRC6a.
  • GPRC6a, wt-AR or mt-AR is transfected into a cell line capable of proliferation, optionally containing an ARE promoter-reporter gene.
  • Some preferred embodiments are comprised of cells from a deposited cancer cell line that expresses or are transfected to express AR, GPR-C6a and an ARE promoter-reporter gene. Docket No. 354.
  • a suitable test system includes a test system whose cells in vitro or in vivo will respond to contact with a naturally occurring androgen or a synthetic agonist at GPR-C6a, wt-AR or mt-AR preferably at concentrations between about 100 ⁇ to about 1 pM or less, more preferably between about 10 ⁇ to 1 pM or at physiologically relevant concentrations.
  • DHT levels of about 10 pM to about 100 pM are used to mimic circulating concentrations in CaP patients of this hormone after maximal androgen blockade (MAB).
  • More preferred in vitro cell-based test systems are comprised of hormone- associated cancer cells.
  • Particularly preferred for such test systems are prostate cancer or breast cancer cells lines including but not limited to low passage or high passage LNCaP, PC-3, CV1 , CWR22, DU145, MCF-7, MCF-10A, T47D, MDA-MB-231 , MDA-MB- 438 or MDA-MB-468.
  • Other preferred in vitro cell-based test systems are comprised of transformed normal cells induced to proliferate, by for example, contacting the test system with mitogen or hormone, transformed normal cells from genetic manipulation of normal cells or immortalized cells from a deposited cell and include THP-1 , COS, CHO, HUVEC, HeLa, HEK293, HEK-293T cells.
  • Other preferred in vitro cell-based test systems are comprised or normal, transformed, immortalized or cancerous endothelial or fibroblast cells.
  • the normal, transformed, immortalized or cancer cells described herein for deriving a suitable test system are transfected with an inducible androgen-responsive promoter-reporter gene or NF- ⁇ response element-reporter gene construct and one or more genes encoding for functional i-AR, GPR-C6a, ERp, ErbB1 (EFGR) or ErbB2 (Her2/neu) or a constitutively active RTK or transcription factor.
  • EFGR ErbB1
  • ErbB2 Her2/neu
  • a suitable in vitro test system comprises low passage (LP) LNCaP cells, optionally transfected with an ARE promoter-reporter gene.
  • LNCaP cells commercially available from American Type Culture Collection (ATCC), are incubated in serum to passage number of 20 to about 35, about 24 to about 32 or about 20 to about 25, preferably about 24 passages, and are used in a suitable test system. Those passaged LNCaP cells are then contacted with a pro-proliferative agent in an effective amount to stimulate proliferation prior to step (a).
  • LP LNCaP cells in step (a) are co-contacted with test compound and pAED.
  • a suitable test system comprises high passage (HP) LNCaP cells, preferably transfected with an ARE promoter-reporter system.
  • HP LNCaP cells from about 80 passages or more, preferably about 84 passages, are used for test compound screening and are contacted with a pro- Docket No. 354.
  • PATENT proliferative agent in effective amount to stimulate proliferation or AR transactivation prior to step (a).
  • HP LNCaP cells in step (a) are contacted with test compound while in androgen-depleted media.
  • a suitable in vitro test system comprises HEK293T cells that are stably transfected with an i-AR ligand-binding domain (LBD) fused to the Gal4 DNA binding domain and which also expresses a reporter gene fused to an Upstream Activator Sequence (Gal4 response elements) both of which may be present in the same gene construct that was used for transfection.
  • LBD i-AR ligand-binding domain
  • Gal4 response elements Upstream Activator Sequence
  • a suitable test system comprises MDA-kb2 cells (ATCC CRL-2713) expressing a gene encoding functional wt-AR and a gene containing an androgen response element upstream to a reporter gene (i.e., an ARE promoter-reporter gene), both of which may be present in the same gene construct that was used for transfection.
  • a reporter gene i.e., an ARE promoter-reporter gene
  • a suitable test system comprises cells from a cell line transfected with a gene construct containing an androgen response element upstream to a reporter gene and a gene construct encoding functional mutant AR, both of which may be present in the same gene construct used for transfection.
  • MtAR-HEK293 cells which are transformed HEK293 fibroblasts that have been transiently co-transfected with and ARE promoter-reporter construct and a cDNA expression vector encoding functional LNCaP mutant AR, are used in a suitable cell-based in vitro test system.
  • a suitable test system comprises MDA-kb2 cells stably transfected with a gene construct containing an MMTV promoter fused upstream of a reporter gene. These cells endogenously express genes for both i-AR (intracellular androgen receptor) and i-GR (intracellular glucocorticoid receptor).
  • i-AR intracellular androgen receptor
  • i-GR intracellular glucocorticoid receptor
  • a suitable test system comprises normal cells having Eft/3 expression or AR-dependent cancer cells having ERp expression, either natively or through transfection (stably or transiently).
  • the potential for a test compound to interact with GPR-C6a or i-AR and to effect transactivation of ERp is of importance considering the well-established anti-proliferative, pro-apoptotic role of ERp in prostate tissue. Therefore, in some preferred embodiments a test or candidate compound is contacted with a suitable test system comprising normal cells expressing ERp or AR-dependent cancer cells expressing wt-AR or mt-AR, preferably also expressing GPR-C6a. In other preferred embodiments a test or candidate compound is contacted with a prostate cancer Docket No. 354. PATENT cell line deficient in AR or AR , but expressing GPRC6a or expressing ⁇ and GPR C6a.
  • a suitable test system is derived from AR 1' cancer cells or transformed normal cells that have been transfected (stably or transiently) with a i-AR expression vector and an androgen inducible reporter construct such as an ARE promoter-reporter, MMT promoter-reporter or a androgen receptor promoter- chloramphenicol acetyl transferase (CAT)-reporter such as GRE 2 E1 bCAT or -286PBCAT.
  • CAT chloramphenicol acetyl transferase
  • CV1 , COS-1 or PC3 cells are transfected with a gene construct encoding i-AR and a construct containing an androgen receptor promoter-reporter gene.
  • a suitable test system comprises cancer cells stably transfected with a promoter-reporter gene construct that is sensitive to estrogenic stimulation.
  • T47D-kBluc cells which endogenously express genes for both forms of the ER (estrogen receptor), namely ERa and ERp, are transfected with a gene construct containing at least three copies of the estrogen response element (ERE) fused upstream of a reporter gene.
  • ERP-HEK293 cells which are HEK293 fibroblasts transiently co-transfected with an ERE promoter- reporter gene construct and a cDNA expression vector encoding functional ERp, preferably full-length human ERp, are used for deriving a suitable cell-based in vitro test system.
  • a suitable test system comprises cells from a cancer cell line expressing constitutively active i-AR that lacks the ligand binding domain as when the amino-terminal domain is fused to a DNA binding domain fragment.
  • CWR-R1 cells are transiently infected with a gene construct encoding constitutively active wild-type receptor, which also contains an inducible promoter-reporter gene such as MMTV promoter-luciferase reporter or ARE promoter-luciferase reporter.
  • a suitable test system comprises cells from a cell line as described herein that is treated with a selective inhibitor of the Erk MAPK pathway.
  • PD098059 which is a selective noncompetitive inhibitor of the Ras- Erk pathway that prevents the activation of MEK-1 by Raf-1 (PD098059 inhibits Raf activation of MEK-2 less effectively), is used prior to contact with a test compound.
  • a suitable test system comprises cells from a cell line as described herein that is treated with a selective inhibitor of the PI3K-Akt pathway.
  • LY295002 is used, which is a selective inhibitor of PI3K prior to contact with a test compound. Docket No. 354.
  • a suitable test system comprises cells from a cell line as described herein that is treated with a compound that directly or indirectly increases intracellular kinase activity or decreases phosphatase activity.
  • a compound that directly or indirectly increases intracellular kinase activity or decreases phosphatase activity examples of these compounds are 8-bromo-cyclic AMP (cyclic AMP analog that acts through PKA), forksolin (adenylate cyclase activator), okadaic acid (phosphatase 1 and 2A inhibitor), vanadate (phosphotyrosine phosphatase inhibitor), growth factors and certain neurotransmitters.
  • Preferred suitable in vivo test systems include castrated SCID mouse xenograft models implanted with hormone-associated tumor cells. In some of these systems hormone support is provided by appropriate subcutaneous implant. In one preferred suitable in vivo test system LuCaP-35V tumor cells are implanted. In another preferred test system LNCaP tumor cells are implanted into castrated SCID mouse. In some of those embodiments pAED is implanted subcutaneously to support AR-dependent cancer cell proliferation.
  • MNU carcinogen N-methyl-N-nitrosourea
  • the MNU-induced tumor model has previously been used to develop tamoxifen therapy in women with breast cancer and is thus appropriate for screening of test compounds potentially useful for the treatment of breast cancer.
  • Substantial evidence suggests this rodent model system mimics human breast cancer since the initiation of cancer occurs primarily at the same site in both humans and rats and the majority of the induced tumors express estrogen and progesterone receptors.
  • rats are co-administered a test compound and a tubulin disrupting agent.
  • Those disrupting compounds interfere with the appropriate function of tubulin in the mitotic spindle by inappropriately stabilizing polymerized tubulin or by inhibiting polymerization.
  • the tubulin disrupting agent is a taxane including, for example, paclitaxel, docetaxel and cabazitaxel. That variation is useful to determine if a test compound acts synergistically with a taxane or allows for reduced or less frequent administration of the taxane for achieving similar efficacy of the taxane with reduced toxicity due to enhanced or synergistic activity..
  • suitable test systems are models for hyperproliferation conditions that are driven by cancer cells whose deposited cell line counterparts would be Docket No. 354.
  • PATENT expected, based upon the insights provided by the invention disclosed herein, to favorably respond (i.e., by decreased proliferation or increased apoptosis) to Src, MEK1/2, MAPK1/3, PI3K or Akt inhibitors at physiologically relevant concentrations.
  • suitable test systems are models for hyperproliferation conditions that are driven by cancer cells whose deposited cell line counterparts would be expected, based upon the insights provided by the invention disclosed herein, to respond to i-Ar inhibitors.
  • test systems model hyperproliferation conditions that include prostate cancer, breast cancer, liver cancer, e.g., hepatocellular carcinoma, central nervous system cancer, e.g., glioma, astrocytoma or oligodenroglioma, lung cancer, e.g., small cell lung cancer or non-small cell lung cancer, colorectal cancer, myeloma, melanoma, lymphoma, thyroid cancer, pancreatic cancer, bone cancer or a cancer that metastases to bone, e.g., metastatic prostate cancer or breast cancer.
  • central nervous system cancer e.g., glioma, astrocytoma or oligodenroglioma
  • lung cancer e.g., small cell lung cancer or non-small cell lung cancer, colorectal cancer, myeloma, melanoma, lymphoma, thyroid cancer, pancreatic cancer, bone cancer or a cancer that metastases to bone, e.g.
  • Monovalent C-linked substituents include optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl and optionally substituted C-linked heteroaryl as these terms are defined herein.
  • Preferred test compounds also include the 5a-androstane, androsten-5-ene or androst-4-ene steroids described herein wherein the monovalent O-linked substituent at C17 is replaced with an optionally substituted amine or N-linked heterocycle.
  • Other preferred test compounds also include the 5a-androstane, androsten-5-ene or androst-4- ene steroids described herein wherein the steroid contains one or more double bonds or one or more additional double bonds, preferably containing 1 , 2, 3 or 4 total numbers of double bonds, excluding compounds inherently unstable or having a hypervalent carbon atom.
  • test compounds are steroid compounds as described herein wherein one or more of the angular methyl groups (i.e., at C18 and/or C19) is replaced with -H or with independently selected monovalent C-linked substituents, including an optionally substituted alkyl group such as ethyl or hydroxymethyl.
  • More preferred test compounds have independently selected monovalent O-linked Docket No. 354.
  • PATENT substituents at C3 and C17, optionally with independently selected monovalent C-linked substituents as second R groups at these positions as these terms are defined herein.
  • Preferred monovalent O-linked substituents in these test compounds are -OH or an ester thereof, independently selected.
  • Monovalent C-linked substituents include optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, including optionally substituted phenyl, and optionally substituted C-linked heteroaryl.
  • Preferred monovalent C-linked substituents in these test compounds are optionally substituted C1 -4 alkyl groups, including methyl, ethyl, propyl, isopropyl, hydroxymethyl, optionally substituted C2-4 alkynyl, including ethynyl, propynyl or chloroethynyl, optionally substituted aryl and optionally substituted heteroaryl as these terms are defined herein.
  • test compounds are reference or control compounds as described herein, or have independently selected -OH or ester groups at C3 and C17 in the a- and ⁇ -configurations, respectively, and additionally contain -H or a monovalent C-linked substituent at C17 in the in the a-configuration, preferably ethynyl or optionally substituted phenyl and optionally substituted monocyclic heteroaryl .
  • a test compound is evaluated in one or more suitable in vitro or in vivo test systems as described herein relative to E-3a-diol for activity in negatively modulating PI3K phosphorylation-mediated activation of its kinase catalytic activity or PIP3-forming activity and for increasing Erk-1 phosphorylation or activity in comparison to Erk-2.
  • a test compound is evaluated in one or more suitable in vitro or in vivo test systems as described herein relative to E-3a-diol for activity resulting from the stimulation of Ga/q signaling.
  • a test compound that activates Ga/q may result in one or more of (i) negatively modulate p1 10a/p85 phosphorylation activity, (ii) negatively modulate tyrosine phosphorylation state of PI3K regulatory subunit p85, (iii) negatively modulate serine phosphorylation state of Raf-1 or (iv) negatively modulate Ras interaction with or activation of p100a.
  • Those downstream effects from Ga/q activation may separately or in some combination thereof provide selection criteria for identifying a candidate compound.
  • a protein concentration, phosphorylation status or capacity for signal transduction is compared relative to one or more other proteins before and after application or administration of a test compound to a suitable test system such that a relative change in amount, concentration or Docket No. 354.
  • PATENT phosphorylation status of a protein indicates an effect of the test compound on that protein's activity or an effect on signal transduction pathways that involve the compared proteins, either directly or through cross-talk between signal transduction pathways.
  • a protein kinase concentration, phosphorylation status or capacity for signal transduction is compared relative to other protein kinases with similar amino acid residue specificity for phosphorylation.
  • the compared protein kinases may all be Ser/Thr protein kinases, as for example when comparing changes in Erk-1/2 phosphorylation activity relative to JNK MAPK activity(ies) or p38 MAPK activity(ies) upon contacting a test compound to a suitable test system.
