WO2011041557A1 - Method of modulating ire1 activity - Google Patents

Abstract

The present invention is directed to compositions and methods of modulating IRE1 RNase activity comprising contacting said IRE1 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 IRE1 oligomer. The invention is also directed to compositions and methods of modulating the unfolded protein response in the cell.

Description

METHOD OF MODULATING IRE1 ACTIVITY

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

61/247,812, filed October 1, 2009. The entire teachings of the above application are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by Grant number

DK047119 from National Institute of Diabetes and Digestive and Kidney Diseases, Grant number DK075311 National Institute of Diabetes and Digestive and Kidney Diseases, Grant number ES08681 from National Institute of Environmental Health Sciences, and Grant number F32-ES014775 from National Institute of

Environmental Health Sciences. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

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.

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

dimerization interface required for activation of the unfolded protein response

(PNAS 103(39): 14343-14348). Autophosphorylation increases the affinity of the IREl nucleotide binding cleft (located in the kinase domain) for nucleotide and binding of the nucleotide in turn promotes dimerization. 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). The dimer interface brings together the two kinase extension nuclease (KEN) domains which form two adjacent sites of ribonuc lease (RNase) activity (Lee et al).

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. There are many diseases associated with dysfunction of protein homeostasis (proteostasis) or the

accumulation of unfolded proteins in the ER. These diseases include, for example, al antitrypsin deficiency and cystic fibrosis. It would therefore be advantageous to develop to methods that are effective for modulation of the UPR.

SUMMARY OF THE INVENTION

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).

Example 1 shows that the binding of a ligand to the binding site in the KEN domain potentiates activation of yeast IREl with ADP.

In some embodiments, 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.

In one embodiment, 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 . In another embodiment, 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 .

In another aspect, 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 .

In another embodiment, 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.

In a further aspect, 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.

In another embodiment, the invention is directed to a method activating the unfolded protein response (UPR) in a patient in need thereof comprising

administering to said patient a therapeutically effective amount of an agent that binds to the Q site of an IREl dimer, wherein binding of the ligand to the Q site enhances the RNase activity of IREl .

In yet another embodiment, 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.

In another embodiment, 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.

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.

In another embodiment, 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 . In some embodiments, the method further comprises the RNase activity of IREl .

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIGs. 1A-E: Small molecule screen for modulators of IREl RNAse activity.

A. Fluorescent-based assay for yeast IREl (amino acids 658-1115) 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. B. Fluorescence timecourse measuring IREl 's RNAse. The activity of IREl (1.0 μΜ) incubated with increasing concentrations of ADP was measured by the cleavage of the fluorescent substrate (25 nM) depicted in FIG. 1 A. C.

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 μΜ. 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). E. Bar graphs depicting the activity of IREl 's (1.0 μΜ) RNase in the presence of various small molecules (25 μΜ) after a 10 minute reaction; the wells containing quercetin (25 μΜ) and ADP (2 mM) are indicated.

FIGs. 2A-E: Quercetin potentiates ADP-mediated IREl RNAse activity

A. Fluorescence timecourse of substrate (25 nM) cleavage by IREl (0.5 μΜ), alone (black) and in the presence of ADP (2 mM, red), quercetin (25 μΜ, blue) or both (green). B. Autoradiograph showing the cleavage of a ³²P-labeled IREl substrate (20 nM), of identical sequence to that depicted in FIG. 1 A, by IREl (0.5 μΜ) incubated with ADP (2 mM) or quercetin (25 μΜ). The full-length substrate and cleavage product are indicated. C. Autoradiograph of IREl (aa 658-1115) (0.5 μΜ) incubated with ³²P-ATP for the indicated time in the presence of ADP (30 μΜ) or quercetin (30 μΜ). A Coomassie stain of the same gel is shown below. D. Autoradiograph depicting the time-dependent cleavage of a 32P-labeled IREl substrate (20 nM; as in FIG. 2B) by IREl (0.5 μΜ) in the presence of ADP (2 mM), quercetin (25 μΜ) or both. E. Plot of the initial rate of IREl RNase activity as a function of quercetin concentration in the absence (filled circles) and presence (open squares) of ADP (2 mM).

