[go: up one dir, main page]

WO2011041557A1 - Procédé de modulation de l'activité ire1 - Google Patents

Procédé de modulation de l'activité ire1 Download PDF

Info

Publication number
WO2011041557A1
WO2011041557A1 PCT/US2010/050926 US2010050926W WO2011041557A1 WO 2011041557 A1 WO2011041557 A1 WO 2011041557A1 US 2010050926 W US2010050926 W US 2010050926W WO 2011041557 A1 WO2011041557 A1 WO 2011041557A1
Authority
WO
WIPO (PCT)
Prior art keywords
irel
site
agent
ligand
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2010/050926
Other languages
English (en)
Inventor
David Ron
Frank Sicheri
Heather Harding
R. Luke Wiseman
Yuhong Zhang
Kenneth Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mt Sinai Hospital
New York University NYU
Original Assignee
Mt Sinai Hospital
New York University NYU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mt Sinai Hospital, New York University NYU filed Critical Mt Sinai Hospital
Publication of WO2011041557A1 publication Critical patent/WO2011041557A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • IREl is a type-I transmembrane endonuclease found in the endoplasmic reticulum (ER) (Lee et al. (2008), Structure of the dual enzyme IREl reveals the basis for catalysis and regulation in nonconventional RNA splicing, Cell 132: 89- 100).
  • the N-terminal luminal domain of IREl senses ER stress and transmits the signal to the cytosolic domain.
  • the cytoplasmic domain of IREl is characterized by distinct protein kinase and ribonuclease activities. The only known substrate of the protein kinase activity is IREl itself.
  • IREl In its resting state, IREl exists as a monomer (Lee et al.). It is believed that luminal stress initiates oligomerization of IREl which promotes (trans)- autophosphorylation of the activation loop of IREl (Lee et al.; Zhou et al. (2006). The crystal structure of human IREl luminal domain reveals a conserved
  • Each IREl promoter includes an N-terminal kinase domain and a ribonuclease domain formed by the C- terminal kinase extension.
  • the C-terminal kinase extension is termed the kinase- extension nuclease (KEN) domain (Lee et al. (2008); Ron et al. How IREl Reacts to ER Stress, Cell 132: 24-25).
  • KEN kinase- extension nuclease
  • the IREl endonuclease activity is based on recognition of an RNA stem loop structure found twice in substrates HAC1 mRNA in yeast or XBP1 mRNA in metazoans. Cleavage of HAC1 or XBP1 mRNA occurs at both sites resulting in an mRNA fragment whose two ends are ligated in a unique splicing event (Ron et al.; Lee et al.).
  • the spliced HAC1 or XBP1 mRNAs encode transcription factor that activate numerous target genes, including genes involved in the unfolded protein response (UPR).
  • the UPR is a signal transduction cascade that occurs in response to the accumulation of misfolded proteins in the ER.
  • the present invention is based on the surprising discovery that there exists a ligand binding site located at the dimer interface formed by adjacent kinase endonuclease (KEN) domains of the IREl dimer (referred to herein as the Q site).
  • KEN kinase endonuclease
  • Example 1 shows that the binding of a ligand to the binding site in the KEN domain potentiates activation of yeast IREl with ADP.
  • the present invention is directed to methods of modulating the IREl RNase activity comprising contacting said IREl with an agent that binds to the Q site or that inhibits the binding of a ligand to the Q site, wherein the Q site is a ligand binding site located at the dimer interface of the KEN domains of the IREl oligomer.
  • the invention is a method of increasing IREl RNase activity comprising contacting IREl with an agent that binds to the Q site of an IREl dimer, wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains, and wherein binding of the agent to the Q site enhances the RNase activity of IREl .
  • the invention is a method of activating the unfolded protein response (UPR) of a cell comprising administering to said cell an agent that binds to the Q site of an IREl dimer, wherein binding of the agent to the Q site enhances the RNase activity of IREl .
  • UTR unfolded protein response
  • the invention is a method of activating IREl RNase activity comprising contacting said IREl with a ligand of the Q site of an IREl oligomer, wherein said binding stabilizes the oligomer or promotes oligomerization.
  • the invention also encompasses a method of activating the unfolded protein response (UPR) of a cell comprising administering to said cell an agent that enhances the activity of natural ligand of the Q site, wherein binding of the natural ligand to the Q site enhances the RNase activity of IREl .
  • URR unfolded protein response
  • the present invention encompasses a method of inhibiting IREl RNase activity comprising contacting said IREl with an agent that inhibits the binding of a ligand to the Q site of an IREl oligomer, wherein inhibition of said binding suppresses RNase activity.
  • the invention is a method of inhibiting the unfolded protein response of a cell comprising contacting said cell with an agent that inhibits the binding of a ligand to the Q site of an IREl oligomer, wherein inhibition of said binding suppresses RNase activity and the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.
  • KEN kinase extension
  • the invention is directed to a method activating the unfolded protein response (UPR) in a patient in need thereof comprising
  • the invention is a method of treating a patient suffering from a condition associated with the accumulation of unfolded proteins in the endoplasmic reticulum, or a protein conformational disease comprising administering to said patient a therapeutically effective amount of an agent that binds to the Q site of an IREl oligomer and wherein said binding enhances the RNase activity of IREl, and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.
  • KEN kinase extension
  • the invention is a method of treating a condition mediated by plasma cells, cancer or an autoimmune disease in a patient in need thereof comprising administering a therapeutically effective amount of an agent that inhibits the binding of a ligand to the Q site of an IREl oligomer wherein said inhibition suppresses RNase activity and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.
  • an agent that inhibits the binding of a ligand to the Q site of an IREl oligomer wherein said inhibition suppresses RNase activity and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.
  • KEN kinase extension
  • the invention also encompasses an isolated or purified polypeptide-ligand complex comprising an IREl oligomer and an agent bound to the Q-site of the oligomer, wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.
  • KEN kinase extension
  • the invention is a method of identifying an agent that enhances RNase activity of IREl comprising screening one or more candidate agents for ability to bind to the Q site of IREl .
  • the method further comprises the RNase activity of IREl .
  • FIGs. 1A-E Small molecule screen for modulators of IREl RNAse activity.
  • a stem loop RNA substrate incorporating an IREl endoribonuclease site (cleavage between G3-C4, labeled bases) was modified 5' with AlexaFluor 647 (AF647) and 3' with BlackHoleQuencher3 (BHQ). Cleavage alleviates quenching allowing fluorescence.
  • FIG. IB Quantification of the ADP-mediated activation of IREl RNAse shown in FIG. IB.
  • the initial rate of IREl RNase activity was plotted against ADP concentration demonstrating an EC50 of ⁇ 40 ⁇ .
  • D Fluorescence time course reporting on cleavage of the IREl substrate as in FIG. 1A (black) or an altered substrate incorporating deoxy guano sine at position 3 (dG3; red), disrupting cleavage at that site.
  • the maximal signal following cleavage of both substrates was determined by incubation with R Ase A (squares).
  • FIGs. 2A-E Quercetin potentiates ADP-mediated IREl RNAse activity
  • FIGs. 3A-F The structure of IREl crystals bound by quercetin and ADP reveal a novel ligand binding site, the Q-site.
  • B The structure of IREl crystals bound by quercetin and ADP reveal a novel ligand binding site, the Q-site.