  • the compared protein kinases will be homologous in amino acid sequence with at least 50% homology, preferably at least 60% homology, more preferably with at least 80% homology relative to a comparator protein kinase sequence, typically to that protein having the highest abundance in a suitable test system.
  • the protein kinases, whose effects from contacting a test or candidate compound to a suitable test system are compared are of significant homology and substrate specificity in a suitable cell-free test system to be regarded as isozymes, as for example when comparing changes in protein kinase activity of the ERK MAPK isoforms Erk-1 relative to Erk-2 upon or after test or candidate compound contact to the test system.
  • comparison of activity or a property of a G protein is made to one or more other G -proteins.
  • comparisons are made between relative release rate or amounts of released monomer Ga/i and Ga/q resulting from a test compound or candidate that engages a GPCR that couples to either of these G proteins.
  • one or more effects from release of a particular Ga monomer after contacting a test compound or candidate with a suitable test system is compared with the basal or mitogen-stimulated state of the system. For example, changes in the amount of tyrosine phosphorylated Ga/q are compared from contacting a test compound or candidate to a suitable test system relative to a suitable reference compound.
  • comparisons with test or candidate compound are preferentially made using 3a-diol or E-3a-diol as control compounds when the GPCR is GPR-C6a.
  • Erk-1 is not necessary for cell proliferation but is required for disease-modifying anti-inflammatory effects as disclosed herein for treating hyperproliferation conditions. Furthermore, increasing activation of Erk-1 relative to Erk-2 results in re-regulation of the apoptotic program and redirection of prosurvival signaling, including those involving other signal transduction nodes such as Src and i-AR, in cancer cells to signal transduction pathways that promote differentiation.
  • comparisons are made between the relative levels of pErk-1 to pErk-2 within a suitable test system prior to and after contacting the test system with test compound.
  • protein levels of Bcl-2, Bcl-x L or Bim from a suitable test system are determined prior to and after contacting a test compound with the test system.
  • comparisons with test or candidate compound are preferentially made using 3a-diol and/or E-3a-diol as control compounds.
  • test compounds that stabilizes that interaction, but do so to preferentially retain Erk-2 in inactive form in the cytosol will also provide an anti-proliferative effect through cytosolic sequesterization of that isoform.
  • compounds that stabilize the interaction of MEK-1 with Erk-1 in preference to Erk-2 allow Erk-1 to reduce the ability of MEK-1 to properly present inactive Erk-2 in the cytoplasm for activation and subsequent translocation of pErk-2 to the nucleus upon growth factor stimulation. Therefore, a test compound that stabilizes the interaction between Erk-1 and MEK-1 will also be antiproliferative.
  • comparison is made between nucleo- cytoplasmic trafficking of pErk-1 or pErk-2 or subcellular localization of Erk-1 , pErk-1 , Erk- 2 or pErk-2 within a suitable test system prior to and after contacting the test system with test compound.
  • Raf produced within a suitable test system is compared prior to and after contacting the test system with test compound.
  • comparisons of test or candidate compound on those phosphorylation states are made using 3a-diol or E-3a-diol as control compounds.
  • comparisons of test or candidate compound on MEK-1 interaction with Erk-1 and/or Erk-2 are made.
  • Test or candidate compounds that improve protein-protein interaction between Erk-2 and MPK-3 such that phosphatase activity is increased are expected, based upon the insights provided by the invention disclosed herein, to inhibit cytoplasmic pro-survival signaling from Erk-2 and thus would prove useful in treating hyperproliferation conditions.
  • Improved binding with MPK-3 of deactivated Erk-2 is also expected, based upon the insights provided by the invention disclosed herein, to compete with its binding to MEK-1 to slow reactivation of Erk-2 and subsequent nuclear translocation of the reactivated isoform.
  • comparisons of MKP-3 phosphatase activity are compared prior to and after contacting the test system with test or candidate compound.
  • optional comparisons of test or candidate compound are made using 3a-diol or E-3a-diol as control compounds.
  • MKP-3 activity is assayed by monitoring hydrolysis of p-nitrophenol phosphate ester.
  • the membrane androgen receptor can be specifically bound by cell-impermeable androgen-protein conjugate, and when contacted with LNCaP cells superficially behave like E-3odiol.
  • T-BSA can induce Erk phosphorylation (but without apparent isoform selectivity), inhibit cell proliferation and induce PSA secretion.
  • androgen-BSA conjugates on downstream signal transduction are sometimes more like that of 3a-diol.
  • 3a-diol increases Akt signal transduction and T-BSA increases PI3K activity in LNCaP, MCF-7 and T47D. Docket No. 354.
  • E-3a-diol selectively permits Erk-1 phosphorylation in LNCaP to the exclusion of Erk-2 without increasing gene expression of either isoform.
  • T-BSA also increases total pErk levels.
  • E-3odiol and T-BSA have opposite effects on phosphorylation of PI3K with E-3a-diol decreasing tyrosine phosphorylation of the p85 regulatory subunit whereas T-BSA increases that phosphorylation in LNCaP and MCF-7.
  • T-BSA increases that phosphorylation in LNCaP and MCF-7.
  • DU-145 it is only on longer exposure to T-BSA (2h) is decreased p85 tyrosine phosphorylation observed [Papadopoulou et al. (2008)].
  • E-3a-diol binds to the m-AR GPR-C6a to release Ga/q to inhibit P85a PI3K phosphorylation and to activate Ras-Erk signaling to selectively activate Erk-1 in preference to Erk-2. It's therefore postulated that androgen- BSA conjugates unexpectedly behave in similar manner to E-3a-diol by inducing apoptosis through interaction at GPR-C6a, but does so on time scales more consistent with a secondary genomic effect and does not engage in a non-genomic effect that selectively engages Erk-2 intracellular ⁇ such that Erk-1 is preferentially phosphorylated.
  • those conjugates are useful for positive controls for PI3K activation as measured by increased pAKT levels, p85 tyrosine phosphorylation and/or downstream effects on effector proteins in PI3K-Akt signal transduction and for non-specific Erk isoform activation as measured by increased pErk-1 and p-Erk-2 levels with control for total protein levels.
  • a cell-impermeable androgen conjugate with C3 steroid attachment is used as a positive control for screening test compounds for secondary genomic activity that is associated with the non-genomic activity of E-3a-diol due to its engagement of GPR-C6a.
  • Those conjugates include T, DHT, 3a-diol or ⁇ covalently attached through a linker to a protein such as BSA, optionally with fluorophore.
  • Preparation of T and DHT-conjugates are described in De Goeij, A.F. (1986).
  • Example use of T-BSA conjugates is described in Hatzoglou, A. et al. (2005).
  • the ⁇ and 3a- diol conjugates are prepared by contacting a suitably protected hydroxysteroid (free C3- OH) with a hindered base and contacting the resulting alkoxide with a suitable protected bifunctional PEG linker wherein one functional group is a terminal a-halo ether that undergoes nucleophillic substitution by the alkoxide intermediate and the other functional group is a carboxylic acid ester.
  • a suitable protected bifunctional PEG linker wherein one functional group is a terminal a-halo ether that undergoes nucleophillic substitution by the alkoxide intermediate and the other functional group is a carboxylic acid ester.
  • the steroid-0-CH 2 - (OCH2) n -COOH wherein n is preferably 1 , 2, 3 or 4
  • intermediate is covalently attached to BSA in analogous manner described for preparation of T and DHT conjugates.
  • 5a- androstane-protein conjugates suitable as test compounds include conjugates of 17a- ethynyl-5a-adrostan-3-one-173-ol (or 17a-ethynyl DHT, henceforth E-DHT). In preferred Docket No. 354.
  • E-DHT is conjugated to BSA in similar manner as conducted for testosterone and DHT (i.e., through an oxime linker utilizing its 3-one substituent).
  • levels of one or more protein products from transcription by Elk-1 (which is an Erk substrate) or dependent on c-fos expression (with Elk-1 as its transcription factor) are compared prior to and after contacting the test system with test compound.
  • comparisons of test or candidate compound are preferentially made using 3a-diol and/or E-3a-diol as control compounds.
  • Erk does not always enter the nucleus, but may be retained in the cytoplasm, which is dependent in part on the activating MEK isoform. Activated Erk may also be retained in the cytoplasm by to one or more scaffold proteins whose subcellular location influences the outcome of the signal flowing through this signal trandsuction node. It is therefore believed the preferential retention of pErk-1 , selectively in a cytosolic subcellular domain with concommitant sequesterization of ErK-2 in an inaccessible domain for phospho-activation occurs by E-3a-diol acting intracellular ⁇ .
  • comparisons of test compound are made using 3a-diol and/or E-3a-diol as Docket No. 354. PATENT control compounds.
  • the short term effects of pErk-1 acting in the cytosol are expected to occur within the 5-15 min time frame whereas pErk-2 effects acting in the nucleus are expected to occur within the 30-60 min time frame.
  • non-genomic AR signaling effects resulting either from the membrane AR or from i-AR scaffolding effects within the cytosol, will occur in the 5-15 min time frame whereas genomic effects from transactivation activity of i-AR acting in the nucleus will occur in the 30-60 min time frame.
  • G protein-dependent signaling is expected to be rapid (within 5 min) and transient in nature while G protein-independent signaling is expected to occur subsequent to G protein-dependent signaling and be more persistent.
  • transformed HEK-293 are transfected (transiently or stably) with an ARE promoter-reporter construct and T877A mutant AR to provide a suitable in vitro test system.
  • ARE promoter-reporter construct and T877A mutant AR to provide a suitable in vitro test system.
  • E-3a-diol was found to stimulate transcription of the reporter, presumably due to partial agonist activity at mt- AR.
  • Other suitable in vitro test systems for evaluating test compounds for nuclear receptor binding and transactivation activity employ artificial cell constructs that contain intracellular forms of various steroid receptors, as exemplified by Table 7,.
  • nuclear binding assays as described herein for evaluating test compounds show that E- 3a-diol does not bind strongly to the wild type AR (wt-AR), yet appears to bind the T877A mutant receptor, and not at all to PR, GR or ERp, and has a weak affinity for ERa.
  • C4-2B cells are transfected (transiently or stably) with an ARE promoter-reporter construct.
  • C4-2B is a CRPC cell line that express the same mutated AR gene as in LNCaP cells.
  • E-3a-diol as a positive control compound activated the ARE promoters in these mutated / ⁇ -expressing cells, but did not inhibit transactivation of i-AR upon co-contacting the cells with ⁇ -AED or DHT. Instead, E-3a-diol appeared to activate the ARE promoter-reporter even though it inhibited proliferation and increased apoptosis. That result supports the assertion that residual i-AR transactivation activity is required for differentiation-induced apoptosis with E-3a-diol acting as a partial agonist.
  • apoptotic activity of a test or candidate compound is examined on LuCaP-35V tumors implanted in SCID mice in the presence of pAED, which is a suitable in vivo test model for evaluating test compounds for activity against CaP, particularly for CRPC.
  • tumor cells contain wt-AR, which is often found in CRPC.
  • E-3a-diol in one such suitable in vivo test system model (see Example 5) further supports its activity against castrate-resistant prostate cancer in the manner described for the C4-2B cell-based test system (whose cells contain T877A Docket No. 354.
  • wt-AR cannot be activated directly by E-3a-diol (i.e., not a sufficient agonist for wt-AR), but is activated indirectly through the Ras-Erk pathway through cross-talk with GPR-C6a-Ga/q signaling stimulated by binding of E-3a-diol to the GPCR, thus providing the required AR signaling that supports differentiation. It is further believed that if pAED is an agonist for GPR-C6a Ga/i signaling this activity is effectively competed by E-3a-diol binding to the GPCR to induce inverse agonist activity by activating Ga/q signaling.
  • IC 50 E-3a-diol 1 .5 ⁇ ) that would indicate, in the absence of inverse agonist action of E-3a-diol at GPR-C6a to activate Erk- 1 and its sequesterization of Erk-2 in inactive form, direct agonist action at wt-AR to support proliferation would occur.
  • a suitable in vitro test system comprises PC-3 or DU-145 cells.
  • a preferred test or candidate compound influences p21 Cip1 activity to inhibit cyclin D-CDK activity or decreases transcription of a p21 Cip1 -inducible reporter gene transfected into PC-3 or DU-145 cells.
  • a more preferred test or candidate compound induces those cells to undergo apoptosis when contacted in combination with a DNA-alkylating or DNA-intercalating agent or with a tubulin disrupting agent.
  • C4-2B cells are injection into tibiae as exemplified by Example 4.
  • xenograft tumors that arise exhibit an osteoblastic response when grown in the bone environment similar to that seen in patients with CaP bone metastases.
  • results in one such suitable in vivo test system demonstrate that E-3a-diol administered as a positive control compound decreases weight of tumored tibiae in comparison to control tumored tibiae and immunohistochemical analysis of the tumored tibiae showed osteoblastic reaction associated with growth of the C4-2B tumors in the bone and tumor foci between the newly formed woven bone in both E-3a-diol-treated and control tibiae.
  • Example 5 which uses LuCaP-35V cells that contain wt-AR
  • E-3a-diol administered as a positive control compound in this model where C4-2B cells endogenously contain T877A mt-AR, resulted in decreased serum PSA.
  • E-3a-diol in the C4-2B and LuCaP-35V indicate its activity for treating late-stage disease as well as earlier stage disease where progression to "androgen-independence" has not occurred. Furthermore, activity is indicated for wild- type and mutant AR-bearing tumors provided that the mutant-AR remains capable of transactivation. Furthermore, transient increases in PSA in a treated subject may actually be associated with an apoptotic effect on tumor cells due to redirection of aberrant AR signaling towards differentiation.
  • E-3a-diol In another suitable in vivo test system test or candidate compounds are evaluated in for activity towards breast cancer (which often contains i-AR) in mammary tumors induced by carcinogen (see Example 19 as exemplified for E-3a-diol).
  • the number of large tumors >300 mm 3
  • ranged between five and eight for all active groups except anastrozole (four) was highest in the E-3a-diol-docetaxel combination group, and lowest in the tamoxifen comparator.
  • E-3a-diol aggressively shrank established tumors and prevented the appearance of new tumors.
  • the rate of tumor volume reduction and degree of tumor suppression after cessation of treatment was similar for both high dose E-3a-diol monotherapy and tamoxifen.
  • E-3a-diol Treatment with E-3a-diol alone resulted in a rapid reduction in tumor burden, similar to tamoxifen and anastrozole, and the combination of E-3a-diol with docetaxel was more effective than any of the agents used alone as monotherapy.
  • tumors began to grow in all monotherapy groups, but not in animals treated with the combination of E-3a-diol and docetaxel.