FIGs. 3A-F: The structure of IREl crystals bound by quercetin and ADP reveal a novel ligand binding site, the Q-site. A. Structure of IREl (658-1115) crystallized in the presence of both ADP and quercetin shown in ribbon format. The kinase domain N- and C-terminal lobes of each promoter in the dimer are green and orange, respectively, and the KEN domains are blue. The kinase and the KEN domain dimer interfaces are indicated. ADP and quercetin are shown in a ball-and- stick representation. B. 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 α3' helix of the KEN domain are shown with side chain residues in ball and stick format. Both structured regions show significant overlap with the previous crystal structure of oligomeric IREl (PDB - 3FBV). 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 μΜ) 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, and IRE IF 1112L measured as in FIG. 4B. The activity of the different enzymes, defined by the fluorescent signal following 1 h treatment, in the presence of ADP was normalized to 1. D. Domain structure of the human-yeast IREl chimera (hylREl). The rabbit serum used to detect the endogenous and chimeric proteins recognizes the juxta-membrane region as indicated. E. RT-PCR analysis of XBP1 mRNA purified from wild-type MEFs (+/+) or IREl knockout cells transduced with empty vector (Mock) or hylREl WT exposed to tunicamycin (Tm; 2.5 μg/mL; 4h), DTT (2 mM; 1 h) or thapsigargin (Tg; 0.5 μΜ; lh). The position of the unspliced (XBP1U) and spliced (XBP1S) product is indicated. F. RT-PCR analysis of XBP1 mRNA purified from wild-type MEFs (+/+) or IREl knockout cells transduced with empty vector (Mock), hylRElWT, hyIRElS984E, or hyIRElK985A and exposed to 450 μΜ quercetin for 2 h or thapsigargin (Tg; 0.5 μΜ for 2h), as indicated. G. Immunoblot of endogenous IREl or the hylREl chimeras immunoprecipitated from lysates of the cells shown in FIG. 4F. 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 (lower panel) 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 μΜ) incubated in the presence of various flavonols (25 μΜ). The activity of IREl in the absence of flavonol is indicated by the x symbols. C. Timecourse of RNase activity of IREl (0.5 μΜ) incubated in the presence of both ADP (2 mM) and flavonols (25 μΜ). D. Autoradiograph of IREl (0.5 μΜ) incubated with ³²P-ATP in the presence of the known kinase inhibitor staurosporine (25 μΜ, a positive control for inhibition) or the flavonols (25 μΜ), following either 10 or 20 min reaction, as indicated. A Coomassie stain of the 20 min gel is shown below. The asterisk indicates the presence of an activity in the sample containing myrecetin that reproducibly degrades IREl . E. RT-PCR analysis of XBP1 mRNA purified from wild-type MEF (+/+) or IREl knockout cells transduced with hylREl WT following exposure to thapsigargin (Tg; 0.5 μΜ for 2 h) or flavonols (450 μΜ for 2 h), as indicated. F. RT-PCR analysis of XBP1 mRNA purified from wild-type MEF (+/+) or IREl knockout cells transduced with hylRElWT, hyIRElS984, hyIRElK985A or empty vector (Mock) following exposure to thapsigargin (Tg, 0.5 μΜ for 2h) or Luteolin (450 μΜ for 2 h), as indicated.

FIGs. 6A-G: Quercetin increases the population of IREl dimers. A.

Coomassie stained SDS-PAGE of yeast IREl (aa 658-1115, 5 μΜ) incubated with ADP (2 mM), quercetin (25 μΜ) or both in the absence (-DSS) or presence of 200 μΜ disuccinimidyl suberate (+DSS), an irreversible chemical cross-linker. B.

Coomassie stained SDS-PAGE of IRE1K985A and IREIWT incubated with ADP (2 mM), quercetin (25 μΜ) or both in the presence of 200 μΜ DSS. C. Comassie stained SDS-PAGE of IREl (5 μΜ) incubated with ADP (2 mM) and the flavonols (25 μΜ) in the presence of DSS (200 μΜ). D. Sedimentation distribution of yeast IREl (aa 658-1115, 5 μΜ; black) in the presence of ADP (2 mM; red), quercetin (25 μΜ; blue) or both (green), as determined by sedimentation velocity analytical ultracentrifugation. The approximate molecular weight of the two species is indicated. 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 μΜ) or both. G. Coomassie stained SDS-PAGE of IREIWT and IRE1D723A (5 μΜ) incubated with ADP (2 mM), quercetin (25 μΜ) or both in the presence of 200 μΜ DSS.

