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WO2020232445A1 - Modulateurs des voies de biosynthèse des nucléotides de pyrimidine - Google Patents

Modulateurs des voies de biosynthèse des nucléotides de pyrimidine Download PDF

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Publication number
WO2020232445A1
WO2020232445A1 PCT/US2020/033458 US2020033458W WO2020232445A1 WO 2020232445 A1 WO2020232445 A1 WO 2020232445A1 US 2020033458 W US2020033458 W US 2020033458W WO 2020232445 A1 WO2020232445 A1 WO 2020232445A1
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compound
cancer
inhibitor
aspects
cell proliferation
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Inventor
Caius G. Radu
Evan ABT
Ethan ROSSER
Arnon Lavie
Matthew DURST
Soumya PODDAR
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University of California Berkeley
University of California San Diego UCSD
University of Alabama at Birmingham UAB
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University of California Berkeley
University of California San Diego UCSD
University of Alabama at Birmingham UAB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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

Definitions

  • pyrimidine nucleotide biosynthesis which consists of nucleoside salvage (NSP) and de novo (DNP) pathways which converge to generate uridine monophosphate (UMP), the common precursor for all pyrimidine nucleotides (Okesli et al, 2017).
  • NSP nucleoside salvage
  • DNP de novo
  • UMP uridine monophosphate
  • the NSP scavenges preformed nucleosides from the extracellular environment, shuttling them into the cell via nucleoside transporters where they are
  • UCKs uridine-cytidine kinases
  • UC 2 is thought to be the primary NSP kinase, given its 20-fold higher catalytic efficiency compared with UCK1 (Van Rompay et al, 2001).
  • the DNP consists of a six-step process that utilizes glutamine, aspartate, bicarbonate and glucose to produce UMP through the action of the trifunctional enzyme CAD, electron transport chain-linked dihydroorotate dehydrogenase (DHODH), and bifunctional UMP synthase (UMPS).
  • DHODH in particular has been the subject of significant research interest in anticancer settings (Madak et al, 2019; Sykes et al, 2016; Santana-Codina et al, 2018; Lolli et al, 2018).
  • the disclosure is directed, inter alia, to the exploitation of nucleotide biosynthetic pathways in treating cancer and to other important ends.
  • the disclosure provides methods for identifying compounds that inhibit cancer cell proliferation by: (i) contacting the compounds with (a) cancer cells and salvage pathway condition-specific growth media and de novo pathway condition -specific growth media: and/or (b) cancer cells and salvage pathway condition-specific growth media; and/or (c) cancer cells and de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound; thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is de novo nucleotide pathway
  • the common metabolite is uridine monophosphate (UMP).
  • the disclosure provides methods of treating cancer in patients in need thereof using compounds that inhibit cancer cell proliferation, wherein the compounds are identified and selected by (i) contacting the compounds with (a) cancer cells and salvage pathway condition- specific growth media and de novo pathway condition-specific growth media, and/or (b) cancer ceils and salvage pathway condition-specific growth media; and/or (c) cancer cells and de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; (ii) identifying inhibition of cancer cell proliferation by the compound; thereby identifying the compound that inhibits cancer cell proliferation; (iii) administering the compound that inhibits cancer cell proliferation to a patient in need thereof.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is de novo nucleotide pathway
  • the common metabolite is uridine monophosphate (UMP).
  • the cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma.
  • the disclosure provides methods of treating cancer in patients in need thereof by administering a therapeutically effective amount of OSU-03012, TAK-632, JNK-IN-8, CNX- 774, or motesanib.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • compositions comprising: (i) dihydroorotate dehydrogenase and (ii) TAK-632 or OSU-03012.
  • the composition is a complex.
  • the composition is a co-crystal.
  • (i) and (ii) are bonded together via one or more hydrogen bonds.
  • FIGS. 1A-1F provide the identification of UMP-DNP and -NSP modulators in a small molecule protein kinase inhibitor library.
  • FIG. 1 A provides the phenotypic screening strategy, where the impact of 430 protein kinase inhibitors on cell proliferation was evaluated in
  • FIGS. IB and 1C are waterfall plots ranking library compounds based on NSP (FIG. IB) or DNP (FIG. 1C) pathway selectivity scores determined as described in Fig. S3B.
  • FIGS. 1D-1F provide a summary of NSP and DNP selectivity scores across library compounds annotated as JNK (FIG. ID), PDK1 inhibitors (FIG. IE), or RAF inhibitors (FIG. IF).
  • FIGS. 2A-2D show that JNK-IN-8 inhibits nucleoside transport in vitro and in vivo.
  • FIG. 2A shows that nucleoside uptake can be prevented by inhibition of either nucleoside transporters or kinases.
  • FIG. 2D shows the quantification of [ 18 F]CFA tumor uptake in FIG. 2C (%ID/g:
  • FIGS. 3A-3H show that OSU-03012 and TAK-632 inhibit DHODH and activate the DNA replication stress response pathway.
  • FIG. 3A shows the UMP biosynthesis via the de novo and salvage pathways.
  • FIG. 3B provides a propidium iodide cell cycle analysis of MIAPACA2 PD AC cells treated ⁇ 5 mM TAK-632 or ⁇ 5 mM OSU-03012 and supplemented with 50 mM orotate (OA) or 10 mM rU (N.S.: no supplement). Insert indicates % S-phase cells.
  • FIG. 3D provides an in vitro DHODH enzyme assay performed in the presence of OSU-03012 or TAK-632.
  • FIG. 3E provides a correlation between DHODH inhibitor (1 mM NITD-982) and OSU-03012 (3.17 mM) or TAK-632 (3.17 mM) response across a panel of 25 PD AC cell lines determined using CTG following 72 h treatment. Response calculated as doubling time normalized proliferation inhibition. Pearson correlation coefficient is indicated.
  • FIG. 3F provides an immunoblot analysis of MIAPACA2 cells treated ⁇ 1 mM PDK1 inhibitor GSK-2334470 (GSK) ⁇ 1 mM OSU-03012 (OSU) ⁇ 10 mM rU for 24 h.
  • FIG. 3G is an immunoblot analysis of MIAPACA2 cells treated ⁇ 1 mM RAF inhibitor LY3009120 (LY) ⁇ 5 mM TAK-632 (TAK) ⁇ 10 mM rU for 24 h.
  • FIGS. 4A-4B show that OSU-03012 and TAK-632 bind DHODH and show the crystal structure of DHODH with OSU-03012 (FIG. 4A) or TAK-632 (FIG. 4B). 2mFo-DFc electron density for OSU-03012 (carbons in yellow) or TAK-632 (carbons in green) contoured at 1 s. Dashed black lines represent hydrogen bonds between the ligands and DHODH. Interacting residues as predicted by LigPlot are shown and labeled.
  • FIGS. 5A-5B provide validation of UMP as a critical, convergent metabolic node in cancer cells.
  • FIG. 5 A shows that UMP can be produced by a de novo pathway (DNP) from glucose, glutamine, bicarbonate and aspartate or from extracellular uridine (rU) by a nucleoside transporter and kinase-dependent salvage pathway (NSP).