  • Tumors from E-3a-diol treatment animals showed increased immunohistochemical markers of apoptosis, increased expression of pro-apoptotic genes, decreased expression of anti-apoptotic genes and decreased levels of androgen and estrogen nuclear hormone receptors.
  • Anastrozole is a standard of treatment for reducing endogenous estradiol in patients with breast cancer that have failed first-line therapy. Although active in the MNU model, anastrozole was inferior to E-3a-diol in the study.
  • E-3a-diol may have a positive effect on bone, having been shown to increase bone mineral density relative to vehicle in an intra-tibial prostate cancer xenograft model (Examples 4-5), which may be relevant to breast cancer considering the importance of bone metastases in this disease [Ye, L. et al. (2009)].
  • E-3a-diol does not have appreciable hepatic, hematopoietic, or cardiopulmonary toxicity or deleterious effects on bone at what are currently believed to be pharmacologically relevant doses and thus may be used as a negative control compound for these toxicities in suitable in vivo test systems.
  • tubulin disrupting agent docetaxel in combination with the test compound is determined using the C4-2B tibia tumor model (Example 16 as exemplified for E-3a-diol when administered as a positive control compound) in castrated SCID beige mice. In that study the benefit of the combination therapy was also indicated by a positive effect on bone mineral density.
  • Example 16 and 17 may be modified in order to evaluate a test or candidate compound found to have one or more effects on protein phosphorylation states disclosed herein for E-3a-diol in combination with a cytotoxic agent or other cancer chemotherapeutic compound known to be effective in treating a hyperproliferation condition in which AR signaling exists or is capable of occurring.
  • a preferred test or candidate compound will inhibit proliferation or induce apoptosis of tumor cells in a suitable in vivo test system for a hyperproliferation condition dependent on the presence of functional i-AR.
  • a preferred test or candidate compound exhibits synergistic or enhanced tumor inhibition activity when used in conjunction with a cancer chemotherapeutic compound in those test systems.
  • the suitable test system in an in vitro, xenograft or carcinogen-induced model for breast or prostate cancer.
  • a synergistic or enhanced effect is observed when contacting the test compound in these test system in conjunction with an ERa or /-AR inhibitor.
  • a synergistic effect is observed when using the test or candidate compound in conjunction with a DNA alkylating or intercalating agent.
  • a test or candidate compound identified as a candidate compound provides a positive effect on bone mineral density in qualitatively similar manner to E-3a-diol when contacted to a suitable in vivo test system, preferentially in the presence of a cancer chemotherapeutic compound.
  • a test or candidate compound additionally affects the phosphorylation state of i-AR in qualitative manner observable for E-3a-diol.
  • a test compound contacted to a suitable in vivo test system effects PSA secretion in qualitatively similar manner described for E-3a-diol for identification as a candidate compound.
  • a test compound or candidate when contacted to a suitable in vitro test system will induce prostate cancer cells to secrete PSA in conjunction with the anti-proliferative or pro-apoptotic Erk sequesterization effects described herein. Docket No. 354.
  • a test or candidate compound contacted to a suitable test system comprising LP LNCaP cells will inhibit their proliferation or induce apoptosis when stimulated with an androgen, such as pAED or DHT, for identification as candidate compound.
  • an androgen such as pAED or DHT
  • a test or candidate compound contacted to a suitable test system comprising castrate-resistant CaP cells, such as HP LNCaP, LuCaP or C2-4B cells will inhibit their proliferation or induce apoptosis in an androgen-depleted environment for its identification as candidate compound.
  • a test or candidate compound contacted to a suitable test system comprising cancer cells having functional i-AR will exhibit one or more in vitro gene transcription effects described herein.
  • an examination of E-3a-diol- treated HP LNCaP cultures showed a dramatic increase in secreted PSA in the media.
  • this rise in PSA was not accompanied by an increase in proliferation; rather the proliferation of HP LNCaP was inhibited.
  • Those findings described herein are similar to the observations in the aforementioned LuCaP-35V tumor treatment study. The data is therefore consistent with a switch in the transactivation program of i-AR culminating in selective ARE promoter engagement that promotes differentiation.
  • a test or candidate compound will affect gene transcription by switching the gene expression patterns observed for DHT or 3a-diol that supports proliferation or survival to one that supports differentiation in the manner observed for E- 3a-diol. Examples of induced changes in gene expression by contacting E-3a-diol to LNCaP cells are shown in Tables 2-5 (Example 2).
  • test or candidate compound will affect one or more genes in qualitatively similar manner to that indicated in Table 6. Particularly preferred are test or candidate compounds that effect expression of one or more of AR, IRX5, JUN, CD44 and JAG1 (decreased expression), and RIS1, TIMP2, RUNX1, CASP10 and LOX (increased expression).
  • [445] A method to identify a candidate compound, the method comprising (or consisting essentially of or consisting of) (a) contacting a test compound with a suitable test system; (b) determining phosphorylation states of Erk-1 and Erk-2 resulting from step (a); and (c) selecting a test compound that positively modulated phosphorylation state of Erk-1 activation loop relative to Erk-2 or negatively modulated the phosphorylation state of Docket No. 354.
  • preferred test systems constitute cells or tissue in vivo or cells in tissue culture or constitute cell extracts wherein signaling through target molecules of interest, e.g., Erk-1 , Erk-2, PI3K proteins, GPR-C6a and/or AR, is(are) functional and phosphorylation changes or other biological responses as described herein, as for example for E-3a-diol and 3a-diol, in response to the test (or control) compound can be measured.
  • Other preferred test systems are cell-free artificial test systems prepared by reconstituting a signal transduction pathway with one or more proteins of that pathway in a suitable buffer with a downstream substrate that is capable of phosphorylation by at least one of the signal transduction pathway proteins
  • test compound to be contacted with a suitable test system will usually be in a vehicle, composition or formulation that is compatible with the test system, e.g., the test compound can be in a solution or suspension or it can be administered as a solid or liquid formulation to an animal such as a rodent (e.g., mouse or rat), or, for clinical assessment of the test compound, it can be administered to a human patient.
  • Said contact is followed by measurement of target molecule phosphorylation states (e.g., Erk-1 , Erk-2) in cells or tissue samples taken from the animal or patient after administration of the test compound to the animal or patient.
  • target molecule phosphorylation states e.g., Erk-1 , Erk-2
  • test systems will typically be contacted with a positive, negative, placebo and/or vehicle control or reference compound to confirm proper functioning of the test system in vitro or in vivo.
  • the test compound and any control or reference compound or composition will be contacted with the test system (i) under conditions where the test system is functional, e.g., cells in tissue culture are maintained under standard growth conditions, (ii) for one or more sufficient periods of time, e.g., for about 5 sec.
  • type of biological response is measured preferably from within 5 sec to 30 min after contacting the test system with test compound.
  • type of biological response is preferably measured from within 60-120 min. after said contact.
  • Typical test compound final concentrations with the test system will be in a range, e.g., about 0.01 nM to about 20 mM or usually about 1 nM to about 10 mM, which can include one or more of about 0.05 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 200 nM, 1 mM, 2 Docket No. 354. PATENT mM and 10 mM. Concentrations of other compounds, e.g., components or excipients in the formulation that contains the test compound will typically be tested at the same or nearly the same concentrations they are at when the test compound is contacted with the test system.
  • the phosphorylation state of Erk-1 activation loop is positively modulated with respect to Erk-2 by the test compound increasing the amount of pErk-1 produced in cells of a suitable test system in comparison to the amount of pErk-2 produced. In those embodiments sometimes Erk-1 protein level is positively modulated without discernable increase in pErk-2 protein level. In other embodiments the phosphorylation state of Erk-2 activation loop is negatively modulated by the test compound decreasing the amount of pErk-2 produced when cells of a suitable test system are stimulated to proliferate in comparison to the amount of pErk-2 that would have been produced in absence of the test compound.
  • the phosphorylation status of Erk-1 activation loop is positively modulated such that the ratio of intracellular pErk-1 to pErk-2 is increased due to contact of test compound with cells of a suitable test system. In those embodiments sometimes pErk-1 is produced and pErk-2 remains at basal levels or is not produced.
  • the suitable test system is a suitable in vitro cell-based test system.
  • the cells within the suitable test system or suitable in vitro cell-based test system are cancer cells dependent on i-AR signaling for survival or proliferation.
  • These embodiments include androgen-associated cancer cells from a deposited cancer cell line known to contain functional i-AR protein.
  • Those embodiments also include hyperproliferating cells not typically dependent on i-AR signaling, but which become increasingly so as a result of an emerging resistance mechanism.
  • effects on cells contacted with test compound are compared to those of control cells (i.e., cells that are sham contacted or are contacted with test compound that is a positive or negative control).
  • control cells i.e., cells that are sham contacted or are contacted with test compound that is a positive or negative control.
  • Those include changes in phosphorylation state of MAPK3 (Erk-1 ) vs. MAPK1 (Erk-2) or phosphorylation state of PI3K in test cells compared to control cells, i.e., modulation of MAPK3 vs. MAPK1 cells compared to control cells.
  • test compounds selected for step (c) that have been contacted with test cells positively modulated the phosphorylation state of Erk-1 or negatively modulated the phosphorylation state of Erk-2 relative to the other Erk isoform without qualitatively or quantitatively similar effects observed for sham contact with control cells.
  • the selected test compound will positively modulated the phosphorylation state of Erk-1 or negatively modulated the phosphorylation state of Erk-2 relative to the other Docket No. 354.
  • Erk isoform qualitatively or quantitatively similar to that observed for E-3a-diol contact with control cells as positive control.
  • test system or the suitable in vitro cell-based test system comprises (or consists essentially of or consists of) cells from a prostate, breast, ovarian cancer or other cancer cell line that is dependent on transactivation of one or more genes with an upstream ARE promoter for survival or proliferation, comprises (or consists essentially of or consists of) cancer cells not having endogenous functional GPR-C6a protein, with or without functional i-AR protein, that are genetically engineered or transfected (stably or transiently) so as to contain functional GPR-C6a protein, cancer cells not having endogenous functional GPR-C6a and i-AR proteins that are genetically engineered or transfected (stably or transiently) so as to contain functional GPR-C6a and i-AR proteins or comprises (or consists essentially of or consists of) transformed normal cells not having endogenous functional GPR-C6a protein or endogenous functional GPR-C6a and i-AR
  • the i-AR protein is a functional wt-AR or a functional mutant AR protein that contains the human amino acid sequence, while in other embodiments the functional wt-AR or mt-AR protein contains the amino acid sequence for a rodent homolog as for example that found in mouse or rat.
  • the gene encoding the androgen-inducible promoter gene and the gene encoding functional i-AR protein are within the same gene construct that is transfected into the cells.
  • functional i-AR gene is engineered into the AR 1' prostate cancer cells such as DU145 and PC-3 or is engineered into transformed normal cells that do not endogenously contain functional i-AR protein.
  • Preferred embodiments use cancer cells containing or genetically engineered to contain full-length human wt-AR protein, while in other preferred embodiments the cancer cells have a T877A mutant AR protein such as the LNCaP mt-AR.
  • the mt-AR protein is mutated in the NH 2 -terminal domain such that an advantage to survival or proliferation is conferred and may also include the T877A mutation.
  • test system or the suitable in vitro cell-based test system comprises (or consists essentially of or consists of) cancer cells or transformed normal cells that are responsive to DHT or pAED endogenously having or transfected (stably or transiently) or genetically engineered to have functional GPR-C6a protein or comprises (or consists essentially of or consists of) AR +/+ cancer cells having a functional GPC-C6a gene or transformed GPC-C6a v ⁇ AR ⁇ ' ⁇ Docket No. 354.
  • PATENT normal cells that are genetically engineered or transfected (stably or transiently) so as to contain functional GPR-C6a and i-AR genes.
  • the prostate cancer cells of step (a) are quiescent.
  • the prostate cancer cells are LP LNCaP cells or HP LNCaP that have been passaged in androgen- and growth factor-depleted media.
  • these or other prostate cancer cells are supported by androgen supplanted to the media in order to provide a suitable test system.
  • the prostate cancer cells are PC-3 or DU-145 cells transfected to contain functional i-AR gene.
  • the cells are contained within suitable in vitro cell-based test systems for modeling CRPC in vitro and preferably include C4-2B or HP-LNCaP cells.
  • the cancer cells are supported under conditions of androgen and growth factor depletion using an i-AR agonist.
  • media support is typically with DHT.
  • T877A mt-AR For cancer cells having or transfected or genetically engineered to have T877A mt-AR, support may be affected not only by wt-AR agonists, but with other natural hormones that are agonists for other nuclear hormone receptors (e.g., estradiol) and further include other natural hormones including DHEA or PAED. In some preferred embodiments these T877A mt-AR cancer cells are stimulated with DHT or pAED.
  • test system or the suitable in vitro cell-based test system are cancer cells or transformed normal cells containing endogenous functional GPR-C6a protein.
  • the cancer or transformed cells of the suitable test system or suitable in vitro test system in step (a) further endogenously contain, or are transfected (stably or transiently) or genetically engineered so as to contain, functional i-AR protein.
  • the cells of the suitable test system or suitable in vitro test system that have endogenous functional GPR-C6a protein and have or are transfected or genetically engineered to have function i-AR protein are further genetically engineered or transfected (stably or transiently) to contain an androgen-inducible reporter gene.
  • the cells of the suitable test system or suitable in vitro test system having or genetically engineered to have functional GPR-C6a and i-AR genes are further transfected (stably or transiently) or genetically engineered to contain an androgen-inducible reporter gene.
  • the cells of the suitable test system or the suitable in vitro cell-based test system are cancer cells wherein the cancer cells are or derived from an androgen signaling-dependent prostate cancer cell line.
  • the androgen-dependent prostate cancer cells typically have minimal baseline AR transactivation activity for a suitable test system when placed into androgen- and growth factor-depleted media and thus in some embodiments are supported by added androgen or another i-AR agonist, (i.e., in these embodiments the suitable test system comprises androgen-dependent prostate cancer in androgen- and growth factor-depleted media supplemented with i-AR agonist).
  • Androgen precursors may also be used if the cancer cells are competent to biotransform the androgen precursor to active androgen.
  • the cells of the suitable test system or the suitable in vitro cell-based test system are cancer cells wherein the cancer cells are an androgen-independent or CRPC prostate cancer cell line or are derived therefrom.
  • those prostate cancer cells typically have sufficient baseline AR transcriptional activity for a suitable test system when placed into androgen-depleted media. In some embodiments this baseline activity is increased by added i-AR agonist.
  • the cells of the suitable test system or the suitable in vitro cell-based test system are cancer cells wherein the cancer cells are transfected (stably or transiently) to contain an androgen-inducible reporter gene.