FIGs. 7A-D: The nucleotide binding cleft and the quercetin binding pocket interact to regulate IREl RNAse. A. Timecourse of IREl RNAse activity incubated without or with lambda phosphatase (λρρ) in the presence of ADP (2 mM) and quercetin (Q; 25 μΜ). B. Comassie-stained SDS-PAGE of IREIWT and

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 μΜ) or both. D.

Quantification of RNase activity for IRE1D797A incubated with quercetin (25 μΜ) and increasing concentrations of ADP. The initial rate of IREl RNAse activity is plotted against ADP concentration (the initial rate of IRE1D797A incubated with quercetin (25 μΜ) alone is shown by the dashed line).

FIG. 8: Quercetin does not alter cleavage specificity of IREl . Bar graph depicting the cleavage of the probe with deoxy-guanosine at position 3 of the loop (dG3 probe, discussed in FIG. ID) by RNase A (a positive control) or IREl (0.5 μΜ) that had been incubated with ADP (2 mM) quercetin (25 μΜ) or both following a 60 min reaction. This indicates that both ADP and quercetin-activated IREl require the 2' hydroxyl at position G3 to catalyze probe cleavage.

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 μΜ) was activated by incubation with ADP (2 mM), quercetin (30 μΜ) 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. An overlay of the nucleotide binding pocket of IRE 1 :ADP:quercetin (pink) and IRE1 :ADP (PDB: 2RIO; green), demonstrating the nearly identical binding of ADP in these two complexes. 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 . Stereo view of a quercetin pair modeled into a putative quercetin binding pocket in the homology model of human IRE la. 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

representation. The overall shape, volume and hydrophobic character of the cavity corresponding to the Q-site is conserved amongst human and yeast IREl . Of the seven amino acid residues that line the Q-site (14 residues in the dimer), five residues (10 residues in the dimer) are conserved between human and yeast IREl . The only notable difference lies at the entrance to the Q-site cavity; in the human IREl model the entrance to the Q-site is partially occluded by residues Glu836 and His963 (corresponding to Ser984 and Serl 115 in yeast IREl, respectively).

However, this should not be a significant impediment to accessing the Q-site, as quercetin can be modeled into this novel binding pocket of human IREl without steric clashes. However, we note that 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 . These findings suggest that quercetin may have different effects on the yeast and human proteins.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows. 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 .

The words "a" or "an" are meant to encompass one or more, unless otherwise specified.

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 β 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. (2009), The unfolded protein response signals through high-order assembly of Irel , Nature 457(7230): 687-93, the contents of each of which are expressly incorporated by reference herein. The amino acid sequences of yeast IREl and mammalian IREl, including human IREl, has been described in the literature, including for example, Zhou et al.

The term "IREl oligomer" encompasses oligomers comprising two or more

IREl proteins. The term "IREl oligomer" expressly encompasses IREl dimers.

The term "Q site" as used herein 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. As discussed in detail above, 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. The term "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.

As used herein, 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. In some embodiments, the Q site activator is an agent that binds to the Q site of an IREl oligomer.

As used herein, 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. In some embodiments, the Q site inhibitor is a competitive or noncompetitive antagonist of the natural ligand.

The terms "ligand of the Q site" and "Q site ligand" 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.

The term "agent" as used herein 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. The term "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 .

As used herein, the terms "inhibiting" or "decreasing" encompasses causing a net decrease by either direct or indirect means. The terms "increasing" or

"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. In some aspects, the complex comprises an IREl Q site activator. In additional embodiments, the complex comprises two molecules of an IREl Q site activator bound to the Q site.

In certain aspects, 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. In one embodiment, the IREl Q site activator binds to the Q site of an IREl oligomer, wherein said binding enhances the RNase activity of IREl . In another embodiment, the IREl Q site activator increases the activity of a natural ligand of the Q site of an IRE oligomer.

In some embodiments, 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. As used herein, the term "flavonoid" encompasses naturally occurring flavonoids as well as synthetic flavonoids. In yet another aspect, 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. Non-limiting examples of 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. In some aspects, 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.

In some aspects of the invention, 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. As shown in FIG. 3E, 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. Corresponding and/or conserved amino acids in homo logs of yeast IRE1 can also be identified using the structure-based alignment method described in Lee et al. (2008), Structure of the dual enzyme Irel reveals the basis for catalysis and regulation in nonconventional R A splicing, Cell 132: 89-100.