  • FIG. 5B provides dose response curves of DHODH inhibitor NITD-982 and nucleoside transport inhibitor dipyridamole (DP A) in JURKAT cells cultured in NSP + DNP (media + 10 mM rU), NSP only (media + 10 mM rU +
  • FIG. 6 shows that UMP-DNP and NSP are interchangeable in sustaining proliferation across a panel of cancer cell lines.
  • Uridine titration in cancer cell lines cultured in media + 10% dFBS ⁇ 1 mM NITD-982 (n 4).
  • Relative proliferation rate (PR) was calculated by normalizing % proliferation values at 72 h to cell line proliferation rate. Proliferation rates were calculated utilizing CTG measurements at the time of treatment (t0) and vehicle-treated controls at 72 h.
  • FIGS. 7A-7F shows a phenotypic screen identifies UMP -NSP and -DNP inhibitors.
  • FIG. 7A provides a CTG analysis of MIAPACA2 cells cultured in NSP+DNP (media +10 mM rU), DNP (media alone), NSP (media +10 mM rU +1 mM NITD-982) or starvation conditions (media + 1 mM NITD-982) for 72 h (me
  • FIG. 7C shows Z’-scores calculated for individual assay plates from experiment in FIG. 1.
  • FIG. 7D shows selectivity scores for BTK inhibitors included in the screen.
  • FIG. 7E shows selectivity scores for VEGFR inhibitors included in the screen, where the hits included CNX-774 and motesanib.
  • FIG. 7F shows validation of hit compounds using 7 d crystal violet proliferation assay. MIAPACA2 cells were treated with 1 mM JNK-IN-8, 1 mM OSU-03012, 1 mM TAK-632 in NSP+DNP, DNP or NSP conditions.
  • FIGS. 8A-8C summarize the evaluation of UMP-NSP and -DNP inhibitor potency and selectivity.
  • FIG. 8A shows calculation of JNK-IN-8, CNX-774, and motesanib IC50 values for JURKAT cells cultured in NSP+DNP, DNP-only, and NSP-only conditions, and EC50 values for gemcitabine (dFdC) rescue.
  • FIG. 8C shows calculation of OSU-03012 and TAK-632 IC50 in JURKAT cells treated for 72 h determined using CTG.
  • FIGS. 9A-9C shows the evaluation of DHODH / inhibitor interactions.
  • FIG. 9A provides the crystallographic data collection and refinement statistics. A high resolution shell in parenthesis; r.m.s., rootmean-square; a.u., asymmetric unit.
  • FIG. 9B provides a stereoscopic image of FIG. 4A (OSU-03012).
  • FIG. 9C provides a stereoscopic image of FIG. 4B (TAK-632).
  • FIG. 10 shows the annexinV/PI flow cytometry analysis of OSU-03012/ ATRi combination. Representative flow cytometry plots from experiment in FIG. 3H.
  • Pyrimidine nucleotide biosynthesis refers to the nucleoside salvage pathway (NSP) and the de novo (DNP) pathway which converge to generate uridine monophosphate (UMP), the common precursor for all pyrimidine nucleotides (Okesli et al, 2017).
  • Nucleoside salvage pathway and“NSP” and“pyrimidine nucleoside salvage pathway” salvages preformed nucleosides from the extracellular environment, shuttling them into the cell via nucleoside transporters where they are phosphorylated by uridine-cytidine kinases (UCKs) to produce UMP.
  • UCKs uridine-cytidine kinases
  • “De novo pathway” and“DNP” and“de novo nucleotide pathway” consists of a six- step process that utilizes glutamine, aspartate, bicarbonate and glucose to produce UMP through the action of the trifunctional enzyme CAD, electron transport chain-linked dihydroorotate dehydrogenase (DHODH), and bifunctional uridine monophosphate synthase (UMPS).
  • DHODH electron transport chain-linked dihydroorotate dehydrogenase
  • UMPS bifunctional uridine monophosphate synthase
  • “Dihydroorotate dehydrogenase” and“DHODH” and“DHOD” refer to a protein which catalyzes the fourth step in the de novo pyrimidine nucleotide pathway. DHODH catalyzes the only oxidation/reduction reaction in that pathway which is the step of converting dihydroorotate to orotate with the aid of flavin cofactor and an electron acceptor.
  • condition-specific growth media refers to in vitro growth media that will allow cancer cells to grow under any given conditions.
  • the condition-specific growth media allows cancer cells to grow under salvage pathway conditions and/or do novo pathway conditions, wherein the salvage pathway and the de novo pathway are convergent metabolic pathways that produce at least one common metabolite.
  • the condition-specific growth media allows cancer cells to grow under pyrimidine nucleoside salvage pathway conditions and de novo nucleotide pathway conditions, wherein these convergent metabolic pathways produce uridine monophosphate as the common metabolite.
  • Condition-specific growth media can readily be determined by the skilled artisan based on the specific cancer cells and specific convergent metabolic pathways being evaluated.
  • the cancer cells are pancreatic cancer cells.
  • OSU-03012 or“AR-12” or“2-amino-N-[4-[ 5-phenanthren-2-yl-3-(trifluoromethyl)- pyrazol-l-yl]phenyl]acetamide” refers to a compound having the structure:
  • TAK-632 or“N-(7-cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)- phenoxy)benzo[d]thiazol-2-yl)cydopropanecarhoxamide” refers to a compound having the structure:
  • TAK-632 Methods for making TAK-632 are described in US Patent No.8,143,258, the disclosure ofwhich is incorporated by reference herein in its entirety.
  • JNK-IN-8 or“3-[[4-(dimethylamino)-1-oxo-2-buten-1-yl]amino]-N [ 3-methyl-4-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide” refers to a compound having thestructure:
  • CNX-774 or“4-[4-[[5-fluoro-4-[3-(prop-2-enoylamino)anilino]pyrimidin-2-yl]amino]phenoxy]-N-methylpyridine-2-carboxamide” refers to acompound having thestructure:
  • a or “an,” as used in herein means one or more.
  • a group such as an alkyl orheteroaryl group,is “substituted with an unsubstituted C 5 -C 20 alkyl,or unsubstituted 2 to 20 membered heteroalkyl
  • the group may contain one or more unsubstituted C 1 -C 20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
  • A“detectable agent” or“detectable moiety” is a compound or composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means.
  • useful detectable agents include 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y. 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, m Ag, m In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr,
  • fluorescent dyes include fluorescent dyes), electron- dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide nanoparticles,
  • SPIO nanoparticle aggregates monocrystalline iron oxide nanoparticles, monocrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate molecules, Gadolinium, radioisotopes, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g.
  • microbubble shells including albumin, galactose, lipid, and/or polymers
  • microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.
  • iodinated contrast agents e.g.
  • a detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.
  • Radioactive substances e.g., radioisotopes
  • Radioactive substances include, but are not limited to, 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y. 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, m Ag, m In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm,
  • Paramagnetic ions that may be used as additional imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g. metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
  • transition and lanthanide metals e.g. metals having atomic numbers of 21-29, 42, 43, 44, or 57-71.
  • These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas.
  • exemplary cancers that may be treated with a compound or method provided herein include pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and Non-Hodgkin's Lymphomas.