  • the upstream promoter in the inducible reporter gene is the ARE promoter for PSA. In other embodiments this promoter is the probasin promoter containing ARE.
  • the reporter gene encodes for luciferase or chloramphenicol acetyl transferase (CAT).
  • the cancer cells are transfected with a gene construct that contains the probasin promoter upstream to a luciferase reporter.
  • the cells of the suitable test system or the suitable in vitro cell-based test system are cancer cells wherein the cancer cells comprise (or consists essentially of or consists of) prostate cancer cells incubated or passaged under hormone- and growth factor-depleted conditions.
  • incubation of the prostate cancer cells is supplemented Docket No. 354.
  • PATENT by androgen or androgen precursor preferably 3a-diol, pAED, testosterone or DHT, or another i-AR agonist.
  • incubation of the prostate cancer cells is LP LNCaP supplemented by pAED.
  • use of supplemented androgen or androgen precursor requires the cancer cells to contain functional T877A mt- AR protein so that supplementation stimulates i-AR signaling or requires cancer cells competent to biotransform androgen precursor to active androgen.
  • test system or the suitable in vitro cell-based test system comprises prostate cancer cells passaged or incubated in androgen- and growth factor-depleted media wherein the androgen- and growth factor- depleted media is charcoal-stripped RPMI serum.
  • test system or the suitable in vitro cell-based test system comprises (or consists essentially of or consists of) LP LNCaP cells co-contacted in step (a) with pAED in an amount effective to activate AR transactivation in the absence of test compound.
  • test system or the suitable in vitro cell-based test system comprises (or consists essentially of or consists of) HP LNCaP cells.
  • test system or the suitable in vitro cell-based test system comprises (or consists essentially of or consists of) C4-2B cells.
  • any one embodiments 1 -16 further comprising (or further consists essentially of or further consists of): (a-2) contacting a test compound with an initial suitable in vitro cell-based test system comprising (i) MDA-kb2 cells transfected to contain a MMTV promoter-reporter gene, (ii) T47D-kBluc cells transfected to contain an estrogen-inducible promoter-reporter gene, (iii) HEK293T cells transfected to contain an androgen-inducible reporter gene and a gene encoding functional i-AR protein, (iv) HEK293T cells containing an estrogen-inducible reporter gene and a gene encoding functional ERp protein, or (v) a combination of (i)-(iv); and (a-1 ) selecting for the conduct or performance of step (a) a test compound from step (a-2) that induces transcription of the reporter(s) in a manner that is qualitatively similar to E-3
  • this step conducted prior to step (a) is used to screen for test compounds that are capable of inducing reporter gene transcription in one or more of the initial suitable in vitro cell-based test system. Such compounds are then selected for determining modulation effects on Erk phosphorylation states according to step (b). In preferred embodiments the test compound induces reporter gene transcription quantitatively similar to E-3a-diol as positive control.
  • the oxidoreductase is capable of operating in oxidative mode with concommitant NAD(P)+ conversion to NAD(P)H towards 3a-hydroxy steroids.
  • the oxidoreductase is present in a suitable in vivo cell- based test system and is preferentially active or has physiologically relevant activity towards the 3a-hydroxy group of 3a-hydroxy steroids.
  • the oxidoreductase is active towards the steroid 3a-diol or is capable of interconverting 3a- diol and DHT.
  • oxidoreductase is an aldo-keto reductase, a short-chain dehydrogenase-reductase (SDR), a hydroxyacyl CoA dehydrogenase or a 17p-hydroxysteroid dehydrogenase (17p-HSD) having 3a- hydroxysteroid oxidoreductase activity.
  • SDR short-chain dehydrogenase-reductase
  • p-HSD 17p-hydroxysteroid dehydrogenase
  • a suitable cell-free test system for determining modulation of oxidoreductase activity operates the enzyme in oxidative mode and is capable of oxidizing naturally occurring 3a-hydroxy steroids to the corresponding 3-one or reducing a 3-one containing steroid to the corresponding 3a- hydroxysteroid.
  • test compound negatively modulates oxidative activity of 17p-HSD Type 10 (17HSD10).
  • the ability of a test compound to negatively modulate this activity is evaluated in a suitable Docket No. 354.
  • PATENT cell free test system by monitoring the production of NADH from oxidation of an alcohol substrate in the absence and presence of test compound.
  • Example assay conditions and alcohol substrates are described in Shafquat, N. et al. (2003), which are incorporated by reference herein.
  • [477] 22 The method of embodiment 20 wherein the test compound inhibits 17p-HSD Type 10 interconversion of 3a-diol and DHT.
  • the identified candidate compound selected from step (c) inhibits the oxidation of 3a-diol to DHT as determined in the cell-free test system at about 10 ⁇ concentration or less, preferably at about 100 nM or less, more preferably at 10 nM or less.
  • the identified candidate compound positively modulates protein-protein interaction with Tfg or positively modulates protein-protein interaction with Erk-2 in comparison to Erk-1 .
  • the protein-protein interaction positively modulated is between Erk-2 and another signal transduction protein, such as a scaffold protein or a phosphatase, to inhibit Erk-2 phosphorylation, enhance pErk-2 de-phosphorylation or inhibit pErk-2 translocation to the nucleus in comparison to the other Erk isoform.
  • protein-protein interaction of Erk-2 with Tfg is enhanced by the selected test or candidate compound.
  • a selected test compound identified as a candidate compound in step (g) has the characteristics of steps (c) and (e).
  • any one embodiments 1 -23 comprising further comprising (or further consisting essentially of or further consisting of): (h) contacting a selected test compound from step (c) or step (e) with cancer cells of a suitable in vivo test system or co-contacting the selected test compound with a positive control cancer chemotherapeutic compound; and (j) selecting the test compound that inhibits cancer cell proliferation statistically significant to cancer cells contacted with vehicle alone, inhibits cell proliferation to similar or greater extent than cancer cells contacted to the positive control alone, or synergistically inhibits cancer cell proliferation when cancer cells are co- Docket No. 354. PATENT contacted with positive control.
  • the in vivo test system is a xenograft animal model resulting from implantation into a rodent such as mice or rats of (1 ) cancer cells from a hormone-associated cancer cell line that is dependent on i-AR signaling for survival or proliferation (2) cancer cells containing functional i-AR or (3) cancer cells that are transfected (stably or transiently) to express or overexpress a gene or genes encoding functional i-AR and/or GPR-C6a or (4) transformed normal cells not containing endogenous i-AR and GPR-C6a proteins that are transfected (stably or transiently) to contain functional i-AR and GPR-C6a proteins.
  • a rodent such as mice or rats of (1 ) cancer cells from a hormone-associated cancer cell line that is dependent on i-AR signaling for survival or proliferation
  • the implanted cells are cancer cells from a hormone-associated cancer cell line that is dependent on i- AR signaling for survival or proliferation.
  • the cancer cells are from a prostate or breast cancer cell line such as LNCaP, LuCaP, C4-2B, CWR22 or MCF-7.
  • a selected test compound identified as a candidate compound in step (j) in addition to the characteristics of step (c) or steps (c) and (e) or steps (c) and (g) or steps (c), (e) and (g) binds to Tfg or selectively binds to Erk- 2 protein in comparison to Erk-1 .
  • a selected test compound identified as a candidate compound in step (j) has the characteristics of steps (c), (e) or steps (c) and (g).
  • a selected test compound identified as a candidate compound in step (j) has the characteristics of steps and (c), (e) and (g).
  • the in vivo test system is a xenograft animal model resulting from implantation of cancer cells from a hormone-associated cancer cell line or transformed cell line that are dependent on AR transactivation for survival or proliferation into an immune-compromised rodent.
  • the cells are from a prostate or breast cancer cell line implanted into castrated SCID mice wherein cancer cell proliferation is optionally supported by androgen or estrogen administration.
  • cancer cells are prostate cancer cells implanted into immune-compromised rodent(s), wherein the immune-compromised rodent(s) are castrated SCID or athymic nu/nu mice, and wherein AR transactivation is supported by androgen supplementation.
  • testosterone supplementation is by administration of a prodrug such as hydroxyl ester of the testosterone 3p-hydroxy group.
  • hydroxyl esters include acetate, enanthate, propionate, isopropionate, isobutyrate, butyrate, valerate, caproate, isocaproate, hexanoate, heptanoate, octanoate, nonanoate, decanoate, undecanoate, phenylacetate, benzoate and hydroxyl esters described in Becker, K.L., Ed. (2001 ), the disclosure of which is incorporated by reference herein.
  • prostate cancer cells are C4-2B or LuCaP-35V cells implanted into castrated SCID mice or CWR22-R1 cells implanted into nu/nu athymic mice.
  • the in vivo test system is a xenograft animal model resulting from implantation into a rodent of cancer cells from a hormone-associated cancer cell line or transformed cell line dependent on AR transactivation for survival or proliferation, wherein the rodent is immune-compromised and wherein the cancer cells implanted into an immune compromised rodent are co- contacted with test compound and a cancer chemotherapeutic compound.
  • chemotherapeutic compounds include tubulin disrupting agents (i.e., compounds that aberrantly stabilize or destabilize tubulin polymers or interfere with tubulin polymerization).
  • Other cancer chemotherapeutic compounds include anti-metabolites (e.g., methotrexate, fluorouracil, azathioprine and mercaptopurine), tyrosine kinase inhibitors, (e.g., imatinib, gefitinib, erlotinib and others reviewed in Shawver, L.K. et al. (2002) and Hsp90 inhibitors (e.g., glendamycin and others reviewed in Neckers, L. (2002)).
  • anti-metabolites e.g., methotrexate, fluorouracil, azathioprine and mercaptopurine
  • tyrosine kinase inhibitors e.g., imatinib, gefitinib, erlotinib and others
  • Additional cancer chemotherapeutic compounds contemplated for this embodiment are DNA damaging agents. These agents include alkylating agents (e.g., the nitrogen mustards cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil and Ifosfamide, the nitrosoureas carmustine, lomustine and streptozocin and the alkyl sulfonate busulfan). Other DNA damaging agents are free-radical generating and DNA crosslinking compounds (e.g., platinum-containing compounds, such as cisplatin, carboplatin and oxaliplatin, and calicheamicins, such as calicheamicin- ⁇ and calicheamicin T).
  • alkylating agents e.g., the nitrogen mustards cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil and Ifosfamide, the nitrosoureas carmustine, lomustine
  • topoisomerase I inhibitors e.g., anthracyclines, which includes daunosamine and tetrahydro-naphthacenedione-based compounds, such as doxorubicin, duanorubicin, valrubicin, idarubicin and epirubicin, which may also act as DNA damaging agents, and Docket No. 354.
  • PATENT camptothecins such as irinotecan and topotecan
  • topoisom erase II inhibitors e.g., anthracyclines, which includes daunosamine and tetrahydro-naphthacenedione-based compounds, such as doxorubicin, duanorubicin, valrubicin, idarubicin and epirubicin, which may also act as DNA damaging agents, and Docket No. 354.
  • PATENT camptothecins such as irinotecan and topotecan
  • anti-tumor antibiotics such as bleomycin, plicamycin and mitomycin, and epipodophyllotoxins, including etoposide and teniposide, which are topoisomerase II inhibitors, may also be co-contacted with a test compound to a suitable test system comprised of (or consisting essentially of or consisting of) cancer cells.
  • a suitable test system comprised of (or consisting essentially of or consisting of) cancer cells.
  • Preferred cancer chemotherapeutic compounds are tubulin disrupting agents, which include taxanes (e.g., paclitaxel, docetaxel and cabazitaxel) and vinca alkaloid compounds (e.g., vinblastine, vincristine, vinorelbine and vindesine).
  • taxanes e.g., paclitaxel, docetaxel and cabazitaxel
  • vinca alkaloid compounds e.g., vinblastine, vincristine, vinorelbine and vindesine
  • the method of embodiment 24 wherein the in vivo test system is a rodent with induced or spontaneous mammary tumors.
  • Induced and spontaneous mammary tumor models are reviewed in Medina, D. (2000) and Medina, D. and Thompson, H. (2000).
  • Preferred mammary tumor models are carcinogen-induced tumors derived from exposure of a rodent (e.g., mouse or rat) to 7,12-dimethylbenz[a]anthracene (DMBA) or N-methyl-N- nitrosourea (MNU).
  • DMBA 7,12-dimethylbenz[a]anthracene
  • MNU N-methyl-N- nitrosourea
  • [494] 34 The method of embodiment 32 or 33 wherein the cancer cells are contacted with test compound or co-contacted with test compound and a cancer chemotherapeutic compound by systemic administration(s) of the compounds to the rodent.
  • the co-contacted cancer chemotherapeutic is administered intraperitoneally.
  • the cancer chemotherapeutic compound is a tubulin disrupting agent.
  • these agents include a taxane compound, such as paclitaxel, docetaxel or cabazitaxel or a vindoline compound such as vinblastine or vincristine.
  • rodents implanted with cells containing functional i-AR derived from a prostate cancer cell line are treated with an anti-androgen which antagonizes binding of DHT to the androgen nuclear hormone receptor, which inhibits the transformation of testosterone to DHT or which inhibits adrenal production of androgens.
  • the anti-androgen is a AR antagonist, 5a-reductase inhibitor, a cytochrome P450 lyase inhibitor or a LH-RH agonist or antagonist and includes MDV3100, flutamide, hydroxyflutamide, nitulamide, bicalutamide, cyproterone, ketoconazole, abiraterone, finasteride, dutasteride, diethylstilbestrol, leuprolide, bruserelin, goserelin and abarelix.
  • PATENT other embodiments rodents implanted with cells containing functional i-AR derived from a breast cancer cell are treated with an anti-estrogen.
  • the anti- estrogen is a ERa receptor antagonist, an anti-aromatase or a LHRH super-agonist including tamoxifen, fulvestrant, letrozole anastrozole and leuprolide.
  • tubulin disrupting agent is a taxane compound.
  • a method to identify a candidate compound comprising (or consisting of or consisting essentially of): (a) contacting a test compound with a suitable test system (a first test system); (b) determining phosphorylation states of Erk-1 and Erk-2 proteins resulting from step (a); (c) determining the phosphorylation state of a Class 1 PI3K resulting from step (a), or
  • step (d) wherein the selected test compound from step (d) is identified as a candidate compound.
  • the suitable test systems are suitable in cell-based in vitro test systems.
  • the method is conducted with the first and second suitable test systems of steps (a') and (a") wherein the test systems are suitable in cell-based in vitro test systems.