Based on the role of IRE 1 in the unfolded protein response (UPR), the method of the present invention can be used to modulate the UPR of a cell. In one embodiment, the invention is directed to a method of enhancing the UPR of a cell comprising administering to said cell a Q site activator. In another embodiment, the invention is directed to a method of inhibiting the UPR of a cell comprising administering to said cell a Q site inhibitor. In some embodiments, 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. In additional embodiments, 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. In another embodiment, the invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a Q site inhibitor.

In some embodiments, 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 β-glucosidase, acid β-galactosidase, iduronate 2-sulfatase, a-L-iduronidase, galactocerebrosidase, acid a -mannosidase, acid β -mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β -galactosidase, N- acetylglucosamine-1 -phosphotransferase, acid sphingmyelinase, NPC-1, acid a- glucosidase, β-hexosamine B, heparin N-sulfatase, a -N-acetylglucosaminidase, a - glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, a -N- acetylgalactosaminidase, a -neuramidase, β -glucuronidase, β-hexosamine A and acid lipase, polyglutamine, a -synuclein, Ab peptide, tau protein transthyretin and insulin.

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. In one embodiment, 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

pallidoluysian atrophy, several forms of spino-cerebellar ataxia, and spinal and bulbar muscular atrophy. Alzheimer's disease is characterized by the formation of two types of aggregates: extracellular aggregates of Αβ 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) are

characterized by aggregation of prion proteins. Exemplary human prion diseases are Creutzfeldt- Jakob Disease (CJD), Variant Creutzfeldt- Jakob Disease, Gerstmann- Straussler-Scheinker Syndrome, Fatal Familial Insomnia and Kuru. In a further embodiment, the protein conformation disease is a loss of function disorder. The terms "loss of function disease" and "loss of function disorder" are used interchangeably herein. 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. In cystic fibrosis, the mutated or defective enzyme is the cystic fibrosis transmembrane conductance regulator (CFTR). One of the most common mutations of this protein is AF508 which is a deletion (Δ) 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. The 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), Schindler disease, Schindler-Kanzaki disease, Sialidosis, Sly syndrome, Tay-Sach's disease and Wolman disease.

As discussed above, 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.

In another embodiment, 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. Exemplary 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.

In a further embodiment, 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. Examples of 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.

In another embodiment, 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-5- oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;

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;

tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel

(TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®; Aventis Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; 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); retinoic acid; esperamicins;

capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as 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.

In another embodiment, 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.

As will be understood by the skilled artisan, the form of an agent

administered to a patient or pharmaceutical composition used according to the methods of treatment described herein depends on the intended mode of

administration and therapeutic application. The 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. The diluent is 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. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Pharmaceutical 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 SEPHAROSE™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

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. Additionally, 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. In general, 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.

Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications. For suppositories, 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. [Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al, Biochem. Biophys. Acta 1368: 201-15, 1998].

As described above, 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. In some aspects, the complex comprises an IRE1 oligomer and a Q site activator. In other aspects, 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. For example, 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 .

The term "candidate agent" encompasses agents with known chemical structure or agents with unidentified structures. The term "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.

Methods of identifying the binding of ligand and a binding site are known in the literature and can be used to identify binding of a candidate agent to the Q site of IREl . Examples of such methods, include, but are not limited to, direct binding and competitive binding assays. In an exemplary direct binding assay, 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. In 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.

125 35 14 32 3

Exemplary radioisotopic labels include I, S, C, P, or H, either directly or indirectly, and the radioisotope detected by direct counting of

radioemmission or by scintillation counting. 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.

As discussed above, 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.

In some embodiments, the screening assay is a cell-based assay. In other embodiments, the screening assay is a cell- free assay. In 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. In one embodiment, 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

accomplished using a technology such as real-time Biomolecular Interaction

Analysis (BIA) (see, e.g., Sjolander S and Urbaniczky C, Anal. Chem. 63:2338-45 (1991) and Szabo A et al, Curr. Opin. Struct. Biol. 5:699-705 (1995)). As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

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. In addition to the IREl recognition loop, the RNA substrate can also comprise a nucleotide sequence that dissociates upon cleavage. In some aspects, the RNA substrate is labeled. In additional aspects, 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 invention is illustrated by the following non-limiting examples.

EXAMPLE 1 : LIGAND ACTIVATION OF IREl

INTRODUCTION:

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). The latter possess two distinct enzymatic activities: a protein kinase, whose only known substrate is IREl itself and a sequence-specific endoribonuclease that cleaves the HAC1 or XBP1 mRNA in yeast and metazoans, respectively (Cox and Walter, 1996; Mori et al, 1996;

Yoshida et al., 2001; Calfon et al., 2002). 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.