  • Electron density (blue-wire mesh) and tube representation of regions of the IREl :ADP: quercetin ternary complex that are unstructured in the IREl : ADP dimer (PDB- 2RIO) are shown in stereo view (two left panels) and superimposed on the structure of the same segments in PBD 3FBV.
  • Top - Amino acids 837-844 including phosphorylated residues S840, S841, and T844 in the activation segment of IREl are shown with side chains displayed in a ball and stick format.
  • Bottom - Amino acids 1036-1042 from the ⁇ 3' helix of the KEN domain are shown with side chain residues in ball and stick format.
  • C Stereo view of the two-fold symmetric quercetin binding pocket (Q-site). The solvent accessible surface of the KEN domain is shown in light blue and dark blue for the two protomers. Quercetin is shown in a ball-and-stick representation.
  • D A comparison of the Q-site from IRE1 :ADP: quercetin (blue) and IRE1 :ADP (PDB - 2RIO, green) shown in a stereo view and depicted as tubes. The residues that line this pocket are shown in ball-and-stick format (the quercetin ligand has been removed from the IRE 1 :ADP: quercetin structure).
  • E Structure of the Q- site with the residues from the two protomers (purple and green) that interact with quercetin (shown as ball-and-stick in the same view as FIG. 3D). An unbiased electron density for each quercetin molecule is shown as orange or gray wire mesh.
  • F Cartoon depicting the spatial arrangement and interactions of IRE 1 and quercetin that define quercetin binding to the Q-site.
  • FIGs. 4A-4G Mutation of residues lining the Q-site interfere with quercetin mediated activation of IRE 1 RNAse.
  • A Coomassie stained SDS-PAGE of wildtype and mutant yeast IREl (aa 658-1115) variants before and after incubation with lambda phosphatase.
  • B Timecourse of IREl RNase activity of IREIWT (black), IRE1S984E (blue), IRE1K985A (red), and IRE1K992L (green) incubated with ADP (A; 2 mM), quercetin (Q; 25 ⁇ ) or both. The concentration of each enzyme was adjusted to approximate the activity of IREIWT in the presence of ADP.
  • C Bar graph comparing IREl RNase activity of IREIWT, IRE1S984E, IRE1K985A,
  • IRE1K992L IRE1K992L
  • IRE IF 1112L measured as in FIG. 4B.
  • Tm 2.5 ⁇ g/mL; 4h
  • DTT 2 mM; 1 h
  • thapsigargin Tg; 0.5 ⁇ ; lh
  • the hylREl chimera migrates slower than endogenous IREl due to the larger size of the yeast kinase and endonuclease domains.
  • the anti-eIF2a immunoblot from the flow- through of the immunoprecipitation reaction serves as a loading control.
  • FIGs. 5A-F The hierarchy of IREl activation by flavonols supports a model of ligand binding in the syn orientation of the prime ring.
  • A Chemical structures of flavonol-based analogs of quercetin. Quercetin and other relevant flavonols are shown with the 3- and 3 '-moieties in the syn orientation; except morin, which is shown in the anti conformation due to clash between the 3- and 2'- hydroxyls.
  • B Timecourse of RNase activity of IREl (0.5 ⁇ ) incubated in the presence of various flavonols (25 ⁇ ). The activity of IREl in the absence of flavonol is indicated by the x symbols.
  • C Chemical structures of flavonol-based analogs of quercetin. Quercetin and other relevant flavonols are shown with the 3- and 3 '-moieties in the syn orientation; except morin, which is shown in the anti conformation due to clash between the 3- and 2'- hydroxyls.
  • FIGs. 6A-G Quercetin increases the population of IREl dimers.
  • E Coomassie stained SDS-PAGE of IREIWT and IRE1D723A incubated without or with lambda phosphatase.
  • F Fluorescence timecourse of RNase activity of IRE 1 WT (black) and IRE 1 D723A (red) in the presence of ADP (2 mM), quercetin (Q; 25 ⁇ ) or both.
  • G Coomassie stained SDS-PAGE of IREIWT and IRE1D723A (5 ⁇ ) incubated with ADP (2 mM), quercetin (25 ⁇ ) or both in the presence of 200 ⁇ DSS.
  • FIGs. 7A-D The nucleotide binding cleft and the quercetin binding pocket interact to regulate IREl RNAse.
  • IRE1D797A incubated with or without lambda phosphatase.
  • C Fluorescence timecourse of RNase activity of IREIWT (filled symbols) and IRE1D797A (open symbols) incubated with ADP (2 mM), quercetin (Q; 25 ⁇ ) or both.
  • D Fluorescence timecourse of RNase activity of IREIWT (filled symbols) and IRE1D797A (open symbols) incubated with ADP (2 mM), quercetin (Q; 25 ⁇ ) or both.
  • FIG. 8 Quercetin does not alter cleavage specificity of IREl .
  • FIG. 9 ADP potentiation of quercetin depends on the presence of divalent cations. Fluorescence timecourse of IREl RNase in the absence (filled symbols) and presence of EDTA (50 mM; open symbols) is shown. IREl (0.5 ⁇ ) was activated by incubation with ADP (2 mM), quercetin (30 ⁇ ) or the combination thereof, as indicated.
  • FIG. 10A and B Structural similarly of the IREl-ADP-quercetin ternary complex and the IRE1-ADP binary complex.
  • A. The IREl : ADP: quercetin ternary complex and the IREl :ADP binary complex (PDB ID 2RIO) are superimposed using a secondary structure matching algorithm (Krissinel, E.,and Henrick, K. (2004). Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr 60, 2256- 2268.) and depicted as blue and green tubes. Protein structural features that are now visible in the ternary complex and absent from the binary complex are colored in pink. B.
  • SSM Secondary-structure matching
  • ADP molecules bound in the nucleotide binding clefts are shown in ball-and-stick representation with the same color scheme as the protein.
  • Magnesium and strontium ions coordinated by the ADP phosphates are shown as blue and pink spheres.
  • Unbiased mFo-DFc electron density in the region close to the ADP and the liganded metal ions are shown as a blue wire-mesh.
  • FIG. 11 Homology modeling of a potential quercetin-binding pocket in human Irel .
  • the solvent accessible surface of the KEN domain is shown on the left and right for the two protomers.
  • the quercetin pair is shown in a ball and- stick representation, respectively. Residues lining the putative quercetin binding pocket are shown in ball-and-stick
  • quercetin can be modeled into this novel binding pocket of human IREl without steric clashes.
  • Ser984 a residue that coordinates quercetin in the IREl :ADP: quercetin crystal structure, is Glu836 in human IREla - a substitution that strongly attenuates quercetin responsiveness of yeast IREl .
  • the present invention is directed to compositions and methods for modulating the ribonuclease (RNase) activity of IREl, methods of modulating the unfolded protein response (UPR) of a cell, methods of treatment and methods of identifying agents that modulate RNase activity of IREl .
  • RNase ribonuclease
  • UPR unfolded protein response
  • IREl is an ER transmembrane endoribonuclease and kinase. As discussed above, the active form of IREl is a dimer and activation can be initiated by autophosphorylation. In Saccharomyces (S.) cerevisiae, the UPR is controlled by IRElp. Mammals express two homologues of the S. Cerevisiae IRElp, IRE la and IREi . IREla is expressed in all cells and tissues whereas IREl ⁇ is primarily found in intestinal epithelial cells. As used herein, the term "IREl" encompasses any IREl, including those of S. Cerevesiae and mammalian IREl, unless otherwise indicated.
  • the IREl of S. Cerevisiae is also referred to herein as "yeast IREl .”
  • the structure of IREl including the conserved dimerization domain has been discussed, for example, in Lee et al. (2008), Structure of the dual enzyme Irel reveals the basis for catalysis and regulation in nonconventional RNA splicing, Cell 132: 89-100, Zhou et al. (2006), The crystal structure of human IREl luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response, PNAS 103(39): 14343-14348 and Korennykh et al.
  • IREl oligomer encompasses oligomers comprising two or more
  • IREl proteins The term "IREl oligomer” expressly encompasses IREl dimers.
  • Q site refers to the ligand binding site located at the kinase extension nuclease (KEN) domain of the IREl dimer.
  • the Q site is located at the dimer interface of adjacent KEN domains of the IREl oligomer.
  • IREl also possesses a nucleotide binding cleft which is located at the kinase domain. It is to be understood that the Q site is distinct from the nucleotide binding site located at the kinase domain.