  • the suitable cell-based intro test systems are comprised of mammalian cancer cell or transformed normal cells endogenously having or genetically engineered to be GPR-C6a +/+ AR +/+ .
  • AR may be wild-type or encode a mutant intracellular AR protein such as Docket No. 354.
  • the GPR-C6a AR +/+ cells harbor a disabling PTEN mutation.
  • test system or systems independently are suitable cell-based in vitro test systems.
  • the method is conducted with the first and second suitable test systems of steps (a') and (a") wherein the test systems are suitable cell-based in vitro test systems, more preferably with those test systems being comprised of cells from the same cell line.
  • [505] 40 The method of embodiment 38 or 39 wherein the suitable cell-based test system or systems comprises (or consists essentially of or consists of) cells from a prostate, breast, ovarian cancer or other cancer cell line dependent on transactivation of one or more genes with an upstream ARE promoter for survival or proliferation or comprises (or consists essentially of or consists of) cancer cells or transformed normal cells genetically engineered or transfected to contain functional i-AR gene.
  • the suitable cell-based in vitro test system of step (a) or the suitable in vitro cell-based test systems of steps (a') and (a") consists essentially of cells from or derived from the same prostate or breast cancer cell line or the same transformed cells.
  • the suitable in vitro test system of step (a) or the suitable in vitro cell-based test system of step (a') or (a") comprises (or consists essentially of or consists of) cancer cells that are responsive to DHT or pAED or are i-AR' ⁇ cancer cells or transformed normal cells not having functional endogenous i-AR protein that are genetically engineered or transfected to contain functional i-AR gene.
  • the first and second test system comprises (or consists essentially of or consists of) cancer cells that are responsive to DHT or pAED or are i-AR ' ' ' cancer cells or transformed normal cells not having functional endogenous i-AR protein that are genetically engineered or transfected to contain functional i-AR gene.
  • Preferred methods use steps (a') and (a") wherein the first and second in vitro cell-based test systems consists essentially of the same line of i-AR +/+ cancer or transformed cells.
  • Preferred methods use steps (a') and (a") wherein the first and second in vitro cell- based test systems consists essentially of the same line of GPR-C6a +/+ cancer or transformed cells.
  • the cancer cells or transformed normal cells express functional endogenous GPR-C6a and i-AR genes or are genetically engineered or transfected (stably or transiently) to express functional GPR-C6a and i-AR genes.
  • [511] 43 The method of embodiment 38, 39, 40 or 41 wherein the cancer cells or transformed normal cells of the suitable in vitro cell-based test system of steps (a) or the cancer cells or transformed normal cells of the suitable cell-based test systems of steps (a') and (a") contain endogenous GPR-C6a protein or are genetically engineered or transfected (stably or transiently) so as to have functional GPR-C6a protein.
  • test systems or cell-based in vitro test system(s) independently are comprised of (or consists essentially of or consists of) prostate cancer cells that were incubated or passaged under hormone- and growth factor-depleted conditions.
  • cells of suitable test system(s) or in vitro cell-based in vitro test system(s) are as described in embodiment 10.
  • prostate cancer cells are HP LNCaP cells.
  • the method of any one of embodiments 38-48 wherein the suitable test system or the suitable cell-based in vitro test system of step (a) or the first and second suitable test systems or the first and second suitable cell-based in vitro test systems of steps (a') and (a") comprises (or consists essentially of or consists of) C4-2B cells incubated or maintained in hormone- and growth factor-depleted medium.
  • any one of embodiment 38-53 further comprising (or further consisting essentially of or further consisting of): (a-2) contacting a test compound with an initial suitable in vitro cell-based test system comprising (i) MDA-kb2 cells transfected to contain a MMTV promoter-reporter gene, (ii) T47D-kBluc cells transfected to contain an estrogen-inducible promoter-reporter gene, (iii) HEK293T cells transfected to contain an androgen-inducible reporter gene and a gene encoding functional i-AR protein, (iv) HEK293T cells containing an estrogen-inducible reporter gene and a gene encoding functional ERp protein, or (v) a combination of (i)-(iv); and (a-1 ) selecting for the conduct or performance of step (a) or steps (a') and (a") a test compound from step (a-2) that induces transcription of the reporter(s)
  • the steps (a-2) and (a-1 ) is conducted prior to step (a) or steps (a') and (a") and is used to screen for test compounds that are capable of inducing reporter gene transcription in one or more of the initial suitable in vitro cell-based test system.
  • the test compounds eliciting reporter gene transcription are then selected for determining modulation effects on Erk phosphorylation states according to step (b) or (b') or tyrosine p85 phosphorylation states according to step (c) or (c').
  • the test compound induces reporter gene transcription quantitatively similar to E-3a-diol as positive control.
  • test compounds eliciting reporter gene transcription are then selected for determining modulation effects on Erk and p85 phosphorylation (i.e., step a-1 is conducted prior to steps (a), (b) and (c), or steps (a'), (a"), (b') and (c')
  • [524] 55 The method of any one of embodiments 40-54 wherein the functional i-AR is wt-AR, T877A mt-AR or an i-AR ligand-binding domain (LBD) fused to AR Gal4 DNA binding domain.
  • the functional i-AR is wt-AR, T877A mt-AR or an i-AR ligand-binding domain (LBD) fused to AR Gal4 DNA binding domain.
  • any one of embodiments 38-55 further comprising (or further consisting essentially of or further consisting of): (d) determining modulation of redox activity of a functional oxidoreductase protein with the test compound selected from step (c) or step (c') in the same test system of step (a), (a') or (a"), a different suitable in vitro Docket No. 354. PATENT cell-based test system, or a suitable cell-free in vitro test system; and (e) selecting the test compound that additionally negatively modulates oxidoreductase 3a-hydroxy steroid oxidation or steroid 3-one reduction activity.
  • oxidoreductase is as described in embodiment 19.
  • oxidoreductase is an aldo-keto reductase, a short-chain dehydrogenase-reductase (SDR), a hydroxyacyl CoA dehydrogenase or a 17p-hydroxysteroid dehydrogenase (17p-HSD) having 3a- hydroxysteroid oxidoreductase activity.
  • SDR short-chain dehydrogenase-reductase
  • hydroxyacyl CoA dehydrogenase or a 17p-hydroxysteroid dehydrogenase 17p-hydroxysteroid dehydrogenase having 3a- hydroxysteroid oxidoreductase activity.
  • p-HSD 17p-hydroxysteroid dehydrogenase having 3a- hydroxysteroid oxidoreductase activity.
  • a suitable cell-free test system for determining modulation of oxidoreductase activity is as described in embodiment 20.
  • test compound negatively modulates oxidative activity of 17p-HSD Type 10 (17HSD10).
  • the ability of a test compound to negatively modulate this activity is evaluated in a suitable cell free test system by monitoring the production of NADH from oxidation of an alcohol substrate in the absence and presence of test compound.
  • Example assay conditions and alcohol substrates are given by embodiment 21 .
  • test compound inhibits 17p-HSD Type 10 interconversion of 3a-diol and DHT.
  • identified candidate compound selected from step (c) or step (c') inhibits the oxidation of 3a-diol to DHT as determined in the cell-free test system at about 10 ⁇ concentration or less, preferably at about 100 nM or less, more preferably at 10 nM or less.
  • any one of embodiments 38-59 further comprising (or further consisting essentially of or further consisting of): (f) determining binding interaction, optionally by SILAC, of test compound selected from step (c), step (c') or step (e) to the scaffold protein Tfg or to Erk protein(s), optionally in the presence of Tfg, Erk-1 and Erk-2 or another scaffold protein or a MAPK phosphatase, and (g) selecting a test compound that in addition to the characteristics of step (c) or steps (c) and (e) binds to Tfg or selectively binds to Erk-2 protein in comparison to Erk-1 or (g') selecting a test compound that in addition to the characteristics of step (c') or steps (c') and (e) binds to Tfg or selectively binds to Erk-2 protein in comparison to Erk-1 .
  • the identified candidate compound positively modulates protein-protein interaction with Tfg or positively modulates protein-protein interaction with Erk-2 in comparison to Erk-1 .
  • PATENT interaction positively modulated is between Erk-2 and another signal transduction protein, such as a scaffold protein or a phosphatase, to inhibit Erk-2 phosphorylation, enhance pErk-2 de-phosphorylation or inhibit pErk-2 translocation to the nucleus in comparison to the other Erk isoform.
  • protein-protein interaction of Erk-2 with Tfg is enhanced by the selected test or candidate compound.
  • a selected test compound identified as a candidate compound in step (g) or step (g') has the characteristics of steps (c) and (e) or steps (c') and (e), respectively.
  • any one embodiments 38-60 comprising further comprising (or further consisting essentially of or further consisting of): (h) contacting a selected test compound from step (c), step (c') or step (e) with cancer cells of a suitable in vivo test system or co-contacting the selected test compound with a positive control cancer chemotherapeutic compound; and (j) selecting the test compound that inhibits cancer cell proliferation statistically significant to cancer cells contacted with vehicle alone, inhibits cell proliferation to similar or greater extent than cancer cells contacted to the positive control alone, or synergistically inhibits cancer cell proliferation when cancer cells are co- contacted with positive control.
  • the in vivo test system is a xenograft animal model as described in embodiment 24.
  • a selected test compound identified as a candidate compound in step (j) has the characteristics of steps (c) and (e) or step (g).
  • a selected test compound identified as a candidate compound from step (j) has the characteristics of steps and (c') and (e) or step
  • (g')- [536] 62 The method of embodiment 61 wherein the in vivo test system is a xenograft animal model resulting from implantation of cancer cells from a hormone-associated cancer cell line or transformed cell line that are dependent on AR transactivation for survival or proliferation into an immune-compromised rodent.
  • the cells are as described in embodiment 25. Docket No. 354.
  • cancer cells are prostate cancer cells implanted into immune-compromised rodent(s), wherein the immune-compromised rodent(s) are castrated SCID or athymic nu/nu mice, and wherein AR transactivation is supported by androgen supplementation.
  • testosterone supplementation is by administration of a prodrug as described in embodiment 29.
  • prostate cancer cells are C4-2B or LuCaP-35V cells implanted into castrated SCID mice or CWR22-R1 cells implanted into nu/nu athymic mice.
  • the in vivo test system is a xenograft animal model resulting from implantation into a rodent of cancer cells from a hormone-associated cancer cell line or transformed cell line dependent on AR transactivation for survival or proliferation, wherein the rodent is immune-compromised and wherein the cancer cells implanted into an immune compromised rodent are co- contacted with test compound and a cancer chemotherapeutic compound.
  • chemotherapeutic compounds include tubulin disrupting agents, anti-metabolites, tyrosine kinase inhibitors, DNA damaging agents, topoisomerase inhibitors, and anti-tumor antibiotics.
  • PATENT including tubulin disrupting agents, anti-androgens and anti-estrogens administered to rodents implanted with cells containing functional i-AR derived from a prostate cancer or breast cancer cell line are as described in embodiment 34.
  • tubulin disrupting agent is a taxane compound.
  • a method of screening for a low toxicity Erk modulator comprising (or consisting essentially of or consisting of): (a) contacting a test compound with a suitable test system of a previous claim such as claim 1 , 2, 3, 4, 38, 39, 40 or 41 or a suitable test system comprising (or consisting essentially of or consisting of) (ii) mammalian cells that expresses a gene or genes encoding functional Erk-1 , Erk-2 and G protein-coupled receptor C6a (GPR-C6a) proteins, or mammalian cells that expresses a gene or genes encoding functional Erk-1 , Erk-2, G protein-coupled receptor C6a (GPR-C6a) and functional PI3K proteins, or a suitable in vitro cell-free test system containing an artificial Erk substrate, and one or more components in the mammalian Ras-Erk signal transduction pathway needed for the Ras-Raf-MEK-Erk or Raf-MEK-Er
  • step (e) administering the test compound of step (d), now identified as a candidate compound, to an animal in a suitable in vivo test system to obtain a treated animal and determining toxicity to the treated animal; and (f) selecting the candidate compound from step (e) that has a therapeutic index of 2 or greater, wherein the selected candidate compound from step (f) is identified as a low toxicity Erk modulator.
  • downstream effector protein of the GPR-C6a receptor whose activity or phosphorylation state is indicative of an activity of that receptor or of released Ga/q subunit from that receptor is PI3K or Akt.
  • downstream effector protein of the GPR-C6a receptor whose activity or phosphorylation state is indicative of the GTP/GDP Galpha/q bound or phosphorylation states is PI3K.
  • positive modulation of Ga/q tyrosine phosphorylation state or positive modulation of released GTP-bound Ga/q from GPR-C6a is indicative of inverse agonist activation of GPR-C6a.
  • negative modulation of the tyrosine phosphorylation state of the p85a or ⁇ 85 ⁇ regulatory subunit of a PI3K is indicative of inverse agonist activation of GPR-C6a.
  • the test compound identified as a candidate compound for administration in step (e) positively modulates the phosphorylation state of the activation loop of the Erk-1 isoform with no substantial modulation of the activation loop of the Erk-2 isoform.
  • step (f) The method of embodiment 75 further comprising (or further consisting essentially of or further consisting of) (g) administering the low toxicity Erk modulator of step (f) to a human having a hyperproliferation condition or a cancer in one or more amounts effective to assess the toxicity and/or efficacy of the low toxicity Erk modulator to treat the hyperproliferation condition in the human so as to obtain a treated human and assessing the toxicity and/or efficacy of the low toxicity Erk modulator on the treated human; and (h) selecting the candidate compound from step (g) that has a therapeutic index of 2 or greater, wherein the selected candidate compound from step (h) is identified as a low toxicity Erk modulator.
  • PATENT kinase regulatory subunit (vii) protein kinase A, (viii) intracellular free cAMP or Ca + concentration, or (ix) a combination of (i)-(viii), wherein the determined modulated activity(ies) or phosphorylation state(s) effected by the candidate compound for administration in step (e) is qualitatively, quantitatively or substantially similar to the modulation(s) determined for E-3a-diol when used as positive control.
  • the downstream GPR-C6a effector protein is a Ga monomer, wherein a test compound identified as a candidate compound for administration in step (e) positively modulates Galpha/q.
  • a test compound identified as a candidate compound for administration in step (e) positively modulates the phosphorylation state of an effector downstream of Erk-1 (i.e., an Erk-1 substrate) without substantial positive modulation of the phosphorylation state of an effector protein specific to Erk-2.
  • Erk-1 substrates are described in Carlson, S.M. et al. (201 1 ) and are incorporated by reference herein.