While the luminal events that initiate IREl signaling are incompletely understood, there is a good correlation between IREl oligomerization,

autophosphorylation and RNase activity (Shamu and Walter, 1996; Bertolotti et al., 2000; Liu et al, 2000). Auto-phosphorylation is required for nucleotide binding and nucleotide binding, in turn, promotes dimerization of IREl 's cytosolic domain in vitro (Lee et al., 2008b). Interestingly, while kinase activity is required for IREl function in vivo (Cox et al, 1993; Mori et al, 1993), this requirement can be bypassed by a mutation that enables non-phosphorylated IREl to bind a non- hydro lyzable ATP analog (Papa et al., 2003). 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). Together, 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.

The aforementioned model, which places nucleotide binding at the core of IREl 's effector function and diminishes the importance of kinase activity, suggests the possibility that ATP mimetic ligands may modulate IREl activity in interesting ways (Korennykh et al, 2009).

The desire to modulate 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. Here we report on the surprising discovery of a novel ligand-binding pocket at the dimer interface of IREl that can be targeted to modulate IREl 's RNAse. RESULTS:

Identification of small molecules that modulate IREl activity:

To detect modulators of IREl we developed a kinetic assay for the sequence- specific RNase activity. An 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).

When purified from bacteria, yeast IREl kinase-endonuclease domain

(residues 658-1115) is phosphorylated (Sidrauski and Walter, 1997; Lee et al, 2008b; Korennykh et al, 2009) (and described below), thus the assay is predicted to report on the ligand-dependent step(s) of IREl activation. Cleavage of the probe by IREl was ADP-dependent with an EC50 of ~40 uM, consistent with the previously reported binding constant of ADP to IREl (KD = 20 μΜ) (Lee et al, 2008b) (FIGs. IB and 1C). Removal of the 2' hydroxyl from the 5' residue at the predicted cleavage site (G3->dG3 of IREl 's recognition loop) blocked cleavage, attesting to the sequence specificity of the assay (FIG. ID).

IREl possesses a known ligand-binding cleft that engages nucleotide.

Therefore we searched for IREl modulators among a collection of small molecules that are known to bind the ATP-binding cleft of various kinases. This exercise led to the identification of several small molecules that modulated IREl 's RNase; the naturally occurring flavonol, quercetin, stood out as a particularly potent activator in this assay (FIG. IE).

Quercetin activates IREl through a site distinct from the nucleotide -binding cleft:

Quercetin is relatively insoluble in aqueous buffers, but at concentrations attainable (30 μΜ) 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

(e.g. Sicheri et al., 1997; Walker et al., 2000). To determine if its ability to activate IREl 's RNase correlated with its affinity towards the nucleotide-binding site of the kinase domain, we compared ADP and quercetin's ability to compete with ATP for that site by assessing their ability to block IREl autophosphorylation. Quercetin was the weaker inhibitor of P incorporation from γ-labeled ATP into IREl, indicating a lower affinity for the nucleotide binding cleft (FIG. 2C).

These findings predict that if quercetin activates IREl exclusively by allosteric effects mediated from the nucleotide-binding cleft, the enhanced RNase activity of quercetin-bound IREl would be inhibited by an excess of ADP; as the weaker activator, but more avid ligand (ADP), displaces the more potent but less avid quercetin from a common binding site. Surprisingly the opposite was observed; addition of saturating amount of ADP (2 mM) to quercetin (30μΜ) enhanced IREl RNase activity (FIG. 2A).

Quercetin potentiation of ADP-mediated RNase activity, observed in the fluorescent based assay was also recapitulated by measuring the cleavage of a ³²P- 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). Analysis of the reaction's initial rate revealed that in the presence of saturating concentrations of ADP (2 mM), the EC50 for quercetin-mediated activation of IREl 's RNase was diminished from >20uM (in the absence of ADP) to 6 uM in its presence (FIG. 2E); in the absence of ADP an accurate EC50 could not be determined, as the analysis was limited by quercetin' s solubility. Together these observations are consistent with simultaneous engagement of ADP and quercetin by IREl and imply the existence of a second site for ligand binding.

A co-crystal structure of IREl, ADP and quercetin defines a novel ligand-binding pocket:

To gain further insight into the potential mechanism of quercetin activation, we determined the crystal structure of IREl in the presence of both quercetin and ADP, using the same construct of yeast IRE 1(658-1115) that had previously been crystallized with ADP alone (with a 24 residue deletion in the activation loop between amino acids 869-892) (Lee et al, 2008b).