  • pancreatic cancer e.g., pancreatic ductal adenocarcinoma
  • brain cancer glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, Medulloblastoma, melanoma
  • Exemplary cancers that may be treated with a compound or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus.
  • Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuro
  • A“cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA.
  • a cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring.
  • Cells may include prokaryotic and eukaroytic cells.
  • Prokaryotic cells include but are not limited to bacteria.
  • Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.
  • Cells may include cancer cells.
  • Control or“control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment.
  • the control is used as a standard of comparison in evaluating experimental effects.
  • a control is the measurement of the growth, activity, or function of cancer cells in the absence of a compound as described herein (including embodiments, aspects, and examples).
  • Cancer model organism is an organism exhibiting a phenotype indicative of cancer, or the activity of cancer causing elements, within the organism.
  • the term cancer is defined above.
  • a wide variety of organisms may serve as cancer model organisms, and include for example, cancer cells and mammalian organisms such as rodents (e.g. mouse or rat) and primates (such as humans).
  • Cancer cell lines are widely understood by those skilled in the art as cells exhibiting phenotypes or genotypes similar to in vivo cancers. Cancer cell lines as used herein includes cell lines from animals (e.g. mice) and from humans.
  • An“anticancer agent” as used herein refers to a molecule (e.g. compound, peptide, protein, nucleic acid) used to treat cancer through destruction or inhibition of cancer cells or tissues. Anticancer agents may be selective for certain cancers or certain tissues.
  • Anti-cancer agent and“anticancer agent” are used in accordance with their plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells.
  • an anti-cancer agent is a chemotherapeutic.
  • an anti-cancer agent is an agent identified herein having utility in methods of treating cancer.
  • an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. Examples of anti-cancer agents include, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g.
  • alkylating agents e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g.,
  • methylmelamines e.g., hexamethly melamine, thiotepa
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g., carmustine, lomusitne, semustine, streptozocin
  • triazenes decarbazine
  • anti -metabolites e.g., 5- azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vinde
  • azatyrosine baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
  • bizelesin breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole;
  • carboxyamidotriazole CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole;
  • collismycin A collismycin B; combretastatin A4; combretastatin analogue; conagenin;
  • crambescidin 816 crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;
  • cyclopentanthraquinones cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
  • cytostatin cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone;
  • dexifosfamide dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;
  • edrecolomab edrecolomab
  • eflomithine emene
  • emitefur epirubicin
  • epristeride estramustine analogue
  • estrogen agonists etanidazole
  • etoposide phosphate exemestane
  • fadrozole fadrozole; trasrabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors;
  • gemcitabine glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide;
  • hypericin ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;
  • imidazoacridones imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin;
  • ipomeanol 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;
  • jasplakinolide kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
  • lentinan sulfate leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
  • masoprocol maspin
  • matrilysin inhibitors matrix metalloproteinase inhibitors
  • menogaril masoprocol
  • matrilysin inhibitors matrilysin inhibitors
  • matrix metalloproteinase inhibitors menogaril
  • merbarone meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;
  • molgramostim monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anti cancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondans
  • parabactin pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate;
  • phosphatase inhibitors picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone;
  • prostaglandin J2 proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
  • oligonucleotides oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors;
  • stipiamide stromelysin inhibitors
  • sulfmosine superactive vasoactive intestinal peptide antagonist
  • suradista suramin
  • suramin suramin
  • swainsonine synthetic glycosaminoglycans
  • tamoxifen methiodide tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turoster
  • hydrochloride acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;
  • cactinomycin calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefmgol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine;
  • dexormaplatin dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;
  • edatrexate edatrexate
  • eflomithine hydrochloride elsamitrucin
  • enloplatin enpromate
  • epipropidine edatrexate
  • eflomithine hydrochloride elsamitrucin
  • enloplatin eflomithine hydrochloride
  • elsamitrucin enloplatin
  • enpromate epipropidine
  • epirubicin hydrochloride erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole
  • fluorocitabine fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;
  • hydroxyurea idarubicin hydrochloride; ifosfamide; iimofosine; interleukin II (including recombinant interleukin II, or rlL.sub.2), interferon alfa-2a; interferon alfa-2b; interferon alfa- nl ; interferon alfa-n3; interferon beta-la; interferon gamma-lb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
  • lometrexol sodium lomustine; losoxantrone hydrochloride; masoprocol; maytansine;
  • mechlorethamine hydrochloride megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran;
  • pegaspargase peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
  • piposulfan piroxantrone hydrochloride
  • plicamycin plicamycin
  • plomestane porfimer sodium
  • porfiromycin prednimustine; procarbazine hydrochloride; puromycin; puromycin
  • hydrochloride pyrazofurin; riboprine; rogletimide; safmgol; safmgol hydrochloride; semustine; pumprazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur;
  • teloxantrone hydrochloride temoporfm
  • teniposide teroxirone
  • testolactone thiamiprine
  • Taxol.TM i.e. paclitaxel
  • Taxotere.TM compounds comprising the taxane skeleton, Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128), Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829, Discodermolide (i.e. as NVP- XX-A-296), ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.
  • Epothilone E Epothilone F
  • Epothilone B N-oxide Epothilone A N-oxide
  • 16-aza-epothilone B 21-aminoepothilone B (i.e. BMS-310705)
  • 21 -hydroxy epothilone D i.e. Desoxyepothilone F and dEpoF
  • 26-fluoroepothilone i.e. NSC-654663
  • Soblidotin i.e. TZT-1027
  • LS-4559-P Pulacia, i.e.
  • LS-4577 LS-4578 (Pharmacia, i.e. LS- 477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 and LU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97
  • NSC-106969 T-138067 (Tularik, i.e. T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e. DDE- 261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, i.e.
  • NSCL-96F03-7 D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e.
  • SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC- 12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e
  • gefhinib IressaTM
  • erlotinib TarcevaTM
  • cetuximab ErbituxTM
  • lapatinib TykerbTM
  • panitumumab VectibixTM
  • vandetanib CaprelsaTM
  • afatinib/BIBW2992 CI-1033/canertinib, neratinib/HKI-272, CP- 724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasat
  • “Selective” or“selectivity” or the like of a compound refers to the compound’s ability to discriminate between molecular targets (e.g., a compound having selectivity for one metabolic pathway over another metabolic pathway).
  • “Specific”,“specifically”,“specificity”, or the like of a compound refers to the compound’s ability to cause a particular action, such as inhibition, to a particular molecular target or metabolic pathway with minimal or no action to other molecular targets or metabolic pathways.
  • salts are meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogen carbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogen sulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogen carbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogen sulfuric, hydriodic, or phosphorous
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al,“Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the compounds of the disclosure may exist as salts, such as with
  • Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)- tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art.
  • the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • the disclosure provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the disclosure.
  • Prodrugs of the compounds described herein may be converted in vivo after administration.
  • prodrugs can be converted to the compounds of the disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
  • Certain compounds of the disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the disclosure. Certain compounds of the disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the disclosure and are intended to be within the scope of the disclosure.
  • the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In aspects, about means within a standard deviation using measurements generally acceptable in the art. In aspects, about means a range extending to +/- 10% of the specified value. In aspects, about includes the specified value.