  • modulated activity(ies) or phosphorylation state(s) determined or measured for step is(are) (i) phosphorylation state or phosphorylation rate (i.e., time course of pErk-1 formation), or cytosolic concentration of pErk-1 , (ii) phosphorylation state or phosphorylation rate of Erk-2, or the nuclear concentration of pErk-2, (iii) phosphorylation state or phosphorylation rate for an Erk-1/2 artificial substrate or downstream effector protein, (iv) activity of or expression of a gene encoding an apoptosis-associated protein, (v) nucleo-cytoplasmic translocation of pErk-1 or pErk-2 (vi) cytosolic activity of pErk-1 , (vii) nuclear activity of pErk-2, (viii) proliferation of the mammalian cells in the suitable test system, (ix) differentiation or gene expression indicative differentiation of mammalian
  • the rate of phosphorylation of an artificial Erk substrate by pErk-1 , pErk-2 or pErk-1/2 substrate is determined in a suitable cell-free test system by phosphorylation of myelin basic protein or a protein or peptide that is capable of phosphorylation by Erk-1 , Erk-2 or Erk-1/2 that is conjugated to a peptide or contains an amino acid sequence capable of binding to the Erk CD domain wherein the suitable cell- free test system additionally comprises an Erk-binding scaffold protein.
  • a candidate compound identified for administration in step (e) has no discernable effects when the cell-free test system contains no Erk scaffold proteins, but exhibits negative modulation of pErk-2 protein levels when contacted to cells of a suitable cell-based test system.
  • differentiation or gene expression in mammalian cells in a suitable test system that is indicative of differentiation include PSA gene expression in mammalian prostate cancer cells, or expression of a suitable reporter gene genetically engineered into prostate cancer cells, in the absence of cell proliferation.
  • the mammalian prostate cancer cells are LNCaP cells or mammalian cancer cells expressing genes encoding functional wt-AR or mt-AR protein.
  • phosphorylation states or phosphorylation rates for an Erk- 1/2 downstream effector protein that are determined include one or more s6 kinases (i.e., p90rsk, RSK-1 or RSK-2), MSK or transcription factors, including Elk-1 , c-Myc, c-Fos, SRF or CREB.
  • activit(ies) for one or more apoptosis-associated proteins, including Bim, Bad or Bcl-2 or effects on gene expression for genes encoding one or more of those proteins are determined.
  • a candidate compound for administration in step (e) increases pErk-1 levels relative to pErk-2 within 5-30 min of contacting the compound to cells of a suitable test system, more preferably without increasing p-Erk-2 10% or more above basal levels in quiescent cells. More preferably the increase in p-Erk-1 is preceded by increased intracellular Ca2+ concentrations with an increase due to intracellular mobilization particularly preferred.
  • a candidate compound for administration in step (e) decreases Bcl-2 gene expression when contacted to cells of a suitable test system wherein the cells are mammalian cancer cells.
  • modulations of kinase, receptors or Ga protein activities or protein phosphorylation states are preferably determined relative to one or more suitable negative and positive control test compounds.
  • positive control compounds for GPR-C6a Ga/q activation include E-3a-diol which is also a negative control compound for GPR-C6a Galpha/i activation.
  • Positive control compounds for GPR-C6a Galpha/i activation include 3a-diol which is also a negative control compound for GPR-C6a Ga/q activation.
  • Other positive control compounds for GPR-C6a-Galpha/i activation include T, DHT, T-BSA.
  • mammalian cells endogenously express a gene encoding functional intracellular androgen receptor (i-AR) protein.
  • Preferred mammalian cells include cancer cells, with prostate and breast cancer cells particularly preferred.
  • [566] 84 The method of embodiment 75 wherein serum albumin or one or more liver enzymes selected from the group consisting of alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ glutamyl transpeptidase are not toxicologically or clinically significantly increased, preferably in about 50% or more of treated animals in one or more groups of treated animals or in about 70% to about 90% of treated animals.
  • rodent serum albumin or one or more of rodent alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ - glutamyl transpeptidase are not increased more than about 2.5-fold or about 3-fold compared to normal values.
  • the rodent is a mouse or rat and about 70% or more of the treated mice or rats or in about 70% to about 95% of the treated mice or rats have none of the recited proteins increased more than about 2.5 fold..
  • step (e) The method of embodiment 75 wherein the treated animals of step (e) have a cancer, precancer or hyperplasia.
  • the treated animals of step (e) have prostate cancer breast cancer, ovarian cancer, endometriosis, liver cancer, a central nervous system cancer, a lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), colorectal cancer, myeloma, melanoma, lymphoma, thyroid cancer, pancreatic cancer or bone cancer or a cancer that metastasized to bone or liver.
  • a lung cancer e.g., small cell lung cancer, non-small cell lung cancer
  • colorectal cancer myeloma, melanoma, lymphoma, thyroid cancer, pancreatic cancer or bone cancer or a cancer that metastasized to bone or liver.
  • the treated aminals of step (e) have prostate cancer, benign prostatic hypertrophy, breast cancer, ovarian cancer, endometriosis, hepatocellular carcinoma, glioblastoma, astrocytoma or oligodendroglioma.
  • the treated animals of step (e) have prostate or breast cancer, including metastatic prostate cancer or metastatic breast cancer, or cancer that has metastasized to bone or liver.
  • test system of step (a) is a suitable in vitro system comprised of mammalian cells derived from a tumor or transformed cell line.
  • mammalian tumor cells are LP LNCaP cells or CRPC cells derived therefrom including HP LNCaP cells.
  • step (i) contacting a test compound of step (a) with a suitable cell-free test system comprising p-Erk-1 , pErk-2, or p-Erk-1/2 and a suitable artificial substrate of the Erk Docket No. 354. PATENT isoform(s) in the absence of Erk scaffold proteins;
  • step (ii) contacting a test compound of step (i) with a suitable cell-free test system comprising p-Erk-1 , pErk-2, or p-Erk-1/2 and the suitable Erk isoforms substrate(s) of step (i) and at least one Erk scaffold proteins, wherein the suitable cell-free test systems of steps (i) and (ii) are substantially the same except for the presence or absence of the Erk scaffold protein(s);
  • step (iv) determining the amounts or rates of pErk-1 , pErk-2 or pErk-1/2 phosphorylation of the suitable artificial substrate of steps (i) in the absence of the test compound of step (i), wherein step (iv) serves as the vehicle control;
  • the amounts of increased pErk-1 relative to pErk-2 kinase activities are additionally determined in the presence of an Erk ATP binding site- dependent inhibitor subsequent to contacting a suitable test system with the test compound of step (a), wherein the selective negative modulation of p-Erk2 kinase activity of the selected candidate compound for conducting step (e) is attenuated or abrogated (i.e., both isoforms become negatively modulated).
  • step (i) contacting a test compound of step (a) with a suitable in vivo test system comprising mammalian cancer cells;
  • step (iv) determining the amounts or rates of formation of pErk-1 and pErk-2 proteins relative to total Erk protein of the suitable artificial substrates of steps (i) in the absence of the test compound of step (i), wherein step (iv) serves as vehicle control; Docket No. 354.
  • increased pErk-1 protein relative to pErk-2 kinase activities is additionally determined in the presence of an inhibitor of Ras-Erk, TNFa-NF- ⁇ , PI3K- Akt, GPCR, ⁇ -arrestin, Raf-1 , Raf-B, Tpl-2, RTK or Src signal transduction wherein the selective negative modulation of p-Erk2 protein level by the candidate compound selected for conducting step (e) is attenuated or abrogated (i.e., both isoforms become negatively modulated).
  • [588] 92 The method of embodiment 75 further comprising (or consisting essentially of or consisting of): (j) determining binding interaction of the test compound selected from step (c) to the scaffold protein Tfg in the presence of Erk-1 and Erk-2); and (k) selecting the test compound that binds to Tfg and selectively binds to Erk-2 protein in comparison to Erk-1 for administration in step (e).
  • the binding interaction to the scaffold protein Tfg of step (j) is conducted in the presence of another scaffold protein or a MAPK phosphatase
  • [593] 1A A method to identify a candidate compound, the method comprising (a) contacting a test compound with a suitable test system (a first test system); (b) determining phosphorylation states of Erk-1 and Erk-2 proteins resulting from step (a); (c) determining the phosphorylation state of a Class 1 PI3K using the suitable test system of step (a) or using a different suitable test system (a second test system), after contacting the test compound with the first test system or the second test system; wherein the first and second suitable test systems are in vitro cell-based test systems comprising cells derived from a prostate or breast cancer cell line having endogenous GPR-C6a and intracellular AR proteins or transformed normal cells not having endogenous GPR-C6a and intracellular AR proteins that have been transfected to contain those proteins; and (d) Docket No.
  • step (a) The method of embodiment 1 A further comprising (a-2) contacting a test compound with a suitable cell-based test system prior to step (a) wherein the prior suitable test system comprises (i) MDA-kb2 cells transfected to contain a MMTV promoter-reporter gene, (ii) T47D-kBluc cells transfected to contain an estrogen-inducible promoter-reporter gene, (iii) HEK293T cells transfected to contain an androgen-inducible reporter gene and a gene encoding functional i-AR protein, or (iv) HEK293T cells containing an estrogen-inducible reporter gene and a gene encoding functional ERp protein; and (a-1 ) selecting a test compound from step (a-1 ) that induces transcription of at least one of the reporter for conducting step (a).
  • the prior suitable test system comprises (i) MDA-kb2 cells transfected to contain a MMTV promoter-reporter gene, (i
  • [596] 4A The method of embodiment 1 A further comprising (e) contacting a selected test compound from step (d) with prostate or breast cancer cells of a suitable in vivo test system; (f) determining cancer cell proliferation in the suitable in vivo test system resulting from step (e); and (g) selecting a test compound from step (f) that inhibits cancer cell proliferation statistically significant to cancer cells contacted with vehicle alone.
  • [600] 8A The method of embodiment 5A wherein prostate cancer cells are CWR22-R1 cells implanted into castrated nu/nu athymic mice supplemented by testosterone.
  • [604] 12A The method of embodiment 4A further comprising (h) determining a minimum effective amount of a compound selected from step (g) for treating breast or prostate cancer in a mammal; (i) administering the minimum effective amount to healthy mammals so as to provide treated mammals; (j) determining liver enzymes levels for alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ glutamyl transpeptidase of the treated mammals; and (k) selecting a compound from step (i) that did not increase liver enzymes levels for any one of the liver enzymes selected from the group consisting of alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ glutamyl transpeptidase more than about 2-fold compared to normal values in about 70% or more of the treated mammals, wherein the compound of step (k) identified as a candidate compound is a low toxicity Erk-1 modulator.
  • [605] 13A The method of embodiment 12A further comprising (I) determining the minimum effective amount of a compound selected from step (k) to elicit more than about a 2-fold increase levels in any of the liver enzymes selected from the group consisting of alanine transaminase, aspartate transaminase, alkaline phosphatase and ⁇ -glutamyl transpeptidase; and (m) determining the therapeutic index of a compound based upon the effective amounts of steps (h) and (I); and (n) selecting the compound from step (I) having a therapeutic index of at least 5.
  • Example 1 Test Compound Binding Partner Determination by SI LAC Method
  • the Pierce NHS-activated agarose slurry (26200) and SILAC RPMI media (89984) were purchased from Thermo Scientific (USA).
  • the HEPES buffer was purchased from Mediatech (USA) and the dialyzed fetal bovine serum was purchased from Sigma-Aldrich.
  • E-3a-diol-benzyl amine was coupled to the NHS-activated bead according to standard protocol described in van Sommeren, A.P.G. et al. (1993), which is incorporated by reference herein.
  • control agarose beads which were subjected to the same treatments as the E- 3a-diol-agarose beads, were also prepared by omitting the E-3a-diol-amine and quenching the NHS-activated beads with ethanolamine.
  • SILAC Media Preparation and Cell Culture Conditions The mouse leukemic monocyte macrophage cell line RAW 264.7 (ATCC CRL-2278) cells were grown for at least 6 cell divisions in media supplemented with 5% dialyzed FBS, in a humidified air atmosphere with 5% C0 2 . All standard SILAC media preparation and labeling steps were followed as previously described in Ong, S.-E. and Mann, M. (2006), which is incorporated by reference herein, and in particular the methodology for SILAC media preparation and stable isotopic labeling described therein.
  • a base media was divided into two portions and either "light" forms of L-arginine and L-lysine or "heavy" L-arginine- 13 C 6 15 N 4 and L-lysine- 13 C 6 15 N 2 were added to generate the 2 SILAC labeling RPMI media.
  • Each growth medium contained the full complement of amino acids and were sterile filtered through a 0.22 ⁇ filter (Millipore).
  • Affinity enrichment mixtures were incubated overnight (4 °C, 16 h) on an end-over-end rotator, the beads pelleted by bench top centrifugation at (3 min, 1000 x g) and the supernatant aspirated.
  • BC experiments beads were combined at the first wash for subsequent washing steps.
  • each tube in a set was washed with 50mM Tris buffer (pH 8) at least twice to remove excess soluble small molecule competitor and then combined for the later washing steps.
  • beads were pelleted (3 min, 1000 x g) and the final wash was aspirated (leaving -25 ⁇ of Docket No. 354. PATENT buffer).
  • SILAC protein affinity enriched pull-downs were reduced and alkylated on bead by re-suspending in 200 ⁇ _ 8M urea, then treated with 10 ⁇ of 100 mM TCEP at 37 °C for 30 minutes. 20 ⁇ _ of 100 mM iodoacetamide was then added and the solution held in the dark for 30 min. with slow turning. Ammonium bicarbonate solution (600 ⁇ _ of 25 mM) was added to create a 2M urea solution and 16 ⁇ _ of trypsin (20 ⁇ g diluted with 40 ⁇ _ resuspension solution) was added.
  • the vials were incubated (37 °C) overnight on a thermomixer and then enzymatic digestion was stopped by the addition of 100% formic acid solution (30 ⁇ _). The vials were allowed to sit at room temperature for 5 min and then spun (3 min, 1000 x g) to pellet the beads. The supernatant was removed to a separate vial for LC/MS/MS analysis.
  • LC-MS/MS Analysis The LC-MS/MS analysis followed a standard protocol for the MuDPIT technique described in the referenced protocol. [Schieltz, D.M. and Washburn, M.P. Cold Spring Harb. Protoc. (2006), incorporated by reference herein]. Briefly, LC-MS data was obtained on a quaternary Agilent 1 10 series HPLC coupled to an LTQ ion trap mass spectrometer (ThermoElectron) equipped with a nano-LC electrospray ionization source. The LTQ was controlled by XcaliburTM data system software (ThermoElectron). LC-MS mobile phase buffers were composed in water with 0.1 % formic acid with the following additional modifiers: A (5% ACN), B (80% ACN), C (500mM ammonium acetate, 5% ACN).