  • Q site encompasses the ligand binding site in the KEN domain of yeast IREl as well as the ligand binding site in homologous IREl proteins from species other than yeast.
  • a "Q site activator” is an agent that binds to the Q site of an IREl oligomer or an agent that increases the biological activity of a natural ligand of the Q site, wherein the binding of the agent or natural ligand to the Q site enhances the RNase activity of IREl .
  • An agent that enhances the RNase activity of IREl after binding to the Q site can be the natural ligand or an agent other than the natural ligand that is capable of binding to the Q site and enhancing RNase activity of IREl .
  • An IREl Q site activator that increases the activity of the natural ligand of the Q site is capable of increasing the binding of the natural ligand to the Q site relative to said binding in the absence of the Q site activator.
  • the activity of the natural ligand can be increased, for example, by increasing transcription of the natural ligand, decreasing degradation of the natural ligand and/or increasing the affinity of the natural ligand for the Q site.
  • the Q site activator is an agent that binds to the Q site of an IREl oligomer.
  • a "Q site inhibitor” is an agent that inhibits the RNase activity of IREl by decreasing the biological activity of a natural ligand of the Q site.
  • the activity of the natural ligand of the Q site is decreased, for example, when the binding of the natural ligand to the Q site is decreased relative to said binding in the absence of the Q site inhibitor.
  • the activity of the natural ligand can be decreased, for example, by decreasing transcription of the natural ligand, increasing degradation of the natural ligand, and/or inhibiting the binding of the natural ligand to the Q site.
  • the Q site inhibitor is a competitive or noncompetitive antagonist of the natural ligand.
  • ligand of the Q site encompass an agent that is capable of binding to the IREl Q site.
  • a natural ligand of the Q site is an endogenous ligand that binds to the IREl Q site in a living organism.
  • agent encompasses a chemical compound, a mixture of chemical compounds, a biological macromolecule (including, for example, a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues.
  • contacting when used in the context of IREl and a ligand of IREl refers to bringing IREl and its ligand in contact under conditions suitable for binding of the ligand to IREl .
  • the terms “inhibiting” or “decreasing” encompasses causing a net decrease by either direct or indirect means.
  • enhancing means to cause a net gain by either direct or indirect means.
  • the binding of a ligand to the Q site of an IREl oligomer forms a complex comprising an IREl oligomer and a ligand bound to the Q-site of the oligomer.
  • the complex comprises an IREl Q site activator.
  • the complex comprises two molecules of an IREl Q site activator bound to the Q site.
  • the invention is directed to a method of activating the ribonuclease (RNase) activity of IREl .
  • the RNase activity of IREl can be activated by administering an IREl Q site activator.
  • the IREl Q site activator binds to the Q site of an IREl oligomer, wherein said binding enhances the RNase activity of IREl .
  • the IREl Q site activator increases the activity of a natural ligand of the Q site of an IRE oligomer.
  • the IREl Q site activator is an agent that binds to the Q site and is a flavonoid.
  • Flavonoids include, for example, flavonols, flavones, flavanones, anthocyanidins, and isoflavones.
  • flavonoids encompasses naturally occurring flavonoids as well as synthetic flavonoids.
  • the agent that binds to the Q site is a flavonol or a flavone. Flavones are characterized by a backbone of 2-phenyl-l-benzopyran-4-one. Exemplary flavones are apigenin and luteolin.
  • Flavonols are characterized by a 3- hydroxyflavone (3-hydroxy-2-phenylchromen-4-one) backbone.
  • Exemplary flavonols are quercetin, rutin (a glycosylated form of quercetin), kaempferol, myricetin, and isorhamnetin.
  • Exemplary IREl Q site activators also include flavones or flavonols characterized by a 5-hydroxyl moiety on the flavone or 3 -hydroxyflavone backbone, respectively.
  • flavonols that posses a 5-hydroxyl moiety on the 3 -hydroxyflavone core are quercetin, kaempferol, isorhamnetin, luteolin, galangin, morin and taxifolin.
  • a non-limiting example of a flavone that possesses a 5-hydroxyl moiety on its flavone core is apigenin.
  • Exemplary IRE1 Q site activators also include flavones or flavonols characterized by a 4 '-substitution on the flavones or 3 -hydroxy flavone backbone, respectively.
  • the 4 '-substitution is a moiety capable of hydrogen bonding with an amino acid residue of the Q site of IRE1.
  • An example of a moiety capable of hydrogen bonding is a hydroxyl moiety.
  • Non- limiting example of flavonols that posses a 4 '-hydroxyl moiety on the 3 -hydroxy flavone backbone are quercetin, kaempferol, isorhamnetin, fisetin, morin and taxfolin.
  • a non-limiting example of a flavone that possesses a 4 '-hydroxyl moiety on its flavones core is apigenin.
  • the IRE1 Q site activator is a flavonol. In another aspect of the invention, the IRE1 Q site activator is quercetin.
  • the Q site is made up of amino acid residues from IRE1 promoters of the IRE1 oligomer.
  • the agent that binds to the Q site can interact with one or more of the amino acid residues of the Q site. Examples of interactions between an agent and amino acid residue include hydrophobic interactions and hydrogen bonding interactions.
  • the Q site of the yeast IRE1 oligomer includes the amino acid residues serine 984 (S984), lysine 985 (K985), glutamine 988 (E988), lysine 992 (K992), proline 1077 (P1077), leucine 1108 (LI 108) and phenylalanine 1112 (Fi l l 2).
  • a Q site ligand can interact with one or more amino acid residues selected from the group consisting of S984, K985, E988, K992, P1077, LI 108 and Fl 112 of yeast IRE1, or corresponding or conserved amino acid residue in a homolog of yeast IRE1 (also referred to herein as a "homologous residue").
  • An amino acid residue that corresponds to an amino acid residue of yeast IRE1 refers to an amino acid residue of a protein homologous to yeast IREl (for example, a mammalian IREl) that corresponds to the specific residue in yeast (for example, K985 in yeast IREl corresponds to K837 in human IREla).
  • a conserved amino acid or a conserved residue refers to an amino acid residue which is found to be common between two proteins and/or occupies a particular position with a peptide motif.
  • the two different proteins can be homologous proteins derived from different species, e.g., yeast and human. It will be apparent to those skilled in the art that the numbering or position of amino acids in proteins homologous to yeast IREl (such as an in a mammalian IREl) can be different from that in yeast IREl .
  • Corresponding and/or conserved amino acids in homo logs of yeast IREl can be identified by comparison of the amino acid sequences, for example using commercially available homology modeling software packages or conventional sequence alignment packages.
  • the method of the present invention can be used to modulate the UPR of a cell.
  • the invention is directed to a method of enhancing the UPR of a cell comprising administering to said cell a Q site activator.
  • the invention is directed to a method of inhibiting the UPR of a cell comprising administering to said cell a Q site inhibitor.
  • the invention is directed to a method of enhancing the UPR of a cell in a patient in need thereof comprising administering to said patient a Q site activator in a therapeutically effective amount.
  • the invention is directed to a method of inhibiting the UPR of a cell in a patient in need thereof comprising administering to said patient a Q site inhibitor in a therapeutically effective amount.
  • the invention also encompasses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a Q site activator.
  • the invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a Q site inhibitor.