  • the terms“treating”, or“treatment” refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation.
  • the term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease.
  • treating is preventing.
  • treating does not include preventing.
  • Treating” or“treatment” as used herein also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • treatment includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
  • Treating” and “treatment” as used herein include prophylactic treatment.
  • Treatment methods include administering to a subject a therapeutically effective amount of an active agent.
  • the administering step may consist of a single administration or may include a series of administrations.
  • the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof.
  • the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.
  • chronic administration may be required.
  • the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.
  • the treating or treatment is no prophylactic treatment.
  • the term“prevent” refers to a decrease in the occurrence of disease symptoms in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.
  • “Patient” or“subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
  • a patient is human.
  • A“effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition).
  • An example of an“effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • A“reduction” of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • A“prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist.
  • A“function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
  • the therapeutically effective amount can be initially determined from cell culture assays.
  • Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.
  • therapeutically effective amounts for use in humans can also be determined from animal models.
  • a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
  • therapeutically effective amount refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above.
  • a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as“-fold” increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side- effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • administering means oral administration, administration as a suppository, topical contact, parenteral, intralesional, intrathecal, or intranasal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • a slow-release device e.g., a mini-osmotic pump
  • Parenteral administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • the administering does not include administration of any active agent other than the recited active agent.
  • compositions described herein are administered at the same time, just prior to, or just after the administration of one or more additional therapies.
  • the compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).
  • the compositions of the disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • bound atoms or molecules may be direct, e.g., by covalent bond or linker (e.g. a first linker or second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).
  • covalent bond or linker e.g. a first linker or second linker
  • non-covalent bond e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).
  • the term“capable of binding” as used herein refers to a moiety (e.g. a compound as described herein) that is able to measurably bind to a target.
  • a moiety e.g. a compound as described herein
  • the moiety is capable of binding with a Kd of less than about 10 mM, 5 mM, 1 mM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.
  • pathway or“signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.
  • extra-cellular components e.g. proteins, nucleic acids, small molecules, ions, lipids
  • ATR kinase inhibitor refers to an inhibitor of ataxia telangiectasia and rad3-related (ATR) kinase, a DNA damage response kinase, with potential antineoplastic activity.
  • ATR a serine/threonine protein kinase, plays a key role in DNA repair, cell cycle progression, and survival, and is activated by DNA damage caused during DNA replication- associated stress.
  • Exemplary ATR kinase inhibitors include berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • VE-821 or“3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2- carboxamide” refers to a compound having the structure:
  • NU6027 or“4-cyclohexylmethoxy-2,6-diamino-5-nitrosopyrimidine” refers to a compound having the structure:
  • AZ20 or“4[4-[(3R)3-methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]- pyrimidin-2-yl]-1H-indole” refers to a compound having the structure:
  • an analog is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called“reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
  • Contacting is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture. In aspects contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.
  • at least two distinct species e.g. chemical compounds including biomolecules or cells
  • the term“activation”,“activate”,“activating”,“activator” and the like in reference to a protein-inhibitor interaction means positively affecting (e.g. increasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the activator.
  • activation means positively affecting (e.g. increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator.
  • the terms may reference activation, or activating, sensitizing, or up- regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease.
  • activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein which is decreased in a disease relative to a non-diseased control).
  • Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein
  • the terms“agonist,”“activator,”“upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein.
  • the agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist.
  • expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.
  • the term“inhibition”,“inhibit”,“inhibiting” and the like in reference to a cancer cell proliferation-inhibitor interaction means negatively affecting (e.g. decreasing) the growth, activity, or function of the cancer cell relative to the growth, activity, or function of the cancer cells in the absence of the inhibitor.
  • inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the cancer cells relative to the concentration or level of the cancer cells in the absence of the inhibitor.
  • inhibition refers to reduction of a disease or symptoms of disease.
  • inhibition refers to a reduction in the activity of particular cancer cells.
  • inhibition refers to a reduction of activity or concentration of cancer cells resulting from a direct or indirect interaction between the inhibitor and the cancer cells.
  • the terms“inhibitor,”“repressor” or“antagonist” or“downregulator” interchangeably refer to a compound capable of detectably decreasing the growth, activity, or function of any given cancer cells.
  • the antagonist can decrease growth, activity, or function of cancer cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist.
  • growth, activity, or function of cancer cells is 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the growth, activity, or function of cancer cells in the absence of the antagonist.
  • expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry,
  • modulator refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule or the physical state of the target of the molecule relative to the absence of the modulator.
  • modulate is used in accordance with its plain ordinary meaning and refers to the act of changing or varying one or more properties.“Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule.
  • the term“associated” or“associated with” in the context of a substance or substance activity or function associated with a disease means that the disease (e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.
  • a disease e.g. a protein associated disease, a cancer (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease)
  • the disease e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease
  • a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and salvage pathway condition-specific growth media and de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound; thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the compound that inhibits cancer cell proliferation is JNK- IN-8, CNX-774, motesanib, OSU-03012, TAK-632, or an analog of any of the foregoing.
  • the cancer cells are pancreatic cancer cells.
  • the cancer cells are pancreatic ductal adenocarcinoma cells.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and salvage pathway condition-specific growth media; wherein the salvage pathway, and its associated de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound; thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the compound that inhibits cancer cell proliferation is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the cancer cells are pancreatic cancer cells.
  • the cancer cells are pancreatic ductal adenocarcinoma cells.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and de novo pathway condition-specific growth media; wherein the de novo pathway, and its associated salvage pathway, are convergent metabolic pathways producing a common metabolite; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound; thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the compound that inhibits cancer cell proliferation is a
  • the compound that inhibits cancer cell proliferation is OSU-03012, TAK-632, or an analog of any of the foregoing.
  • the cancer cells are pancreatic cancer cells.
  • the cancer cells are pancreatic ductal adenocarcinoma cells.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and (a) salvage pathway condition-specific growth media and de novo pathway condition-specific growth media and (b) salvage pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound in (a) and/or (b); thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the cancer cells are pancreatic cancer cells. In aspects, the cancer cells are pancreatic ductal adenocarcinoma cells. In aspects, the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) and (b). In aspects, the compound that inhibits cancer cell proliferation in (a) and (b) is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing. In aspects, the cancer is pancreatic cancer. In aspects, the cancer is pancreatic ductal adenocarcinoma.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) but not (b).
  • the compound that inhibits cancer cell proliferation is (a) but not (b) is OSU- 03012, TAK-632, or an analog of any of the foregoing.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and (a) salvage pathway condition-specific growth media and de novo pathway condition-specific growth media and (b) de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound in (a) and/or (b); thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the cancer cells are pancreatic cancer cells. In aspects, the cancer cells are pancreatic ductal adenocarcinoma cells. In aspects, the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) and (b). In aspects, the compound that inhibits cancer cell proliferation in (a) and (b) is OSU-03012, TAK-632, or an analog of any of the foregoing. In aspects, the compound that inhibits cancer cell proliferation in (a) and (b) is a dihydroorotate dehydrogenase inhibitor. In aspects, the cancer is pancreatic cancer. In aspects, the cancer is pancreatic ductal adenocarcinoma.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) but not (b).