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

IREl :ADP:quercetin crystal structure, namely amino acids 837 to 844

corresponding to the N-terminal portion of the phosphorylated kinase domain activation loop and amino acids 1036 to 1042 corresponding to a flexible region in KEN domain thought to contain putative nuclease specificity determinants (FIG. 3B). The structure of these regions corresponds well to an alternative crystal structure of the IRE1 cytosolic domain (PDB: 3FBV, Korennykh et al., 2009).

Apart from ADP at the nucleotide binding cleft of IRE 1 (which is indistinguishable between the IRE1 :ADP and IRE1 :ADP:quercetin crystals, FIG. 10B), 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 (FIG. 3D) 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).

To test the importance of the contacts predicted by the crystal structure to quercetin mediated activation of IRE1 's RNase, we examined several mutations in residues predicted to interact with the bound ligand (FIG. 3F). Like the wildtype, IRE1S984E, IRE1K985A, IRE1K992L and IRE IF 1112L mutants were well expressed in E coli and purified as soluble phosphorylated proteins (as evidenced by enhanced mobility following incubation with lambda phosphatase, FIG. 4A) and exhibited wildtype levels of ADP-mediated RNase activity (FIGs. 4B-C). All four mutations abolished quercetin mediated activation of IREl's RNase (FIGs. 4B-C), validating the importance of the contacts revealed in the crystal structure to quercetin-mediated activation and supporting the crystal-based model of the ligand's disposition in the Q-site.

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). In an effort to circumvent these difficulties, we stably transfected IREl knockout cells (Calfon et al., 2002) with 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). However, in cells lacking endogenous IREl, exposure to quercetin promoted hylREl -dependent splicing of endogenous XBP1 (FIG. 4F). Mutations that blocked quercetin-mediated activation of yeast IREl in vitro also blocked activation in vivo (FIGs. 4F and 4G). These observations indicated that activation of the human-yeast chimera requires binding to the Q-site and that the membrane bound enzyme may access and cleave its endogenous substrate in response to activation by quercetin in vivo.

In the crystal structure quercetin is modeled with the 3'-hydroxyl facing the bulk solvent (FIGs. 3C and 3E), but the resolution of the X-ray data cannot unambiguously rule out an alternative orientation whereby the monocyclic phenyl ring is rotated 180 degrees. Therefore, we compared the ability of structurally diverse flavonols to activate IREl's RNAse in vitro (FIG. 5 A).

Among the flavonols tested, quercetin was the most potent activator of IRE 1 RNAse in vitro, both in the absence (FIG. 5B) and presence of saturating

concentrations of ADP (FIG. 5C). Kampferol, a quercetin derivative lacking the 3'- hydroxyl, was next in potency. Kampferol is a weak inhibitor of IRE 1 autokinase activity (FIG. 5D), suggesting that its ability to activate IREl 's RNase is derived mainly through binding the novel Q-site. Isorhamnetin, luteolin and morin were weaker activators demonstrating only modest potentiation with ADP. While the feeble activation of IREl 's RNase by isorhamnetin and luteolin, (when added alone) could be attributed to their low affinity for the nucleotide-binding cleft, the modest potentiation with ADP argues for lower affinity to the novel Q-site. This argument is even stronger in the case of morin, whose ability to attenuate IREl 's autokinase activity is similar to quercetin. The weak potentiation with ADP of isorhamnetin and morin (relative to quercetin) 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). Similarly, 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

rationalized by the hydrogen bond with S984, predicted from the crystal structure, the role of the 5-hydroxyl is unclear.

The structure activity relationship in vivo was similar to that observed in vitro, but produced some surprises: 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:

As the Q-site is located at the dimer interface of the KEN domain and as dimerization is a pre-requisite for RNase activity (Lee et al., 2008b; Korennykh et al, 2009), we sought to determine if quercetin binding affects the stability of the dimer thereby explaining the activation of IREl's RNase.

On SDS-PAGE IREl (658-1115) has the mobility predicted of a monomer

(53 kDa), which is unaltered by the crosslinker disuccinimidyl suberate (DSS). Upon addition of ADP, a faint slower migrating, DSS-dependent band that corresponds in mobility to a predicted dimmer was observed. The intensity of this slower migrating band was increased by incubation with quercetin and was increased further upon addition of both quercetin and ADP (FIG. 6A).