  • Fused silica microcapillary columns (100 ⁇ i.d. x 365 ⁇ o.d.) were pulled to generate 5 ⁇ tips using a Model P-2000 C0 2 laser puller (Sutter Instrument). Biphasic columns were packed with 10 cm of 5 ⁇ Aqua C18 reverse phase resin (RP; Phemomenex) followed by 3 cm of Partisphere strong cation exchange resin (SCX; Whatman). Loading/desalting tips were prepared by packing 4 cm of RP resin into a 250 ⁇ silica microcapillary fitted with a 2 ⁇ inline microfilter (Upchurch Scientific). Column packing was performed using a high pressure loading device (600 psi helium). Columns and tips were equilibrated in buffer A shortly before use.
  • RP Aqua C18 reverse phase resin
  • SCX Partisphere strong cation exchange resin
  • Tandem mass spectra were searched using the Sequest algorithm (3.0) against the mouse database (ipi.MOUSEv368.fasta) from the European Bioinformatics Institute.
  • the mass window for peptides searched was given a tolerance of 3 Da. between the measured average mass and the calculated average mass and the b and y ions were included. All samples were searched with a static mod of +57 Da. for cys residues.
  • DTASelectTM (v2.0.25) was used to render Sequest output files. For tryptic rendering, default parameters were used, along with constraints for tryptic ends and exclusion of protein subsets.
  • Example 2 Gene Transcription Effects in LNCaP Cells from Contact with 17-E- 3cc-diol.
  • PCR arrays The PCR arrays, cDNA synthesis reagent (Cat# C-03), and PCR Master Mix (Cat# PA-MI) were purchased from SuperArray Bioscience Corporation. PerfectPureTM RNA (Fisher Cat# 29003 19) was used to extract RNA from the cells.
  • the cDNA was synthesized on iCyclerTM (Biorad) and the arrays were processed on real time PCR machine (iCycler IQ, Biorad). The results were analyzed using the software provided online by SuperArrayTM.
  • LNCaP clone FCG cells were grown in RPMI 1640 medium (ATCC Cat# 30-2001 ) containing 2 mM L-glutamine modified to contain: 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate.
  • the cells were transferred to poly D-lysine coated 96 well plates in RPMI 1640 medium phenol red free (Gibco Cat# 11 835-055) containing 2 mM L-Glutamine modified to contain: 10% Charcoal / Dextran- treated FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1 .5 g/L sodium bicarbonate and contacted with 30 nM E-3a-diol and 30 nM DHT or 10 nM ⁇ .
  • RNA from the cells was extracted with PerfectPureTM RNA kit (5 Prime, Inc., Gaithersburg, MD) following the manufacturer's protocol including a DNase step. cDNA synthesis was then performed using CO-3 reagent following the manufacturer's protocol. After proceeding with PCR array preparation following the SuperArrayTM protocol the PCR plates were run on a real time PCR machine using the settings recommended by the SuperArray protocol for the equipment used.
  • CDK4 expression was decreased in 2 of 7 Cancer Pathway arrays (-2.93 and -2.35 fold).
  • Caspase-9 expression was upregulated in 1 of 3 apoptosis arrays (5.31 fold).
  • a few genes were present on different pathway arrays.
  • CDK2 expression was downregulated in 1 of 7 cancer pathway arrays (-2.71 fold) and 1 of 2 signal transduction arrays (-2.06 fold).
  • These and other genes that were up-regulated (increased expression) or down-regulated (decreased expression) by at least 50% compared to controls in at least 3 of 6 repeats are listed in Table 2.
  • E-3a-diol modulates various androgen-regulated cell cycle and signal transduction genes including CDK2, CDKN1A, KLK2, IGFBP-3, TMEPAI, ODC1, GREB1 and AR.
  • E-3a-diol affected the expression of genes involved in various phases of the cell cycle including Gi phase and Gi/S transition including CCNE1, CDK4 and CDKN1B as well as cell cycle checkpoint and cell cycle arrest (ATM & CHEK2).
  • Bcl2 and CFLAR (Caspase 9) were the most notable.
  • Bcl-2 expression was down- regulated in most samples treated with E-3a-diol.
  • E-3a-diol also down-regulated ABCG2 and ABCC5, both members of the ABC transporter family involved in drug resistance to chemotherapy.
  • Example 3 Gene Transcription Effects from Contacting E-3cc-diol with a Xenograft Model using LuCaP Cells. An Example Suitable In Vivo Test System using wt-AR CaP cells for Determining Gene Expression Effects.
  • RNA from group 1 control LuCaP- 35V
  • RNA from group 2 LiCaP-35V+E-3a-diol
  • Each pool contained Docket No. 354.
  • PATENT an equal amount of RNA from three different tumors from the specific group.
  • a reference standard RNA for use in two-color oligo arrays was prepared as described in Coleman, I.M. et al. (2006). Total RNA was amplified using the Ambion MessageAmpTM aRNA Kit (Ambion Inc, Austin, TX).
  • Amplified amino-allyl aRNA from each pooled sample was labeled with Cy3 fluorescent dye (reference amino-allyl aRNA was labeled with Cy5) and hybridized to Agilent 44K whole human genome expression oligo microarray slides (Agilent Technologies, Inc., Santa Clara, CA).
  • Immunohistochemical (IHC) analysis used 5- ⁇ sections of paraffin-embedded subcutaneous and intra-tibial tumors were used and was performed by standard procedure as described in Kiefer J. A. et al. (2004), using an anti-human AR mouse monoclonal antibody (1 :60 dilution, BioGenex, San Ramon, CA). For analysis, conducted according to the procedure described in Lai J.S. et al. (2004), a quasi-continuous score was created by multiplying each nuclear intensity level (1 for no stain, 2 for faint stain, 3 Docket No. 354. PATENT for intense stain) by the corresponding overall percentage of cells at that intensity for the entire tumor, and then summing the results.
  • IHC Immunohistochemical
  • IHC for PSA expression in intra-tibial tumors was done using an anti-human PSA rabbit polyclonal antibody (Dako, 3 ⁇ g/mL, Carpinteria, CA).
  • the i-AR regulated genes of Table 6 are classified into the following categories. Metabolism: Fatty Acids (FADS1 , FABP5, AZGP1 , AMACR, LONPL, LIPG), Polyamines (LOX, SAT, ODC1 ) Purines (GDA, AMPD3); Mitochondrial and Ribosomal Proteins: RBM24, MRPL33, RPL21 , RPLP1 ; Stress, Apoptosis: IL1 R1 , NFKBIZ, SAA2, SAA3, GPX3, CASP10, NMES1 (C15orf48), SUM02, MICAL1 , RAB32, MT1 G; Adhesion, morphology: Rho signaling (CDC42EP2, PVRL3, POPD3, ARHGEF10, RHOBTB3 GRHL2)CDH26, CLDN8; Development: Wnt, Notch, Hox (PBXN02, PXDN, LGALS3, CTBP1 , LFNG)
  • E-3a-diol Still other genes upregulated by E-3a-diol are involved in development. Hox- and Wnt-related gene expression is normally associated with immature cells and cancer cell proliferation. However, Wnt is also required for epithelial differentiation Docket No. 354. PATENT and Hox for functional differentiation in mature cells. Thus upregulation of those genes in the context of G1 arrest is believed to promote differentiation that leads to apoptosis. In addition, the HOX gene IRX5 is down-regulated and forced down- regulation in LNCaP is known to induces apoptosis. Finally, Downregulation of v- JUN and JAG1 will relieve inhibition to differentiation. Other genes known or implicated in G1 arrest and apoptosis subsequent to it include PPM1 E, TIMP2, CASP10, which are upregulated by E-3a-diol.
  • Example 4 Anti-Proliferative Effect of E-3cc-diol in Xenograft Model using C4-2B cells.
  • C4-2B cells were directly injected into murine bones (intra-tibial) to determine the effect of E-3a-diol on bone CRPC tumors as described in Koreckij, T.D. et al. (2009).
  • C4- 2B cells were used since their xenograft tumors exhibit an osteoblastic response when grown in the bone environment similar to that seen in patients with CaP bone metastases.
  • These cells are a CRPC subline of LNCaP cells, which also harbors the T877A mt-AR.
  • These cells were derived from a bone metastasis [Thalmann, G.N. et al. (2000)] and were maintained in vitro under standard tissue culture conditions.
  • Hematoxylin and eosin (H&E) staining of the tumored tibiae showed osteoblastic reaction associated with growth of the C4-2B tumors in the bone and tumor foci between the newly Docket No. 354.
  • PATENT formed woven bone in both E-3a-diol-treated and control tibiae.
  • Example 5 which uses LuCaP-35V cells, E-3a-diol treatment in this model resulted in decreases of serum PSA.
  • Example 5 Anti-Proliferative Effect of E-3cc-diol in Xenograft Model using LuCaP- 35V cells. An Example Suitable In Vivo Test System using wt-AR CaP cells for Determining Anti-Proliferative Effects.
  • LuCaP-35V cells are CRPC subline of the LuCaP 35 CaP xenograft derived from the lymph node metastasis of a patient who had previously undergone an orchiectomy [Corey, E. et al. (2003)]. LuCaP-35V cells harbor wt-AR and are maintained by serial passage in castrated severe combined immunodeficient (SCID) male mice.
  • SCID severe combined immunodeficient
  • mice Male CB-17 SCID mice (Charles River Laboratories, Wilmington, MA) were castrated, and after a 2-week recovery period, half of the mice then received subcutaneous ⁇ pellets (5 mg, 60-day time release; IRA, Sarasota, FL). The other half of animals received placebo pellets. LuCaP-35V was implanted subcutaneously ( ⁇ 20 mg tumor bits) 3 days after implantation of the pellets.
  • Tumor volumes were measured twice weekly, and blood samples were drawn weekly for PSA determinations (IMx Total PSA Assay; Abbott Laboratories, Abbott Park, IL). Exponential growth equations were used for calculations of tumor doubling times. Animals were sacrificed after 4 weeks of treatment when tumors exceeded 1000 mm 3 or if otherwise compromised. Sacrifice PSA index was calculated by dividing the serum PSA levels by the tumor volume. At sacrifice, half of each tumor was processed for paraffin embedding and immunohistochemistry (IHC), and the other half was flash frozen for gene expression analysis and determinations of intratumoral androgen levels.
  • IHC paraffin embedding and immunohistochemistry
  • E-3a-diol significantly increased the tumor doubling times of LuCaP-35V (LuCaP-35V + E-3a-diol, 18.2 ⁇ 6.28 days; untreated LuCaP-35V, 10.44 ⁇ 1 .8 days; P ⁇ .0001 .
  • E-3a-diol treatment resulted in significant increases in serum PSA levels in the treated animals versus control animals bearing LuCaP-35V tumors in the period of 1 to 3 weeks after treatment initiation (P ⁇ .0001 ).
  • PSA levels were not significantly higher in the treated animals at the end of the study. It is believed this is due to the decreases in tumor volume. This explanation is supported by results showing a significantly higher PSA index in the LuCaP-35V + E-3a-diol animals than PSA index in the LuCaP-35V animals, 0.18 ⁇ 0.07 versus 0.07 ⁇ 0.02 ng/mL per cubic millimeter, respectively (P ⁇ .0001 ).
  • C4-2B cells were grown in RPMI 1640 (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Atlanta Biological, Atlanta, GA) under standard tissue culture conditions.
  • Cells were transiently transfected with an androgen response element (ARE) reporter or with a 5.8-kb PSA luciferase plasmid using the Amaxa Nucleofector with solution V on program 27 as per the manufacturer's instructions (Amaxa Biosystems, Inc, Gaithersburg, MD).
  • the hTK renilla- luciferase plasmid was transfected under the same conditions to allow for normalization of transfection efficiencies. Control cells were mock-transfected.
  • C4-2B cells were plated in a six-well plate (200,000 cells per well) in RPMI 1640 medium with 5% charcoal-stripped serum, and E-3a-diol was added at 0, 10, and 50 nM concentrations. Cells were incubated for 48 hours, and luciferase activity was detected with a Dual-Luciferase Reporter AssayTM (Promega, Madison, Wl) using a Tecan GENios PlusTM illuminometer (Phenix Research Products, Hayward, CA).
  • C4-2B cells were plated in a six-well plate (200,000 cells per well) in RPMI 1640 medium with 5% charcoal-stripped serum, and E-3a-diol was added at 0, 10, and 50 nM concentrations. After three days viable cells were counted using the Trypan blue exclusion assay. Results show that E-3a-diol increases AR- mediated transcription using either reporter plasmid in C4-2B cells (expresses mutated AR) AR-mediated transcription was also increased in LuCaP-35V cells (express wild-type AR), but this activation did not reach statistical significance.
  • Example 7 Determining Binding and Transactivation Activity of Nuclear Hormone Receptors by Test Compound as Exemplified for E-3cc-diol.
  • NR binding affinity was accomplished by a highly sensitive in vitro fluorescence polarization (FP)-based competition binding assay using fluorophore-conjugated high affinity NR-ligands (FP Nuclear Receptor Binding Assay System; Invitrogen).
  • FP Fluorescence polarization
  • FluormoneTM high affinity proprietary fluorescent ligand
  • Compounds that behave as NR competitive ligands with respect to the Fluormone/NR interaction will cause a concentration-dependent suppression in the extent of fluorescence polarization (FP) according to their relative binding affinity.
  • the concentration of the test compound that results in half-maximal decrease of the polarization signal corresponds to the IC 50 value, which is a measure of the relative affinity of the test compound for a given NR.
  • E-3a-diol The nuclear receptor binding profile of E-3a-diol is reported in Table 7. DHT and E 2 are included as controls for their respective receptors. E-3a-diol does not bind strongly to the wild type AR, or at all to PR, GR or ERp, and has a weak affinity for ERa.
  • the human MDA-kb2 breast cancer cell line utilized for evaluation of AR and GR transactivation activity was initially developed by the U.S. EPA [Wilson, V. et al. (2002)].
  • This cell line is commercially available as ATCC Cat. No. CRL-2713 and stably expresses an androgen and glucocorticoid-responsive luciferase reporter for detection of AR or GR agonists and antagonists (both AR and GR receptors are expressed endogenously in this cell line).
  • the T47D-kBluc human breast cancer cell line was also initially developed by the U.S.
  • EPA EPA (Ibid.) and stably expresses an estrogen-responsive luciferase reporter for detection of estrogen receptor agonists and antagonists (both ERa and ERp are expressed endogenously in this cell line).
  • This cell line is commercially available as APCC Cat. No. 286 and was used in transactivation assays for ERa and ERp.