  • the compound that inhibits cancer cell proliferation in (a) but not (b) is JNK- IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and (a) salvage pathway condition-specific growth media and (b) de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound in (a) and/or (b); thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the cancer cells are pancreatic cancer cells. In aspects, the cancer cells are pancreatic ductal adenocarcinoma cells. In aspects, the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) and (b). In aspects, the cancer is pancreatic cancer. In aspects, the cancer is pancreatic ductal adenocarcinoma. In aspects, the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) but not (b).
  • the compound that inhibits cancer cell proliferation in (a) but not (b) is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (b) but not (a).
  • the compound that inhibits cancer cell proliferation in (b) but not (a) is OSU-03012, TAK-632, or an analog of any of the foregoing.
  • the compound that inhibits cancer cell proliferation in (b) not (a) is a dihydroorotate dehydrogenase inhibitor.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with cancer cells and (a) salvage pathway condition-specific growth media and de novo pathway condition-specific growth media; (b) salvage pathway condition-specific growth media, and (c) de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound in (a) and/or (b) and/or (c); thereby identifying the compound that inhibits cancer cell proliferation.
  • the salvage pathway is a pyrimidine nucleoside salvage pathway
  • the de novo pathway is a de novo nucleotide pathway
  • the common metabolite is uridine monophosphate.
  • the cancer cells are pancreatic cancer cells.
  • the cancer cells are pancreatic ductal adenocarcinoma cells.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a), (b), and (c).
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) and (b), but not (c).
  • the compound that inhibits cancer cell proliferation is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the disclosure provides methods of treating cancer in patient in need thereof by administering to the patient a therapeutically effective amount of the compound that inhibits cancer cell proliferation in (a) and (c), but not (b).
  • the compound that inhibits cancer cell proliferation in (a) and (c) but not (b) is OSU-03012, TAK-632, or an analog of any of the foregoing.
  • the compound that inhibits cancer cell proliferation in (a) and (c) but not (b) is a dihydroorotate dehydrogenase inhibitor.
  • the cancer is pancreatic cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the method of treating cancer further comprises administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the disclosure provides methods of treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a salvage pathway inhibitor but that is not a de novo pathway inhibitor. In embodiments, the disclosure provides methods of treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a de novo pathway inhibitor but that is not a salvage pathway inhibitor.
  • the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite.
  • the disclosure provides methods of treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a pyrimidine nucleoside salvage pathway inhibitor but that is not a de novo nucleotide pathway inhibitor.
  • the pyrimidine nucleoside salvage pathway and de novo nucleotide pathway are convergent metabolic pathways producing UMP as a common metabolite.
  • the compound that is a pyrimidine nucleoside salvage pathway inhibitor is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is JNK-IN-8. In aspects, the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is CNX-774. In aspects, the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is motesanib. In aspects, the methods further comprise administering to the patient a
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating pancreatic cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a pyrimidine nucleoside salvage pathway inhibitor but that is not a de novo nucleotide pathway inhibitor.
  • the pyrimidine nucleoside salvage pathway and de novo nucleotide pathway are convergent metabolic pathways producing UMP as a common metabolite.
  • the compound that is a pyrimidine nucleoside salvage pathway inhibitor is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is JNK-IN-8. In aspects, the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is CNX- 774. In aspects, the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is motesanib. In aspects, the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactobsib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating pancreatic ductal adenocarcinoma in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a pyrimidine nucleoside salvage pathway inhibitor but that is not a de novo nucleotide pathway inhibitor.
  • the pyrimidine nucleoside salvage pathway and de novo nucleotide pathway are convergent metabolic pathways producing UMP as a common metabolite.
  • the compound that is a pyrimidine nucleoside salvage pathway inhibitor is JNK-IN-8, CNX-774, motesanib, or an analog of any of the foregoing.
  • the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is JNK-IN-8. In aspects, the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is CNX- 774. In aspects, the compound that is a pyrimidine nucleoside salvage pathway inhibitor but not a de novo nucleotide pathway inhibitor is motesanib. In aspects, the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a de novo nucleotide pathway inhibitor but that is not a pyrimidine nucleoside salvage pathway inhibitor.
  • the pyrimidine nucleoside salvage pathway and de novo nucleotide pathway are convergent metabolic pathways producing UMP as a common metabolite.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is a dihydroorotate dehydrogenase inhibitor.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is OSU-03012, TAK-632, or an analog of the foregoing.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is OSU-03012.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is TAK-632.
  • the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating pancreatic cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a de novo nucleotide pathway inhibitor but that is not a pyrimidine nucleoside salvage pathway inhibitor.
  • the pyrimidine nucleoside salvage pathway and de novo nucleotide pathway are convergent metabolic pathways producing UMP as a common metabolite.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is a dihydroorotate dehydrogenase inhibitor.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is OSU-03012, TAK-632, or an analog of the foregoing.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is OSU-03012.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is TAK- 632.
  • the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating pancreatic ductal adenocarcinoma in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound that is a de novo nucleotide pathway inhibitor but that is not a pyrimidine nucleoside salvage pathway inhibitor.
  • the pyrimidine nucleoside salvage pathway and de novo nucleotide pathway are convergent metabolic pathways producing UMP as a common metabolite.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is a dihydroorotate dehydrogenase inhibitor.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is OSU-03012, TAK-632, or an analog of the foregoing.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is OSU-03012.
  • the compound that is a de novo nucleotide pathway inhibitor but not a pyrimidine nucleoside salvage pathway inhibitor is TAK-632.
  • the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of dihydroorotate dehydrogenase inhibitor.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012, TAK-632, or an analog of the foregoing.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012.
  • the dihydroorotate dehydrogenase inhibitor is TAK-632.
  • the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the disclosure provides methods of treating pancreatic cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of a dihydroorotate dehydrogenase inhibitor.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012, TAK-632, or an analog of the foregoing.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012.
  • the dihydroorotate dehydrogenase inhibitor is TAK-632.
  • the methods further comprise administering to the patient a
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, or an analog of any one of the foregoing.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, or AZ20.
  • the disclosure provides methods of treating pancreatic ductal adenocarcinoma in a patient in need thereof by administering to the patient a therapeutically effective amount of a dihydroorotate dehydrogenase inhibitor.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012, TAK-632, or an analog of the foregoing.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012.
  • the dihydroorotate dehydrogenase inhibitor is TAK-632.
  • the methods further comprise administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, or an analog of any one of the foregoing.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, or AZ20.
  • compositions comprising dihydroorotate dehydrogenase and a dihydroorotate dehydrogenase inhibitor.
  • the compositions are co-crystals.
  • the dihydroorotate dehydrogenase and dihydroorotate dehydrogenase inhibitor are ionically bonded together.
  • the dehydrogenase inhibitor are bonded together via one or more hydrogen bonds.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012, TAK-632, or an analog of any of the foregoing.
  • the dihydroorotate dehydrogenase inhibitor is OSU-03012.
  • the dihydroorotate dehydrogenase inhibitor is TAK-632.
  • any of the compounds described herein may be administered to a subject in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.