Quercetin's effects on IREl crosslinking were abolished by the ligand binding-site mutant IRE1K985A, although ADP-dependent crosslinking was still observed (FIG. 6B). Similar results were obtained with other quercetin disrupting mutants (IRE1S984E and IRE1K992L; data not shown), demonstrating that quercetin-mediated crosslinking is dependent on binding to the site identified in the crystal. A hierarchy at which the various flavonols activated IREl 's RNase also correlated with their ability to promote IREl crosslinking, with Kampferol being second to quercetin (FIG. 6C).

Quercetin's effect on the oligomeric state of IREl was further explored by sedimentation velocity analytical ultracentrifugtion (AUC). Non-liganded IREl sedimented as a single -4S species, corresponding to the predicted molecular weight of -50 kDa. In the presence of ADP, IREl sedimented as two distinct species corresponding in molecular weight to a 4S monomer (50 kDa) and a larger 5.5S species (-85 kDa), consistent with the previously demonstrated ADP-dependent dimerization of IRE (Lee et al., 2008b). Quercetin alone increased the population of the higher molecular weight state and addition of ADP increased this further (FIG. 6D), 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).

To further evaluate dimerization's role in quercetin-dependent IREl activation, we assessed quercetin's ability to activate an IREl variant with a well- characterized mutation in the dimer interface. To minimize any affect the mutation might have on ligand binding, we chose D723A, located ~3θΑ from the Q-site. As reported previously, 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

IRE1D723A (FIG. 6F). Furthermore, neither ligand promoted DSS-induced crosslinking of the mutant protein (FIG. 6G). These findings indicate that quercetin- mediated activation of IREl's RNase entails dimerization at the same interface required for ADP-mediated activation, as suggested by the co-crystal structure.

Quercetin-binding at its novel site modifies the nucleotide binding cleft:

Dimerization of IREl 's kinase and endonuclease domain, critical to activation of the RNase, is dependent on engagement of the nucleotide binding cleft by a suitable ligand. Under normal circumstances autophosphorylation must precede such ligand binding, however this requirement can by bypassed by a mutation that alters the architecture of the nucleotide binding cleft (Papa et al., 2003). Given this plasticity, we sought to determine if quercetin-mediated activation of IREl also depends on autophosphorylation.

As expected, dephosphorylation with lambda phosphatase markedly diminished ADPmediated RNase activity. Surprisingly, the dephosphorylated IREl remained strongly responsive to quercetin (FIG. 7A). However, 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). Compared with the wildtype, 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.

While IRE1D797A had no detectable ADP-mediated RNase activity (as previously noted, Lee et al, 2008b), addition of quercetin substantially activates its RNase (FIG. 7C).

Isothermal titration calorimetry had previously indicated that the

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.

DISCUSSION:

The discovery of a novel ligand-binding site in the kinase-endonuclease domain of yeast IREl , reported on for the first time here, provides an interesting perspective on features known from previous studies, to regulate the enzyme. The new findings also suggest the existence of hitherto unanticipated modes of physiological regulation of IREl 's endonuclease activity as well as features of the enzyme that may be exploited for pharmacological manipulation of the UPR.

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. The suggestion, from the crystal structure, that quercetin promotes RNase activity first and foremost by stabilizing the back-to-back active dimeric configuration of the enzyme is further supported by the enhanced population of dimmers observed by analytical ultracentrifugation and by the enhanced

crosslinkability of IREl protomers in the presence of quercetin.

The suggested preeminence of dimerization in quercetin' s mechanism of action fits well with the prevailing notions on how ER stress modulates IREl activity: 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 (Shamu and Walter, 1996) 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). As such higher order oligomers were not observed in this in vitro study of quercetin's interactions with the soluble cytoplasmic domain, it seems likely that quercetin activates IREl by promoting the penultimate step (dimerization) in the cascade described above.

The structure of IREl 's cytoplasmic domain in the IREl : ADP and

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. Unfortunately, we are unable to detect (or exclude) allosteric regulation at the RNase site, as we are unable to measure IREl's affinity for RNA (Km) or its enzymatic activity in the presence of saturating concentrations of RNA substrate (Vmax, data not shown).

Allosteric regulation of the nucleotide binding cleft by ligand at the Q-site is suggested by the surprising behavior of IRE1D797A. D797 is not part of the nucleotide cleft and the mutation likely exerts its effects by preventing de- protonation of the protein substrate in the kinase reaction (and not by directly affecting nucleotide binding) (Lee et al, 2008b; Korennykh et al, 2009).