  • the invention is directed to a method of treating a condition associated with the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and/or a protein conformation disease or condition comprising administering to said patient a and a therapeutically effective amount of a Q site activator.
  • the Q site activator can be part of a pharmaceutical composition.
  • Exemplary proteins that can accumulate in the ER or that are misfolded or unfolded include glucocerebrosidase, hexosamine A, cystic fibrosis transmembrane conductance regulator, aspartylglucsaminidase, a-galactosidase A, cysteine transporter, acid ceremidase, acid a-L-fucosidase, protective protein, cathepsin A, acid ⁇ -glucosidase, acid ⁇ -galactosidase, iduronate 2-sulfatase, a-L-iduronidase, galactocerebrosidase, acid a -mannosidase, acid ⁇ -mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid ⁇ -galactosidas
  • Conditions associated with the accumulation of unfolded proteins include, but are not limited to, alpha- 1 antitrypsin deficiency, cystic fibrosis, autoimmune diseases or disorders.
  • Protein conformational diseases encompass gain of function disorders and loss of function disorders.
  • the protein conformational disease is a gain of function disorder.
  • the terms "gain of function disorder,” “gain of function disease,” “gain of toxic function disorder” and “gain of toxic function disease” are used interchangeably herein.
  • a gain of function disorder is a disease characterized by increased aggregation-associated proteotoxicity. In these diseases, aggregation exceeds clearance inside and/or outside of the cell.
  • Gain of function diseases include, but are not limited to neurodegenerative diseases associated with aggregation of polyglutamine, Lewy body diseases, amyotrophic lateral sclerosis, transthyretin-associated aggregation diseases, Alzheimer's disease and prion diseases.
  • Neurodegenerative diseases associated with aggregation of polyglutamine include, but are not limited to, Huntington's disease, dentatorubral and
  • Alzheimer's disease is characterized by the formation of two types of aggregates: extracellular aggregates of ⁇ peptide and intracellular aggregates of the microtubule associated protein tau.
  • Transthyretin-associated aggregation diseases include, for example, senile systemic amyloidoses and familial amyloidotic neuropathy.
  • Lewy body diseases are characterized by an aggregation of a-synuclein protein and include, for example, Parkinson's disease.
  • Prion diseases also known as transmissible spongiform encephalopathies or TSEs
  • the protein conformation disease is a loss of function disorder.
  • Loss of function diseases are a group of diseases characterized by inefficient folding of a protein resulting in excessive degradation of the protein. Loss of function diseases include, for example, cystic fibrosis and lysosomal storage diseases.
  • cystic fibrosis the mutated or defective enzyme is the cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • AF508 is a deletion ( ⁇ ) of three nucleotides resulting in a loss of the amino acid phenylalanine (F) at the 508th (508) position on the protein.
  • Lysosomal storage diseases are a group of diseases characterized by a specific lysosomal enzyme deficiency which may occur in a variety of tissues, resulting in the build-up of molecules normally degraded by the deficient enzyme.
  • Lysosomal enzyme deficiency can be in a lysosomal hydrolase or a protein involved in the lysosomal trafficking.
  • Lysosomal storage diseases include, but are not limited to, aspartylglucosaminuria, Fabry's disease, Batten disease, Cystinosis, Farber, Fucosidosis, Galactasidosialidosis, Gaucher' s disease (including Types 1, 2 and 3), Gml gangliosidosis, Hunter's disease, Hurler- Scheie's disease, Krabbe's disease, a-Mannosidosis, B-Mannosidosis, Maroteaux- Lamy's disease, Metachromatic Leukodystrophy, Morquio A syndrome, Morquio B syndrome, Mucolipidosis II, Mucolipidosis III, Neimann-Pick Disease (including Types A, B and C), Pompe's disease, Sandhoff disease, Sanfilippo syndrome (including Types A, B, C and D),
  • IRE1 is involved in processing of XBP-1.
  • XBP-1 has been described as playing a role in plasma cell development (see, for example, U.S. Patent Publication No. 200302244228, the contents of which are expressly incorporated by reference herein).
  • the invention therefore encompasses methods of treating a condition mediated by plasma cells comprising administering a Q site inhibitor.
  • Conditions mediated by plasma cells include, but are not limited to, myasthenia gravis, pemphigus vulgaris, systemic lupus erythromatosus, Guillain Barre syndrome, proliferative glomerulonephritis, hemophilia with inhibitory antibodies to factor VIII, hemophilia with inhibitory antibodies to factor IX, autoimmune thrombocytopenia, autoimmune hemolytic anemia and paraneoplastic syndrome.
  • the invention also encompasses method of treating a cancer mediated by abnormal proliferation of plasma cells including, for example, multiple myeloma and plasma cell dyscrasia.
  • the invention is directed to a method of treating an autoimmune disease in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a Q site inhibitor.
  • autoimmune diseases include, systemic lupus erythematosus, rheumatoid arthritis, Goodpasture's syndrome, Grave's disease, Hashimoto's thyroiditis, pemphigus vulgaris, myasthenia gravis, scleroderma, muscular dystrophy, autoimmune hemoloytic anemia, autoimmune thrombocytopenic purpura, polymyositis, dermatomyositiis, pernicious anemia, Sjogren's syndrome, ankylosing spondylitis, vasculitis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease and type I diabetes mellitus.
  • the invention is directed to a method of treating cancer or a tumor in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a Q site inhibitor.
  • cancer that can be treated according to the present invention are breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, basal cell carcinoma, neuroblastoma, hematologic cancer, rhabdomyosarcoma, liver cancer, skin cancer, leukemia, basal cell carcinoma, bladder cancer, endometrial cancer, glioma, lymphoma, and gastrointestinal cancer.
  • the invention is a method of treating cancer or a tumor comprising administering a therapeutically effective amount of a Q site inhibitor in combination with the administration of a chemotherapeutic agent.
  • Chemotherapeutic agents that can be utilized in combination with a Q site inhibitor include, but are not limited to, alkylating agents such as cyclosphosphamide
  • CYTOXAN® alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,
  • trietylenephosphoramide triethylenethiophosphaoramide and trimethylolomelamine
  • nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard
  • nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine
  • antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo
  • defofamine demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium;
  • TAXOL® Bristol-Myers Squibb Oncology, Princeton, N.J.
  • docetaxel TAXOTERE®; Aventis Antony, France
  • chlorambucil gemcitabine
  • 6- thioguanine mercaptopurine
  • methotrexate platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • platinum etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide;
  • daunomycin aminopterin
  • xeloda ibandronate
  • CPT-11 topoisomerase inhibitor
  • RFS 2000 difluoromethylornithine
  • DMFO difluoromethylornithine
  • retinoic acid esperamicins
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the chemotherapeutic agents above.
  • the invention is a method of treating cancer or a tumor comprising administering a therapeutically effective amount of a Q site inhibitor in combination with radiation therapy.
  • Treating” or “treatment” includes the administration of the compositions, compounds or agents of aspects of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • a “therapeutically effective amount” is an amount which, alone or in combination with one or more other active agents, can control, decrease, inhibit, ameliorate, prevent or otherwise affect one or more symptoms of a disease, disorder or condition to be treated.
  • compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluents are selected so as not to affect the biological activity of the pharmacologic agent or composition. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SEPHAROSETM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SEPHAROSETM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • compositions for parenteral administration, pharmaceutical compositions or
  • pharmacologic agents can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • Injectable formulations can be prepared either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can also be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997.
  • the compositions and pharmacologic agents described herein can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1%>- to about 2%>.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Topical application can result in transdermal or intradermal delivery.
  • Transdermal delivery can be achieved using a skin patch or using transferosomes.
  • a skin patch or using transferosomes.
  • the binding of a ligand to the Q site of an IRE1 oligomer forms a complex comprising an IRE1 oligomer and a Q site ligand.
  • Some embodiments of the invention are directed to an isolated or purified polypeptide- ligand complex comprising an IRE1 oligomer and an agent bound to the Q-site of the oligomer.
  • the IRE1 oligomer can, for example, be an IRE1 dimer.
  • the complex comprises an IRE1 oligomer and a Q site activator.
  • the complex comprising an IRE1 oligomer a Q site inhibitor.
  • the IRE1 oligomer of the complex can, for example, an oligomer of yeast IREl .
  • the IREl oligomer can also comprise an oligomer of a mammalian IREl .
  • An isolated complex is a complex separated from the components that naturally accompany it.
  • the complex present in the organism is not isolated, but the same complex, separated or modified by human intervention from some or all of the components that naturally accompany is isolated.
  • a purified complex is a complex has been isolated under conditions that reduce or eliminate other materials or contaminants, such as materials which naturally accompany the complex in nature.
  • the present invention also encompasses a method of identifying agents that modulate RNase activity of IREl comprising screening a candidate agent for the ability to bind to the IREl Q site. In some aspects, the method also comprises the step of measuring RNase activity of IREl .
  • candidate agent encompasses agents with known chemical structure or agents with unidentified structures.
  • candidate agent also encompasses mixtures of agents, for example, extracts from biological samples. Multiple candidate agents can be screened as part of libraries or collections of agents.
  • IREl and/or or the candidate agent can be labeled (for example, radioisotopic, fluorescent and enzymatic labels) such that the binding of a ligand to IREl can identifying by detecting the labeled IREl and/or candidate agent in the complex of IREl oligomer and ligand.
  • the binding of a candidate agent to the Q site of IREl can also be detected using a competitive binding assay.
  • the ability of candidate agent to compete with a known ligand of the Q site of IREl is measured.
  • the ability of the candidate agent to compete with a known ligand of the Q site can be measured by labeling the known ligand.
  • the label can be a radioisotopic or enzymatic label as described above.
  • radioisotopic labels include I, S, C, P, or H, either directly or indirectly, and the radioisotope detected by direct counting of
  • Enzymatic labels include, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • IREl possess a nucleotide binding cleft on the kinase domain.
  • the assay can be conducted in the presence of a ligand of the nucleotide binding cleft of IREl .
  • An example of a ligand of the nucleotide binding cleft is ADP.
  • the screening assay is a cell-based assay. In other embodiments, the screening assay is a cell- free assay.
  • the IREl can be contacted with a candidate agent and the ability of the candidate agent to bind to the IREl Q site is determined.
  • the IREl can be provided as a lysate of cells that express IREl, as a purified or semipurified polypeptide, or as a recombinantly expressed polypeptide.
  • a cell-free assay system further comprises a cell extract or isolated components of a cell. Determining the ability of the candidate agent to modulate the RNase activity of IREl can also be
  • BIOA surface plasmon resonance
  • the binding of a candidate agent to the Q site can also be determined by detecting the formation of a complex between the IREl oligomer and the candidate agent.
  • the complex can be detected using methods well-known in the art including crystallography and/or detection of a label present in the complex.
  • RNase activity can be measured by detecting cleavage of an RNA substrate comprising an IREl recognition loop. Methods of measuring RNase activity can be determined using methods known in the literature and/or using commercial assays. RNase activity of IREl can, for example, be measured as exemplified in Example 1 below.
  • XBP1 mRNA is an example of an RNA that possesses an IREl recognition loop.
  • the RNA substrate can also comprise a nucleotide sequence that dissociates upon cleavage. In some aspects, the RNA substrate is labeled.
  • the RNA substrate comprises a fluorescent label and a moiety that quenches the fluorescent label, wherein cleavage of the substrate removes the quenching moiety and cleavage is detected by detecting fluorescence.
  • An exemplary moiety that quenches the fluorescent label is Black- Hole Quencher 3 (BHQ).
  • the endoplasmic reticulum (ER) unfolded protein response (UPR) couples perturbation of the protein- folding environment in the ER lumen to rectifying changes in transcription and translation (Bernales et al., 2006) (Ron and Walter, 2007).
  • the oldest and most conserved branch of the UPR is initiated by IREl, an ER localized type-I transmembrane protein whose luminal domain senses ER stress and transmits the signal to the cytosolic effector domain (Cox et al., 1993; Mori et al, 1993; Tirasophon et al, 1998; Wang et al, 1998).
  • IREl recognizes an RNA stem loop structure, found twice in its substrates.
  • HAC1/XBP1 mRNA cleavage occurs at both sites, releasing an mRNA fragment and initiating an unconventional splicing event that is completed when the two ends of the mRNA are ligated.
  • the spliced HAC1/XBP1 mRNAs encodes a transcription factor that activates numerous UPR target genes.
  • the crystal structure of the active form of yeast IRE1 provides further clues to the coupling of kinase and nuclease activity:
  • the cytosolic portion of IRE 1 is a single folded domain that forms an extensive back-to-back dimer.
  • the phosphorylated activation loop of the kinase portion is partially disordered and ADP is bound in the nucleotide-binding cleft (Lee et al, 2008b; Korennykh et al, 2009).
  • these observations support a model whereby a luminal stress signal initiates oligomerization in the plane of the membrane promoting (trans)-autophosphorylation of IREl 's activation loop.
  • the resulting conformational change favors nucleotide binding, which, in turn, stabilizes the back-to-back dimer.
  • the dimer interface extends through the length of IRE 1, juxtaposing the Kinase Extension Nuclease (KEN) domain located at the C-terminus of each IREl protomer, which thereby attain an active conformation for sequence- specific RNA cleavage.
  • KEN Kinase Extension Nuclease
  • IREl activity is fuelled in part by evidence that chronic ER stress (and aspects of the cellular response to it) contribute to the time and use-dependent attrition of tissues with high secretory capacity, for example, the insulin-producing beta cells of the pancreas (Kaufman, 2002; Powers et al, 2009). Furthermore, in addition to promoting the first step in XBP1 splicing,
  • phosphorylated mammalian IREl is able to activate Jun N-terminal kinase, by recruiting the scaffold protein TRAF2 (Urano et al., 2000). This latter branch of the UPR is believed to contribute to some of the deleterious consequences of ER stress, such as insulin resistance (Ozcan et al, 2004). Therefore, ligands that drive a wedge between IREl kinase and RNase activity would be of special interest.