  • the compositions are suitable for formulation and administration in vitro or in vivo. Suitable carriers and excipients and their formulations are known in the art and described, e.g., Remington: The Science and Practice of Pharmacy, 21st Ed, Lippicott Williams & Wilkins (2005).
  • “Pharmaceutically acceptable excipient” and“pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the disclosure without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethy cellulose, polyvinyl pyrrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents
  • Solutions of the pharmaceutical compositions can be prepared in water suitably mixed with a lipid or surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Solutions can be administered, e.g., parenterally, such as subcutaneously or intravenously (e.g., infusion or bolus).
  • a lipid or surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Solutions can be administered, e.g., parenterally, such as subcutaneously or intravenously (e.g., infusion or bolus).
  • compositions can be delivered via intranasal or inhalable solutions.
  • the intranasal composition can be a spray, aerosol, or inhalant.
  • the inhalable composition can be a spray, aerosol, or inhalant.
  • Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers, if required, may be included in the formulation.
  • Various commercial nasal preparations are known in the art.
  • Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • oral pharmaceutical compositions will comprise an inert diluent or edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the percentage of the compositions and preparations may, of course, be varied and may be between about 1 to about 75% of the weight of the unit.
  • the amount of nucleic acids in such compositions is such that a suitable dosage can be obtained.
  • aqueous solutions for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • Aqueous solutions in particular, sterile aqueous media, are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
  • Sterile injectable solutions can be prepared by incorporating the compounds in the required amount in the appropriate solvent followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium. Vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients, can be used to prepare sterile powders for reconstitution of sterile injectable solutions. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated. Dimethyl sulfoxide can be used as solvent for rapid penetration, delivering high concentrations of the active agents to a small area. [0102] The dosage and frequency (single or multiple doses) of the compounds and
  • compositions administered to a subject can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated, kind of concurrent treatment, complications from the disease being treated or other health-related problems.
  • Other therapeutic regimens or agents can be used in conjunction with the methods and recombinant proteins described herein. Adjustment and manipulation of established dosages (e.g., frequency and duration) are within the ability of the skilled artisan.
  • the effective amount can be initially determined from cell culture assays.
  • Target concentrations will be those concentrations of compounds that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.
  • effective amounts of compounds for use in humans can also be determined from animal models.
  • a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
  • Dosages of the compounds may be varied depending upon the requirements of the patient.
  • the dose administered to a patient should be sufficient to affect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the art. Dosage amounts and intervals can be adjusted individually to provide levels of the compounds effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is effective to treat the clinical disease or symptoms demonstrated by the particular patient. This planning should involve the careful choice of recombinant proteins by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects.
  • the compounds are administered to a patient at an amount of about 0.001 mg/kg to about 500 mg/kg.
  • the compounds are administered to a patient in an amount of about 0.01 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg, or 300 mg/kg. It is understood that where the amount is referred to as "mg/kg,” the amount is milligram per kilogram body weight of the subject being administered with the recombinant proteins.
  • the compounds are administered to a patient in an amount from about 0.01 mg to about 500 mg per day, as a single dose, or in a dose administered two or three times per day.
  • Embodiment P1 A method for identifying a compound that inhibits cancer cell proliferation, the method comprising: (i) contacting the compound with (a) cancer cells and salvage pathway condition-specific growth media and de novo pathway condition-specific growth media; and'or (b) cancer cells and salvage pathway condition-specific growth media; and/or (c) cancer cells and de novo pathway condition-specific growth media; wherein the salvage pathway and de novo pathway are convergent metabolic pathways producing a common metabolite; and (ii) identifying inhibition of cancer cell proliferation by the compound; thereby identifying the compound that inhibits cancer cell proliferation.
  • Embodiment P2 The method of Embodiment P1, wherein (b) does not comprise de novo pathway condition-specific growth media; and wherein (c) does not comprise salvage pathway condition-specific growth media.
  • Embodiment P3 The method of Embodiment P1 or P2, wherein: (a) comprises cancer cells and pyrimidine nucleoside salvage pathway condition-specific growth media and de novo nucleotide pathway condition-specific growth media, (b) comprises cancer cells and pyrimidine nucleoside salvage pathway condition-specific growth media; and (c) comprises cancer cells and de novo nucleotide pathway condition-specific growth media.
  • Embodiment P4 The method of any one of Embodiments P1 to P3, wherein the method comprises contacting the compound with (a) and (b).
  • Embodiment P5. The method of any one of Embodiments P1 to P3, wherein the method comprises contacting the compound with (a) and (c).
  • Embodiment P6 The method of any one of Embodiments P1 to P3, wherein the method comprises contacting the compound with (b) and (c).
  • Embodiment P7 The method of any one of Embodiments P1 to P3, wherein the method comprises contacting the compound with (a), (b), and (c).
  • Embodiment P8 A method of treating cancer in a patient in need thereof, the method comprising: (i) identifying the compound that inhibits cancer cell proliferation according to any one of Embodiments P1 to P7; (ii) selecting the compound that inhibits caner cell proliferation in (b) or (c); and (iii) administering to the patient a therapeutically effective amount of the compound that inhibits caner cell proliferation in (b) or (c).
  • Embodiment P9 The method of Embodiment P8, wherein the compound that inhibits caner cell proliferation in (b) does not inhibit cancer cell proliferation in (c).
  • Embodiment P10 The method of Embodiment P8 or P9, comprising selecting the compound that inhibits caner cell proliferation in (b); and administering to the patient a therapeutically effective amount of the compound that inhibits caner cell proliferation in (b).
  • Embodiment P 11 The method of any one of Embodiments P8 to P10, wherein the compound that inhibits caner cell proliferation in (b) is JNK-IN-8, CNX-774, or motesanib.
  • Embodiment P12 The method of Embodiment P8, wherein the compound that inhibits caner cell proliferation in (c) does not inhibit cancer cell proliferation in (b).
  • Embodiment P13 The method of Embodiment P8 or P12, comprising selecting the compound that inhibits caner cell proliferation in (c); and administering to the patient a therapeutically effective amount of the compound that inhibits caner cell proliferation in (c).
  • Embodiment P14 The method of Embodiment P8, P12, or P13, wherein compound that inhibits caner cell proliferation in (c) is OSU-03012 or TAK-632.
  • Embodiment P15 The method of any one of Embodiments P1 to P13, wherein the cancer is pancreatic cancer.
  • Embodiment P16 The method of Embodiment P15, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
  • Embodiment P17 A method of treating pancreatic cancer in a patient in need thereof, the method comprising administering a therapeutically effective amount of a de novo nucleotide pathway inhibitor.
  • Embodiment P18 The method of Embodiment P17, wherein the de novo nucleotide pathway inhibitor is a dihydroorotate dehydrogenase inhibitor.
  • Embodiment P19 The method of Embodiment P17 or P18, wherein the de novo nucleotide pathway inhibitor is OSU-03012 or TAK-632.
  • Embodiment P20 The method of any one of Embodiments P17 to P19, wherein the de novo nucleotide pathway inhibitor is not a pyrimidine nucleoside salvage pathway inhibitor.
  • Embodiment P21 A method of treating pancreatic cancer in a patient in need thereof, the method comprising administering a therapeutically effective amount of a pyrimidine nucleoside salvage pathway inhibitor.