  • the resulting level of expression of the reporter luciferase gene in the extracts was measured by the extent of luciferase enzymatic activity as relative light units (RLU) in a Pecan Genios Pro (illuminometer mode) when incubated with an appropriate luciferase substrate (luciferase assay system; Promega Cat. No. E1501 ). Results are expressed as EC50 values (in nM concentration units).
  • E-3a-diol The ability of E-3a-diol to transactivate the same panel of nuclear receptors is reported in Table 8. As shown, DHT and E-3a-diol transactivate the T877A mutant AR with similar potency. E-3a-diol exhibits weak activity for both ER isoforms. E 2 is also active on the mutant AR, demonstrating the promiscuous nature of the mutant AR.
  • MtAR-HEK293 cells are HEK293 fibroblasts transiently co-transfected with an ARE/luciferase promoter/reporter construct and a cDNA expression vector encoding the full-length LNCaP AR. These cells exhibit virtually undetectable levels of endogenous sex steroid receptors. a MDA-kb2 cells are stably transfected with a promoter/reporter Docket No. 354.
  • PATENT constructs sensitive to sex steroid receptor stimulation (MMTV promoter) fused upstream of a luciferase reporter gene. These cells endogenously express both AR (androgen receptor) and GR (glucocorticoid receptor).
  • the MDA-kb2 cells are engineered to detect activated AR stimulation of androgen response elements (AREs) that are normally present on androgen-responsive genes such as PSA.
  • AREs androgen response elements
  • the MBA-kb2 assay induced an 8- to 10-fold increase in luciferase at the optimum DHT concentration.
  • b T47D-kBluc cells are stably transfected with a synthetic promoter/reporter construct sensitive to estrogenic stimulation, consisting of 3 copies of the estrogen response element (ERE) fused upstream of a luciferase reporter gene. These cells express endogenously both forms of the ER (estrogen receptor), namely ERa and ERp.
  • c ERp-HEK293 cells are HEK293 fibroblasts transiently co-transfected with an ERE/luciferase promoter/reporter construct and a cDNA expression vector encoding the full-length human ERp. These cells exhibit virtually undetectable levels of endogenous sex steroid receptors. d Possibly an antagonist at higher concentrations and a partial agonist at low concentrations.
  • Example 8 Proliferation Assay by Colorimetric Determination. Example for Determining Anti-Proliferative Effects of Test Compounds, Optionally in the Presence of ⁇ , on Cancer Cells in a Suitable Test System.
  • the MTT proliferation assay is based on the enzymatic reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] in living, metabolically active cells.
  • the reaction is carried out in situ, and the reaction product, a purple-colored formazan soluble in dimethylsulfoxide, is measured colorimetrically.
  • the assay is conducted generally according to the methodology in Romijn, J. C. et al. (2006) using the following procedure.
  • Transformed or cancer cells such as LNCaP cells (6,000 cells per well) are plated out into wells of a poly-D-lysine coated assay plate each containing 200 ⁇ _ RPMI 1640 medium containing 2 mM L-glutamine modified to further contain 10% FBS, 10mM Hepes, 1 mM sodium pyruvate, 4.5 g/L glucose and 1 .5 g/L sodium bicarbonate.
  • the assay plate is incubated overnight at 37°C, 5% C0 2 .
  • Test compounds in 10 mM DMSO to be screened are diluted to 100 ⁇ then to 10 ⁇ in RPMI 1640 medium containing 2 mM L- Glutamine modified to further contain 10% Charcoal/Dextran treated FBS, 10mM Hepes, 1 mM sodium pyruvate, 4.5 g/L glucose and 1 .5 g/L sodium bicarbonate. Media in the wells of the assay plate are replaced with 200 ⁇ _ of the 100 ⁇ or 10 ⁇ test compound solution.
  • a 410 nM androstenediol ( ⁇ ) solution which is prepared from dilution of a 10 mM stock solution in DMSO with Docket No. 354.
  • PATENT the same media that is used to prepared the compound solution, is optionally added to each well for a final concentration of 10 nM ⁇ per well, and otherwise is sham added.
  • the plate is then incubated for 5 days at 37°C, 5% C0 2 .
  • Example 9 Proliferation Assay by Fluorometric Determination.
  • Fluorometric determination relies upon the CyQuantTM assay, which is based on dye fluorescence enhancement upon binding to cellular nucleic acids by addition of a buffer containing the CyQuant-GR dye to lysed cells. This assay does not rely on cellular metabolic activity.
  • LNCaP-FGC, PC3 and DU145 cells are obtained from the American Type Tissue collection (ATTC, Manassas, Virginia).
  • ATCC American Type Tissue collection
  • RPMI/10% fetal bovine serum (FBS) After incubation overnight at 37 °C, 5% C0 2, the media is removed and the cells are washed once with 150 ⁇ -Jwell of phenol red-free RPMI containing 10% charcoal/dextran-treated FBS (CS-RPMI) and optionally containing 10 nM ⁇ .
  • test compounds with anti-proliferative activity a dilution sequence of test compound is analyzed and the data are modeled using a nonlinear regression, sigmoidal dose response, variable slope equation. The curve fit is constrained with a bottom of 0 and a top of 100. IC 50 values are generated by GraphPad Prism 4. Cell proliferation is measured on day five using the CyQuant assay (Invitrogen, Carlsbad CA).
  • test compound on cell growth of LNCaP, PC3 and DU145 cells are assayed by plating 3 x 10 5 cells in 5% CS-RPMI with optional supplementation with 10 nM ⁇ . After 4 days cells are manually counted with a hemocytometer.
  • Example 10 Cell Cycle and Apoptosis Assay. Examples for Determining Effects on Cell Cycle and Apoptosis of a Test Compound in a Suitable Test System Comprising Docket No. 354. PATENT
  • DHT can cause a Gi arrest but not cell death of LNCaP cells at concentrations above 10 "10 M [Lee, C. et al. (1995)].
  • ARE studies see Example 15
  • E-3a-diol had a similar cytostatic effect on LNCaP and to determine if, unlike DHT, exposure to E-3a-diol caused apoptosis in LNCaP cells.
  • LP LNCaP cells (5 x 10 5 ) were seeded in phenol-red free RPMI with 5% charcoal stripped serum (CSS) in 6-well plates and allowed to adhere overnight, then cultured in CSS supplemented with 10 nM AED with or without 50 nM E-3a-diol for four days. At the end of the incubation period, floating and adherent cells were harvested for cell cycle analysis.
  • CSS charcoal stripped serum
  • LP LNCaP cells were re-suspended in a 10 mg/mL solution of 4, 6-diamidino-2-phenylindole (DAPI) and 0.1 % NP-40 in a Tris-buffered saline solution (pH 7.0) and analyzed using an Influx cytometer (Cytopiea, Seattle, WA). Analyses were performed with MultiCycle software (Phoenix Flow Systems, San Diego, CA). Apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit (Calbiochem, La Jolla, CA) according to the instructions of the manufacturer to detect apoptosis.
  • PI and Annexin positive cells were considered apoptotic.
  • Figure 1 LNCaP ( ⁇ ), PC3 ( A) and DU145 ( T)] cells were incubated with E-3a-diol (HE3235) at 1 nM, 10 nM and 50 nM and assayed in duplicate for inhibition of cell growth relative to vehicle.
  • Prostate specific antigen is an androgen-responsive gene product frequently used as a reporter for AR activation
  • examples of reporter gene constructs with PSA as the reporter are provided by Langeler, E.G. et al. (1993); Lee, C. et al. (1995); Arnold, J.T. et al. (2007), all of which are incorporated by reference.
  • Example 12 Androgen-mediated transcription assays. Example for Transfection of an Androgen-Inducible Reporter into Cancer Cells.
  • LP LNCaP cells were transfected with an ARE-promoter reporter construct and incubated with E-3a-diol with or without DHT and ⁇ present.
  • the hTK renilla-luciferase plasmid (2 ng) was transfected under the same conditions to enable normalization of transfection efficiencies.
  • Cells were seeded in 24-well plates in phenol-red free RPMI with 5% CSS to allow adherence overnight, and then DHT, E2 or PAED were added (1 nM or 10 nM final concentrations) with or without 50 nM E-3a-diol in DMSO. Cells were incubated for -48 hours, lysed and subjected to a dual-luciferase assay according to manufacturer's recommendations (Promega, Madison, Wl) using Tecan Genios plus illuminometer. The luciferase activity was measured and signal normalized to transfection efficiencies based on TK transfection.
  • E-3a-diol did not inhibit the activation of the LNCaP mt-AR when the cells were stimulated with either DHT or ⁇ . Instead, E-3a-diol activated mt-AR in this setting. Consistent with this finding, an increase in amount of PSA secretion per cell was observed after incubation with E-3a-diol. PSA rose from 8.5 ng/mL/10 5 cells to 175 ng/mL/10 5 cells by day 4 in LP LNCaP cells culture incubated with 50 nM E-3a-diol.
  • LP LNCaP cells were treated with 1 or 10 nM DHT or ⁇ in charcoal-stripped serum (CSS) and 50 nM E-3a-diol (HE3235). The fold change above background is shown on the Y-axis of Figure 5.
  • Example 13 Anti-Proliferative Effect of E-3cc-diol in Xenograft Model with LP LNCaP cells.
  • mice received ⁇ pellets and were implanted with LNCaP tumor cells and monitored as described below. Once the tumors reached 15 - 25 mm 3 , the mice were paired by tumor volume, and each mouse in a pair was assigned to the vehicle or 4 mg/mouse/day E-3a- diol group. Animals were dosed i.p. with 200 ⁇ E-3a-diol or vehicle once a day for 21 days.
  • PAED pellets (5 mg, 60-day time-release, IRA, Sarasota, FL). Three days later, all mice were injected subcutaneously in the right flank with 100 ⁇ _ of 7.5 x 10 6 LNCaP tumor cells in phenol red-free RPMI mixed 1 :1 with Matrigel (BD, Franklin Lakes, NJ). Tumor volumes were measured weekly and calculated as a2 x b/2 with a being the width and b the length of the tumor in mm (reported as mm 3 ). E-3a-diol was prepared for injection by dilution in CaptisolTM (CyDex, Lenexa, Kansas).
  • the incidence of a measurable tumor was estimated as the relative frequency of mice having a measurable tumor at least once any time in the experiment.
  • the significance of the difference in incidence from vehicle was tested by means of Fisher's exact test, adjusted for multiplicity of comparisons [Westfall, P. H. et al. (1999)].
  • Dose response in the proportion of mice with tumor was tested for significance via exact Cochran Armitage test.
  • Time to first measurable tumor volume is analyzed via Kaplan Meier product limit estimates, with the exact log-rank test applied to test for the significance of the differences [Cantor, A. (1997)]
  • Tumor volumes and tumor growth rates are analyzed non-parametrically via exact Wilcoxon-Mann-Whitney test. Resulting p- values are corrected for multiplicity of comparisons (Westfall, op. cit).
  • Reduction of tumor volume was defined as a reduction in volume of at least 20% of the baseline volume, persisting to the end of the study. Fisher's exact test was applied to test for the significance of the difference in the incidence of such reduction relative to vehicle. Time to first measurable tumor volume was analyzed via Kaplan-Meier product limit estimates, with the exact log-rank test applied to test for the significance of the difference. To detect the difference between active and control group, Fisher's exact test and exact 95% CI for the difference were applied. A tumor of non-measurable volume was a tumor that, with the methodology at hand, measures 0 to the end of the study. The growth rate of a tumor was also analyzed via the mixed model.
  • Example 14 Phosphotyrosine Profiling of HP LNCaP Cells contacted with E- 3cc-diol.
  • SH2 profiling array (TranSignalTM Phosphotyrosine Profiling Array from Panomics Cat# MA3041 ).
  • the profiling array kit includes all the reagents necessary to perform the assay.
  • the membranes included in this kit are spotted in duplicate with 100 ng of 40 different SH2 domain proteins.
  • LNCaP cells at passage 83 were plated at 8 x 10 6 into each of four 10 cm tissue culture dishes in RPMI 1640 medium ATCC Cat# 30-2001 containing 2 mM L- glutamine modified to contain: 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1 .5 g/L sodium bicarbonate for 3 days until the cells reached 80% confluency.
  • Membranes one for each treatment, from the TranSignalTM Phosphotyrosine Profiling Array kit (Panomics, Inc., Redwood City, CA) were washed once in wash buffer for 30 minutes, blocked until the membranes appeared uniformly wetted (about 1 hour). The membranes were then rinsed once in wash buffer. The next day the confluent HP LNCaP cells were washed in pre-warmed PBS.

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Abstract

L'invention concerne des procédés pour identifier des modulateurs à faible toxicité des protéines kinases activées par mitogènes, en particulier MAPK1 et MAPK3. De tels modulateurs sont utiles pour traiter les conditions d'hyperprolifération dont les cancers, par exemple les cancers de la prostate, du sein, du foie et du colon. De tels traitements sont accompagnés de niveaux de toxicité réduits, par ex. des enzymes du foie élevées, qui sont associés aux inhibiteurs MAPK dépendant des sites de liaison ATP.
PCT/US2013/070754 2013-03-08 2013-11-19 Procédés de dépistage de récepteur et de modulateur de kinase Ceased WO2014137420A1 (fr)

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Citations (3)

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US20070082366A1 (en) * 2005-10-11 2007-04-12 Khan Tapan K Alzheimer's disease-specific alterations of the ERK1/ERK2 phosphorylation ratio
US20090208979A1 (en) * 2008-02-14 2009-08-20 Technion Research And Development Foundation Ltd. Method for identifying antipsychotic drug candidates
US20110281748A1 (en) * 2006-09-21 2011-11-17 Prometheus Laboratories Inc. Drug selection for gastric cancer therapy using antibody-based arrays

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Publication number Priority date Publication date Assignee Title
US20070082366A1 (en) * 2005-10-11 2007-04-12 Khan Tapan K Alzheimer's disease-specific alterations of the ERK1/ERK2 phosphorylation ratio
US20110281748A1 (en) * 2006-09-21 2011-11-17 Prometheus Laboratories Inc. Drug selection for gastric cancer therapy using antibody-based arrays
US20090208979A1 (en) * 2008-02-14 2009-08-20 Technion Research And Development Foundation Ltd. Method for identifying antipsychotic drug candidates

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VERNHET ET AL.: "An anti-inflammatory benzamide derivative inhibits the protein kinase C (PKC)-dependent pathway of ERK2 phosphorylation in murine macrophages", THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 28, no. 1, October 1997 (1997-10-01), pages 358 - 365 *

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