  • RESULTS the surprising discovery of a novel ligand-binding pocket at the dimer interface of IREl that can be targeted to modulate IREl 's RNAse.
  • RNA substrate was prepared incorporating the IREl recognition loop from XBP1 and a destabilized stem that dissociates upon cleavage.
  • the RNA probe was modified at the 5' and 3' positions with AlexaFluor647 (AF647) and Black-Hole Quencher 3 (BHQ), respectively, resulting in a quenched IREl substrate. Cleavage relieves the BHQ-dependent quenching of AF647 promoting allowing fluorescence. (FIG. 1A).
  • yeast IREl kinase-endonuclease domain When purified from bacteria, yeast IREl kinase-endonuclease domain
  • IREl possesses a known ligand-binding cleft that engages nucleotide.
  • Quercetin activates IREl through a site distinct from the nucleotide -binding cleft:
  • Quercetin is relatively insoluble in aqueous buffers, but at concentrations attainable (30 ⁇ ) it promoted ⁇ 10 fold more RNase activity than saturating concentrations of ADP (2 mM; FIG. 2A). Quercetin does not alter the specificity of IREl 's RNAse, as reflected by the cleavage of 32P-labeled RNA substrate of identical sequence to the fluorescent reporter (FIG. 2B). Similarly, yquercetin- activated IREl did not cleave the fluorescent substrate lacking the 2 '-hydroxyl of G3 in the putative IREl cleavage site (FIG. 8).
  • Quercetin is known to engage the nucleotide-binding site of diverse kinases
  • Quercetin potentiation of ADP-mediated RNase activity was also recapitulated by measuring the cleavage of a 32 P- radiolabeled RNA stem-loop substrate (FIG. 2D). Furthermore, chelating divalent cations by adding EDTA strongly attenuated ADP's effect, indicating that potentiation is observed when ADP engages the nucleotide-binding cleft in a conventional mode (i.e. complexed with Mg+2; FIG. 9).
  • a co-crystal structure of IREl, ADP and quercetin defines a novel ligand-binding pocket:
  • IREl ADP: quercetin crystals diffracted at 3.4 A resolution (Supplemental Table 1) revealing a protein dimer defined by two discontinuous contact surfaces formed by the kinase domain and KEN domain (FIG. 3A).
  • the structure of IREl in the IREl :ADP: quercetin complex demonstrated a trilobal architecture nearly identical to that of the previously reported IRE1 :ADP complex (FIG. 10A) with two notable exceptions: Two regions that are disordered in the IRE1 :ADP binary complex (PDB: 2RIO) are now visible as ordered structural elements in the
  • the IRE1 :ADP:quercetin complex also demonstrated a unique density at the dimeric interface of the KEN domains attributed to two quercetin molecules (FIGs. 3A and 3C). We refer to this deep solvent exposed symmetric binding pocket as the Q-site.
  • the electron density for quercetin in the Q-site is highly asymmetric and we were able to unambiguously dock the larger bicyclic benzopyran ring and smaller monocyclic phenyl ring of quercetin into the wider and the narrower part of the quercetin electron density, respectively (FIG. 3C).
  • the similarity at the Q site between the structures of IRE 1 : ADP: Quercetin and IREl :ADP suggests that this site is a preformed pocket generated through dimerization.
  • the Q-site is lined by residues S984, K985, E988, K992, P1077, II 108 and Fl 112, with the quercetin pair interacting with residues from both IREl protomers (FIG. 3E), and is predicted to be largely conserved in mammalian IREl (FIG. 11).
  • the primary contacts that dictate quercetin binding are mediated by 1) pi-pi stacking between Fl 112 and the bicyclic benzopyran ring of quercetin; 2) a hydrophobic interaction between K985 and the monocylic quercetin ring (which is rotated 30 degrees relative to the benzopyran ring); and 3) hydrogen bonds between the 4'- hydroxyl of quercetin and the S984 sidechain.
  • Quercetin also engages the other protomer in the IREl dimer through formation of a hydrogen bond between the 7- hydroxyl and the carboxylate moiety of E988, stabilizing the dimeric interface between the two protomers (FIG. 3E).
  • the two quercetin molecules in the Q-site are symmetrically arranged about the two-fold axis of the IREl dimer bridging and strengthening the interaction between the two IREl protomers through propagation of the pi-pi stacking in the pocket mediated by the interaction between the monocyclic rings across the KEN domain dimmer interface (FIG. 3F).
  • Quercetin' s pleiotropic effects in mammalian cells which include induction of ER stress (Ito et al, 1999) and toxicity, obscure the analysis of possible interactions with endogenous mammalian IREl in vivo, whereas inertness in yeast, suggesting poor biovailability, precluded analysis in that species (data not shown).
  • IREl knockout cells Calfon et al., 2002
  • a chimeric IREl construct consisting of residues 1- 549 of human IRE la encoding the lumenal, transmembrane and juxta-membrane domains) and residues 658-1115 of yeast IREl (the kinase and endonuclease domains) (hylREl; FIG. 4D).
  • the chimeric protein failed to splice endogenous XBP1 in response to ER stress (induced by exposing cells to thapsigargin, tunicamycin or DTT, FIG. 4E).
  • quercetin was the most potent activator of IRE 1 RNAse in vitro, both in the absence (FIG. 5B) and presence of saturating
  • the weak potentiation with ADP of isorhamnetin and morin may be attributed to destabilization of the syn- orientation of the 3- and 3'-hydroxyl moieties of quercetin by exposure of a hydrophobic methoxy moiety to the solvent (in the case of isorhamnetin) or a steric clash between the 2'-hydroxyl and the 3-hydroxyl (in the case of morin).
  • the reduced activity of luteolin and apigenin (derivatives of quercetin and kampferol, respectively, lacking the 3-hydroxyl) also supports the syn-orientation for quercetin in the Q-site by demonstrating the importance of the 3-hydroxyl in IREl activation, which likely defines the angular rotation of the two ring systems of quercetin.
  • the importance of planarity in the bicyclic benzopyran ring explains taxifolin's inactivity (lacking the 2-3 double bond).
  • the importance of the 5- and 4'-hydroxyls of quercetin is supported by the inability of fisetin and galangin to potentiate ADP activation, respectively. While the involvement of the 4'-hydroxyl can be
  • Luteolin and galangin were strong activators of XBP1 splicing in IREl knockout cells expressing hylREl (FIG. 5E).
  • the potent activation of XBP1 splicing by luteolin was dependent on binding the novel Q-site, as splicing was strongly attenuated in cells expressing Q-site mutant hylREl chimeras (FIG. 5F).
  • the differences in the relative activity of flavonols in vitro and in vivo may reflect biotransformation, differences in bioavailability or subtle alteration of protein structure in the Q-site of the full-length chimeric protein compared to the bacterially-expressed enzyme used in vitro. Quercetin increases the population of IREl dimers in vitro:
  • IREl oligomers corresponding well with enzymatic activity (FIG. 2A).
  • Significant populations of larger species were not identified by AUC, indicating that in the conditions used here, IREl oligomers consisted primarily of dimers.
  • the observed differences in the sedimentation coefficient of IREl oligomers in the presence of ADP, quercetin or both is consistent with the formation of a more stable dimer by the ternary complex that undergoes less rapid equilibration during sedimentation with less mass- averaging effects (Brown et al, 2008).
  • IRE1D723A was purified from bacteria as a phosphorylated protein in a yield similar to that of wildtype IREl (FIG. 6E), demonstrating that this mutant does not disrupt IREl 's autokinase activity and is likely a well folded protein. Neither ADP, nor quercetin nor a combination thereof activated
  • Quercetin-binding at its novel site modifies the nucleotide binding cleft:
  • dephosphorylation with lambda phosphatase markedly diminished ADPmediated RNase activity.
  • the dephosphorylated IREl remained strongly responsive to quercetin (FIG. 7A).