  • Embodiment P22 The method of Embodiment P21, wherein the pyrimidine nucleoside salvage pathway inhibitor is JNK-IN-8, CNX-774, or motesanib.
  • Embodiment P23 The method of Embodiment P21 or P22, wherein the pyrimidine nucleoside salvage pathway inhibitor is not a de novo nucleotide pathway inhibitor.
  • Embodiment P24 The method of any one of Embodiments P17 to P23, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
  • Embodiment P25 The method of any one of Embodiments P8 to P24, further comprising administering to the patient a therapeutically effective amount of an ATR kinase inhibitor.
  • Embodiment P26 The method of Embodiment P25, wherein the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • the ATR kinase inhibitor is berzosertib, VE-821, ceralasertib, schisandrin B, NU6027, dactolisib, AZ20, caffeine, wortmannin, or an analog of any one of the foregoing.
  • Embodiment P27 The method of any one of Embodiments P8 to P24, further comprising administering to the patient a therapeutically effective amount of an anti-cancer agent.
  • Embodiment P28 A composition comprising dihydroorotate dehydrogenase and TAK- 632.
  • Embodiment P29 The composition of Embodiment P28, wherein TAK-632 and dihydroorotate dehydrogenase are bonded together via a hydrogen bond.
  • Embodiment P30 A composition comprising dihydroorotate dehydrogenase and OSU- 03012.
  • Embodiment P31 The composition of Embodiment P30, wherein OSU-03012 and dihydroorotate dehydrogenase are bonded together via a hydrogen bond.
  • a cell-based metabolic modifier screening platform for the discovery of modulators of convergent pyrimidine nucleotide biosynthetic pathways was designed and implemented. In screening a library of protein kinase inhibitors, multiple compounds were shown to possess previously uncharacterized nucleotide metabolism-modifying activity.
  • the JNK inhibitor JNK- IN-8 was found to be a potent inhibitor of nucleoside transport, a property which was confirmed using nucleoside-analog Positron Emission Tomography (PET) imaging in mice.
  • PET nucleoside-analog Positron Emission Tomography
  • the PDK1 inhibitor OSU-03012 also known as AR-12
  • the RAF inhibitor TAK-632 were shown to inhibit the therapeutically relevant enzyme dihydroorotate dehydrogenase (DHODH) and their affinities were unambiguously confirmed through in vitro assays and co-crystallization with human DHODH.
  • DHODH dihydroorotate dehydrogenase
  • JNK c-Jun N- terminal kinase
  • PDK1 3 -phosphoinositi de-dependent protein kinase 1
  • AR-12 pan-RAF inhibitor TAK-632
  • NSP inhibitors those which inhibited proliferation in NSP-only conditions are NSP inhibitors, while those that inhibited growth in DNP-only conditions are DNP inhibitors.
  • the screen design was validated using the known DHODH inhibitor NITD-982 (Wang et al., 2011) and the FDA-approved nucleotide transport inhibitor dipyridamole (DP A), using CellTiter-Glo (CTG) to evaluate proliferation impairment (FIG. 5B).
  • a library of 430 protein kinase inhibitors was chosen for evaluation, the rationale being twofold. First, it was hypothesized that the synthetic lethality screen may identify compounds that indirectly targeted pyrimidine metabolism by inhibiting regulatory signal transduction pathways. Second, because the majority of kinase inhibitors are ATP-mimetics, and given their resemblance to nucleotides, it was predicted that protein kinase inhibitors may posses additional, non-canonical targets within nucleotide metabolism.
  • JNK inhibitor JNK-IN-8 The JNK inhibitor JNK-IN-8, the BTK inhibitor CNX-774, and the VEGFR inhibitor motesanib were active in the NSP-only condition (FIG. IB).
  • the PDK1 inhibitor OSU-03012 also known as AR-12
  • the pan-RAF inhibitor TAK-632 were identified as eliciting potent and selective inhibition of proliferation in the DNP- only condition (FIG. lD)(Zhu et al., 2004; Okaniwa et al, 2013).
  • OSU-03012 and TAK-632 were unique in their ability to selectively inhibit the DNP, suggesting that their ability to inhibit the pyrimidine DNP was not the consequence of on-target effects (FIGS. 1E-1F).
  • Hit selectivity was confirmed using an orthogonal long-term proliferation inhibition assay (FIG. 7F).
  • JNK-IN-8 inhibits nucleoside uptake in vitro and in vivo
  • JNK-IN-8 was exceptionally potent with IC50 values in the low nanomolar range.
  • the activity of JNK-IN-8 could arise from either the inhibition of nucleoside shuttling across the plasma membrane, achieved by concentrative (CNT) or equilibrative (ENT) nucleoside transporters, or of pyrimidine nucleoside phosphorylation by UCKs.
  • CNT concentrative
  • ENT equilibrative
  • nucleosides rely upon the same equilibrative transporters (ENT1/2) to enter the cell but require different kinases (UCKs in the case of rU and deoxycytidine kinase - dCK- in the case of dC (Le et al, 2017)) for conversion into their respective monophosphate forms and resultant intracellular accumulation (FIG. 2A).
  • the assay revealed that JNK-IN-8 inhibited the uptake of both rU and dC with similar potency (33 nM and 31 nM, respectively), indicating that the compound was inhibiting nucleoside transport (FIG.
  • JNK-IN-8 treatment rescued JURKAT cells from the anti-proliferative effects of gemcitabine, a dCK-dependent nucleoside analog prodrug which relies upon nucleoside transporters to enter the cell, with an EC50 of 15.2 nM. Similar rescue was observed with CNX- 774, motesanib and DPA treatment (FIGS. 8A-8B).
  • OSU-03012 and TAK-632 target de novo UMP biosynthesis and activate the DNA replication stress response pathway
  • TAK-632 and OSU-03012 Two kinase inhibitors, TAK-632 and OSU-03012, were identified as potent and selective inhibitors of the DNP (FIG. 8C). We reasoned that these compounds could target either CAD, DHODH, or UMPS enzymes in de novo pyrimidine biosynthesis (FIG. 3A). Both compounds induced S-phase arrest in MIAPACA2 cells, a phenotype associated with insufficient dNTP biosynthesis to sustain DNA replication and activation of intra-S phase signaling checkpoints. This effect was rescued by orotate (the product of DHODH)
  • DHODH inhibition emerged as a likely mechanism, as it catalyzes one of three committed steps within the DNP and is a druggable protein (Madak et al, 2019). Additionally, both OSU-03012 and TAK-632 posses fluorine substituents which have been shown to stabilize bioactive conformations of DHODH inhibitors (Bonomo et al., 2013; Baumgartner et al, 2006). In an in vitro colorimetric recombinant human DHODH activity assay, TAK-632 and OSU-03012 both inhibited DHODH activity in a dose-dependent manner (FIG. 3D) (Baumgartner et al, 2006). Importantly, the response to TAK-632 or OSU-03012 correlated with the response to a known DHODH inhibitor in this cell line panel (FIG. 3E).
  • OSU-03012 was recently reported to synergize with replication stress response kinase inhibitors in RSK-subtype mutant KRAS cancer models (Yuan et al., 2018). However, after confirming that OSU-03012 binds DHODH, we hypothesized that the observed synergy resulted from DHODH inhibition rather than PDK1 inhibition. Immunoblot analysis of S6K and S6 phosphorylation, PDK1 downstream targets, confirmed that GSK-2334470, a known PDK1 inhibitor, potently blocked PDK1 while OSU-03012 triggered S345 CHEK1 phosphorylation, a replication stress biomarker, only in the absence of rU (FIG.
  • pyrimidine metabolism was identified as an amenable extension for this type of screen, as it consists of convergent (de novo and salvage) pathways, and UMP depletion triggers a quantifiable change in cellular proliferation phenotype.
  • JNK-IN-8 developed as an irreversible inhibitor of c-Jun N-terminal kinases 1, 2, and 3 with low-nanomolar affinity, was the most potent of three NSP inhibitors identified (Zhang et al, 2012). The data show that it is additionally a potent inhibitor of nucleoside transport, both in vitro and in vivo. Thus, JNK-IN-8 should not be used in conjunction with compounds, including cancer-treating antimetabolites, which rely upon nucleoside transport for their research or therapeutic purpose. Furthermore, JNK-IN-8 should not be used in research settings wherein upregulation of the pyrimidine DNP may confound experimental results.
  • OSU-03012 and TAK-632 were identified as inhibitors of the pyrimidine DNP. This work is the first to unambiguously confirm the affinity of OSU-03012 and TAK-632 for DHODH through crystallography studies. Notably, the studies herein show that OSU-03012 and TAK-632 bind in the same hydrophobic tunnel of DHODH as known inhibitors brequinar and teriflunomide (the active metabolite of leflunomide). This indicates that these two protein kinase inhibitors compete with ubiquinone, a redox partner of DHODH which traverses the hydrophobic tunnel to regenerate FMN from FMNH 2 . By competitively inhibiting the binding of ubiquinone, these compounds prevent DHODH from completing its redox cycle effectively abrogating its activity.
  • OSU-03012 has orphan drug designation in the European Union for treatment of tularaemia and cryptococcosis. We hypothesize that its effectiveness in these indications stems from its ability to inhibit DHODH, rather than from‘on-target’ effects against PDK1. Indeed, DHODH inhibitors have demonstrated efficacy against viruses such as dengue virus and respiratory syncytial virus (Bonavia et al, 2011; Yang et al., 2018; Wang et al, 2011). In anticancer settings, OSU-03012 was recently demonstrated to synergize with CHK1 inhibitors in KRAS-mutant cancers (Yuan et al, 2018), which was initially attributed to its ability to inhibit PDK1.
  • the inventors designed and applied a metabolic modifier screen which identified multiple protein kinase inhibitors as having non-canonical targets within pyrimidine metabolism.
  • constructed phenotypic screens designed against other metabolic networks containing convergent nodes may find use in drug discovery campaigns or in repurposing screens using existing compounds.
  • Drugs were prepared in DMSO or H 2 O and diluted fresh in cell culture media for treatments.
  • NITD-982 was synthesized as previously described by Bonavia et al, Proc Natl Acad Sci USA, 108(17), 6739-6744.
  • Protein Kinase Inhibitor Phenotypic Screen A library of 430 protein kinase inhibitors was arrayed in polypropylene 384-well plates at 200x concentrations covering a 7-point concentration range (corresponding to lx concentrations: 5mM, 1.65mM, 550nM, 185nM, 61.5nM, 20.6nM, 6.85nM). 25m1 per well of condition-specific growth media (DNP+NSP:
  • Radioactive probe uptake assays were conducted as previously described (Campbell et al, 2011). Briefly, 5 x10 5 CCRF-CEM cells were resuspended in 1 mL of media per well in 12-well plates. After 1 h, cells were incubated with the indicated amounts of tritiated probe ⁇ JNK-IN-8 for an additional hour. Cells were then harvested and washed twice with ice-cold PBS. Radioactivity was measured using a beta- counter (Perkin-Elmer).
  • PET Positron Emission Tomography
  • AnnexinV/PI Treated PD AC cells were washed with PBS and incubated with AnnexinV and propidium iodide diluted in 1x Annexin binding buffer. 20,000 events were collected per sample.
  • PD AC cells were plated in 6-well cell culture plates at 1x10 4 cells/well and treated as described. Following treatment cells were fixed by incubating in 4% PFA in PBS for 15 m at room temperature. Plates were subsequently washed with PBS and stained with 0.1% crystal violet in H 2 O for 15 m at room temperature.
  • the vector was transformed into C41(DE3) cells for productions.
  • Cells were grown at 37°C in 2xYT medium supplemented with 100 mg/mL ampicillin (Amp), treated with 0.1 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) at an OD600 nm of 0.6-0.8, and then cultured for an additional 18h at 18°C.
  • Cells were harvested by centrifugation, washed with 200 mM NaCl and 25 mM Tris pH 7.5, and pelleted at 5000 rpm for 20 minutes before storage at -20°C. 6.7g/L of cell pellet was obtained.
  • DHODH was purified according to known purification conditions (Baumgartner et al, 2006). Cell pellet was resuspended in lysis buffer (50 mM Tris pH 7.5; 600 mM NaCl; 0.33% w/v Thesit; 10% Glycerol; 1 mM PMSF) and lysed by sonication on ice.
  • lysis buffer 50 mM Tris pH 7.5; 600 mM NaCl; 0.33% w/v Thesit; 10% Glycerol; 1 mM PMSF
  • Lysed cells were centrifuged at 58,500 RCF for 45 minutes at 4°C, and the supernatant was filtered through a 0.45 mM filter and loaded onto a 5-mL His-Trap column pre-equilibrated with buffer A (50 mM Tris pH 7.4; 600 mM NaCl; 0.05% w/v Thesit; 10% Glycerol).
  • buffer A 50 mM Tris pH 7.4; 600 mM NaCl; 0.05% w/v Thesit; 10% Glycerol.
  • the column was washed with buffer A for 70 mL, buffer A with 25 mM imidazole for 50 mL, and buffer A with 50 mM imidazole for 50 mL.
  • the protein was eluted with buffer A with 250 mM imidazole.
  • the eluted fraction was diluted 1: 1 with Buffer A.
  • DHO dihydroorotate
  • DDAO dodecyldimethyl-N-amineoxide
  • Membranes were washed with TBST-T and incubated with HRP -linked secondary antibodies prepared at a 1 :2500 dilution in 5% nonfat dry milk in TBS-T. HRP was activated by incubating membranes with a mixture of SuperSignal Pico and SuperSignal Femto ECL reagents (100: 1 ratio). Exposure of autoradiography film was used for detection.

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Abstract

L'invention concerne, entre autres, des composés et des compositions qui modulent les voies de biosynthèse des nucléotides de pyrimidine, telles que la voie de sauvetage des nucléosides de pyrimidine et la voie de novo de pyrimidine, et leur utilisation dans le traitement du cancer.
PCT/US2020/033458 2019-05-16 2020-05-18 Modulateurs des voies de biosynthèse des nucléotides de pyrimidine Ceased WO2020232445A1 (fr)

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