  • dephosphorylation with lambda phosphatase was incomplete (FIG. 7B), we sought to explore the relationship between quercetin-mediated activation and phosphorylation further with a mutation that abolishes kinase activity without (directly) altering the structure of the nucleotide binding cleft.
  • D797A alters a residue essential to deprotonation of the kinase substrate and abolishes all measureable kinase activity (Lee et al., 2008b).
  • bacterially expressed IRE1D797A exhibits enhanced mobility on SDS- PAGE that is unaltered by treatment with lambda phosphatase (FIG. 7B), consistent with an unphosphorylated form of the enzyme.
  • IRE1D797A is unable to bind ADP (Lee et al, 2008b), remarkably, addition of ADP attenuated the quercetin-mediated activation of IRE1D797A (FIG. 7D), suggesting that both accessibility and functionality of the nucleotide binding cleft of the unphosphorylated IRE1D797A had been altered by binding of ligand to the novel Q- site.
  • the crystal structure of IREl with ADP and quercetin reveals two ligand binding sites occupied by different ligands: ADP in the nucleotide binding cleft of the kinase and a pair of quercetin molecules nestled at the back-to-back dimer interface of the KEN domain.
  • the two quercetin molecules interact broadly with one another and make numerous symmetrical contacts with residues from both IREl protomers.
  • Luminal signals initiated by dissociation of an inhibitory ligand (Bertolotti et al, 2000) (Liu et al, 2000) or by direct binding of misfolded proteins to IREl 's luminal domain (Credle et al., 2005) promote interaction between IRE1 protomers in the plane of the ER membrane.
  • the resulting transautophosphorylation enhances affinity for nucleotide (likely ADP) (Sidrauski and Walter, 1997; Papa et al, 2003) whose engagement at the nucleotide binding cleft promotes conformational change(s) that favor back-to-back dimerization of the cytoplasmic effector domain (Lee et al., 2008b).
  • the dimer may be further incorporated into higher order structures that favor cooperativity (Aragon et al., 2009; Korennykh et al, 2009).
  • quercetin activates IREl by promoting the penultimate step (dimerization) in the cascade described above.
  • IREl ADP: quercetin crystals is nearly identical with notable further structuring in both the kinase activation loop and the RNA binding site in the IREl : ADP: quercetin structure.
  • the structure of these two regions in the ternary complex is similar to that observed previously in the large oligomer of IREl (Korennykh et al., 2009), suggesting that quercetin binding mimics the impact of oligomerization and potentially alters enzymatic activity.
  • IRE1D797A which lacks detectable autokinase activity and is thus refractory to activation by ADP (Lee et al., 2008b), is nonetheless activated by quercetin.
  • this pocket may engage endogenous ligand(s) and that perhaps quercetin itself or related flavonols, which are produced in plants may represent such ligands for plant IRE Is.
  • the hydrophobic nature of the Q-site may provide an important pathway-specific feedback component to the known links between phospholipid metabolism and yeast IREl (Nikawa and Yamashita, 1992) (Cox et al., 1997).
  • mammalian IREl also possess a pocket analogous to the Q-site that may be accessed by quercetin-like compounds (FIG. 11).
  • spliced XBP1 which would be enhanced in response to IREl RNase activators, could drive a wedge between potentially salubrious signaling from IREl to XBP1 and potentially damaging IREl effector functions linked to its kinase activity (e.g. TNK activation), favoring survival and function of ER stressed cells.
  • drugs that access the novel site and inhibit RNase activity could be used to neuter the IREl branch of the UPR with beneficial effects in fighting cancers, like multiple myeloma, which rely on that pathway for their survival.
  • Quercetin, ADP, and flavonols were purchased from Sigma-Aldrich. The initial small molecule screen was performed with the Kinase Inhibitor Library (BioMol International). RNA probes were purchased from Invitrogen either with or without modifications on the 5' (AlexaFluor647) or 3' (Black Hole Quencher 3). IREl (658-1115) was cloned into the pGEX.Smt3 vector (derived from H6.Smt3, a kind gift of Chris Lima). All mutations in IREl were prepared by site-directed mutagenesis and sequenced to confirm incorporation of the appropriate mutation.
  • BL21 E. coli expressing GST. Smt3. IREl (658-1115) were induced with 1 mM IPTG and grown overnight at 18 deg C. Bacterial lysates were prepared by disruption in an EmulsiFlex- C3 (Avestin, Inc.) and cleared by centrifugation.
  • GST.Smt3.IREl (658-1115) was purified on a GSTrap 4B (GE Healthcare) affinity column and eluted with 40 mM glutathione (Sigma). The protein was further purified on a Mono Q (GE Healthcare) anion exchange column using an Akta FPLC (GE Healthcare). IREl (658-1115) was cleaved from GST.Smt3 by incubation with 2 ⁇ g of Ulpl per mg of IREl (658-1115) overnight at 4 deg C.
  • the cleaved protein was gel filtered into 20 mM Hepes pH 7.5, 200 mM NaCl on a Superdex 75 column (GE Healthcare) and frozen at -80 deg C in 10% glycerol at a concentration of ⁇ 10 ⁇ .
  • IREl RNAse activity was measured by incubation of purified IRE 1(658-
  • RNAse activity measurements were measured on a TecanF500 (Tecan US) using an excitation filter of 612 nm (bandpass 10 nm) and emission filter of 670 (bandpass 25 nm).
  • IREl autophosphorylation was measured by incubation of 0.5 ⁇ IREl
  • IRE1 Bacterial expressed IRE1 (658-1115) lacking residues 869-892 was purified as previously described (Lee et al, 2008b). Crystals of IREl were grown in the presence of Mg2+- ADP and quercetin in a hanging drop. Crystals flash- frozen in cryo-protectant (10% PEG 8K, 50 mM Na-Cacodylate pH 6.5, 300 mM KC1, 100 mM SrOAc and 30 % glycerol) were used for data collection at the NE-CAT beamline. Data processing was performed using MOSFLM and SCALA. Molecule replacement was performed using Phaser. Initial coordinated and refinement restraints for ADP and quercetin were obtained from HIC-up server. The final model was obtained using iterative cylces of manual building in Coot and automated refinement using Refmac5 and CNS. Model validation was performed using
  • Transduced fluorescent cells were FAC sorted and tested for expression of the chimeric protein by immunoprecipitation followed by immunoblotting with a poly- clonal serum directed to the cytosolic juxta-membrane region of mammalian IRE la, common to the endogenous IRE la (of wildtype MEFs) or the chimeric proteins (Bertolotti et al, 2000). Transduced cells were exposed to the indicated
  • RNA concentrations of flavonols, or tunicamycin, thapsigargin or dithiothreitol (DTT) and RNA was procured and XBP1 splicing measured by a ratio-metric PCR assay.
  • IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92-96.
  • Ligand-independent dimerization activates the stress-response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum. J Biol Chem 275, 24881-24885.
  • transcription factor with a basic- leucine zipper motif is required for the unfolded protein- response pathway.
  • IREl encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae. Mol Microbiol 6, 1441-1446.
  • Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
  • XBPl A Link Between the Unfolded Protein Response, Lipid Biosynthesis and Biogenesis of the Endoplasmic Reticulum. J Cell Biol 167, 35-41.
  • XBPl mRNA is induced by ATF6 and spliced by IREl in response to ER stress to produce a highly active transcription factor.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions et des procédés de modulation de l'activité RNase de IRE1 consistant à mettre en contact ladite IRE1 avec un agent qui se lie au site Q ou qui inhibe la liaison d'un ligand au site Q, le site Q étant un site de liaison de ligands situé à l'interface dimérique des domaines KEN de l'oligomère IRE1. L'invention porte également sur des compositions et des procédés de modulation de la réponse aux protéines mal repliées dans la cellule.
PCT/US2010/050926 2009-10-01 2010-09-30 Procédé de modulation de l'activité ire1 Ceased WO2011041557A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US24781209P 2009-10-01 2009-10-01
US61/247,812 2009-10-01

Publications (1)

Publication Number Publication Date
WO2011041557A1 true WO2011041557A1 (fr) 2011-04-07

Family

ID=43826655

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/050926 Ceased WO2011041557A1 (fr) 2009-10-01 2010-09-30 Procédé de modulation de l'activité ire1

Country Status (1)

Country Link
WO (1) WO2011041557A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011154908A1 (fr) * 2010-06-08 2011-12-15 National University Of Ireland, Galway Manipulation de hsp70 et interactions de protéine ire1alpha
WO2018108991A3 (fr) * 2016-12-13 2018-07-26 Ecole Polytechnique Federale De Lausanne Méthodes de traitement de maladies du peptide bêta-amyloïde
US10702571B2 (en) 2015-12-03 2020-07-07 The University Of North Carolina At Pembroke Materials for cathepsin B enhancement and methods of use
CN113136348A (zh) * 2020-01-20 2021-07-20 暨南大学 高产紫杉叶素的酿酒酵母工程菌及其构建和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050250182A1 (en) * 2002-04-22 2005-11-10 University Of Michigan Novel genes, compositions, and methods for modulating the unfolded protein response
US20070202544A1 (en) * 2003-10-09 2007-08-30 Fumihiko Urano Methods For Diagnosing And Treating Endoplasmic Reticulum (er) Stress Diseases
US20090232738A1 (en) * 2008-01-14 2009-09-17 President And Fellows Of Harvard College Methods for modulating de novo hepatic lipogenesis by modulating XBP-1 activity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050250182A1 (en) * 2002-04-22 2005-11-10 University Of Michigan Novel genes, compositions, and methods for modulating the unfolded protein response
US20070202544A1 (en) * 2003-10-09 2007-08-30 Fumihiko Urano Methods For Diagnosing And Treating Endoplasmic Reticulum (er) Stress Diseases
US20090232738A1 (en) * 2008-01-14 2009-09-17 President And Fellows Of Harvard College Methods for modulating de novo hepatic lipogenesis by modulating XBP-1 activity

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KORENNYKH ET AL.: "The unfolded protein response signals through high-order assembly of Ire1.", NATURE., vol. 457, no. 7230, February 2009 (2009-02-01), pages 687 - 693, XP002636404, DOI: doi:10.1038/NATURE07661 *
NATSUME ET AL.: "Protective effect of quercetin on ER stress caused by calcium dynamics dysregulation in intestinal epithelial cells.", TOXICOLOGY EPUB, vol. 258, no. 2-3, 30 January 2009 (2009-01-30), pages 164 - 175, XP026002548, DOI: doi:10.1016/j.tox.2009.01.021 *
THANGASAMY ET AL.: "Quercetin: A Potential Complementary and Alternative Cancer Therapy.", COMPLEMENTARY AND ALTERNATIVE THERAPIES IN THE AGING POPULATION, September 2008 (2008-09-01), pages 563 - 585 *
WISEMAN ET AL.: "Flavonol activation defines an unanticipated ligand-binding site in the kinase- RNase domain of IRE1.", MOL CELL., vol. 38, no. 2, 23 April 2010 (2010-04-23), pages 291 - 304, XP002636403, DOI: doi:10.1016/J.MOLCEL.2010.04.001 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011154908A1 (fr) * 2010-06-08 2011-12-15 National University Of Ireland, Galway Manipulation de hsp70 et interactions de protéine ire1alpha
US10702571B2 (en) 2015-12-03 2020-07-07 The University Of North Carolina At Pembroke Materials for cathepsin B enhancement and methods of use
WO2018108991A3 (fr) * 2016-12-13 2018-07-26 Ecole Polytechnique Federale De Lausanne Méthodes de traitement de maladies du peptide bêta-amyloïde
CN113136348A (zh) * 2020-01-20 2021-07-20 暨南大学 高产紫杉叶素的酿酒酵母工程菌及其构建和应用
CN113136348B (zh) * 2020-01-20 2022-06-03 暨南大学 高产紫杉叶素的酿酒酵母工程菌及其构建和应用

Similar Documents

Publication Publication Date Title
Holder et al. Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase
Huang et al. Identification of a cellularly active SIRT6 allosteric activator
You et al. Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives
Smith et al. Identification of the first specific inhibitor of p90 ribosomal S6 kinase (RSK) reveals an unexpected role for RSK in cancer cell proliferation
Frech et al. High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity
Piccolo et al. Inositol pentakisphosphate promotes apoptosis through the PI 3-K/Akt pathway
Hsu et al. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2
Laudadio et al. Natural alkaloid berberine activity against Pseudomonas aeruginosa MexXY-mediated aminoglycoside resistance: in silico and in vitro studies
Farkas et al. Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux
Mori et al. An overview on the potential antimycobacterial agents targeting serine/threonine protein kinases from Mycobacterium tuberculosis
Liang et al. MALT1 as a promising target to treat lymphoma and other diseases related to MALT1 anomalies
Lopez et al. S-Glycosyl primary sulfonamides− A new structural class for selective inhibition of cancer-associated Carbonic Anhydrases
US8546074B2 (en) Sir2 activity
Le et al. The ADP-binding kinase region of Ire1 directly contributes to its responsiveness to endoplasmic reticulum stress
WO2011041557A1 (fr) Procédé de modulation de l'activité ire1
Cai et al. High-throughput screening strategies for the development of anti-virulence inhibitors against Staphylococcus aureus
Czarny et al. A small-molecule screening platform for the discovery of inhibitors of undecaprenyl diphosphate synthase
Zhou et al. Tanshinones induce tumor cell apoptosis via directly targeting FHIT
Xiong et al. Phenylalanine 90 and 93 are localized within the phenol binding site of human UDP-glucuronosyltransferase 1A10 as determined by photoaffinity labeling, mass spectrometry, and site-directed mutagenesis
Cuncic et al. Bis (N, N-dimethylhydroxamido) hydroxooxovanadate inhibition of protein tyrosine phosphatase activity in intact cells: comparison with vanadate
Larsen et al. An aminoquinazoline inhibitor of the essential bacterial cell wall synthetic enzyme GlmU has a unique non-protein-kinase-like binding mode
Lackovic et al. Inhibitors of Leishmania GDP-mannose pyrophosphorylase identified by high-throughput screening of small-molecule chemical library
Jäger et al. Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5
Yue et al. Structure-guided design, synthesis, and antivirulence assessment of covalent Staphylococcus aureus sortase A inhibitors
Jenkins et al. Identification of small-molecule inhibitors of human angiogenin and characterization of their binding interactions guided by computational docking

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10821262

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10821262

Country of ref document: EP

Kind code of ref document: A1