IRE1D797A, which lacks detectable autokinase activity and is thus refractory to activation by ADP (Lee et al., 2008b), is nonetheless activated by quercetin.

Remarkably, addition of ADP to quercetin bound IRE1D797A attenuates its RNase activity, suggesting a profound allosteric switch at the nucleotide binding cleft induced by quercetin binding, which bypasses the restriction on nucleotide binding by non-phosphorylated IREl and alters the consequences of nucleotide binding to favor inhibition of RNase. The efficient activation of the human-yeast chimeric, hylREl by quercetin in vivo, indicates that the RNase can be activated in the context of the full-length protein through the Q site. It is tempting to speculate that 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. Alternatively, 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). Furthermore, modeling suggests that mammalian IREl also possess a pocket analogous to the Q-site that may be accessed by quercetin-like compounds (FIG. 11). Therefore, interaction between products of lipid or sterol metabolism and mammalian IREl at the Q-site site may help explain the emerging links between lipid metabolism and the UPR in mammals (Sriburi et al., 2004) (Lee et al., 2008a).

The findings described here also suggest a hitherto unanticipated mode for accessing the UPR pharmacologically (independently of the nucleotide binding cleft that is common to many kinases). Drugs that may bind a homologous Q site in mammalian IREl and recapitulate quercetin activation could serve as potent modulators of the protein-folding environment in the ER. This may affect so-called proteostasis networks and alter the fate of misfolding-prone medically-important mutant proteins (e.g. lysosomal hydrolases, Powers et al, 2009).

Furthermore negative feedback by 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. Conversely, 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. MATERIALS AND METHODS :

Plasmids and reagents

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.

Purification of IRE 1(658-1115)

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 2μg 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 μΜ.

Biochemical Assays for IRE 1(658-1115)

IREl RNAse activity was measured by incubation of purified IRE 1(658-

1115) 0.5-1μΜ with 25 nM of the quenched RNA probe at room temperature in a 384 well low- volume round bottom plate (Corning) with a final reaction volume of 20 nL. IREl reaction buffer (20 mM Hepes pH 7.5 50 mM KOAc 1 mM MgOAc and 1 mM DTT) was used for all RNAse activity measurements. Time-dependent fluorescence was measured on a TecanF500 (Tecan US) using an excitation filter of 612 nm (bandpass 10 nm) and emission filter of 670 (bandpass 25 nm). An analogous protocol was followed to measure the IREl -dependent cleavage of 20 nM of 32P-labeled RNA substrate, prepared by 5' end-labeling with T4 polynucleotide kinase, following the reaction by denaturing acrylamide gel electrophoresis.

IREl autophosphorylation was measured by incubation of 0.5 μΜ IREl

(658-1115) with 0.1 mCi 32P-ATP (MP Biomedicals) for the indicated time in 20 mM Hepes pH 7.5 50 mM MgC12 2 mM MnC12 1 mM DTT at room temperature. Incorporation of ³²P was followed by SDS-PAGE and radiography. Crosslinking was performed by incubation of 5 μΜ IREl (658-1115) with 200 μΜ disuccinimidyl suberate (DSS; Pierce) for 30 min at room temperature in IRE1 reaction buffer. The reaction was quenched by the addition of 50 mM Tris pH 7.5. The samples were then boiled in Laemmili buffer and separated by SDS-PAGE. Crystallization of IRE 1

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

PROCHECK. Structural images presented in figures were prepared in Chimera. The statistical parameters are provided in Supplemental table 1.

Quercetin mediated XBP1 spicing in mammalian cells by human-yeast

chimeric IREl

Cells lacking both IRE la and IREip (Calfon et al., 2002) were transduced with GFP-marked retroviruses expressing human-yeast IREl chimeric proteins. 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

concentrations of flavonols, or tunicamycin, thapsigargin or dithiothreitol (DTT) and RNA was procured and XBP1 splicing measured by a ratio-metric PCR assay.

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Supplemental Table 1

Data collection statistics

Beamline NE-CAT 24-ID-C

Resolution 65.51 - 3.2 (3.37 - 3.2)

Space group P65

Unit cell parameters a=b=131.02 A, c=175.82 A,

a=b=90°, g=120

total no. of reflections 77529

unique no. of reflections 27637

Rmerge 11.0 % (38.7 %)

Mean I/s(I) 8.3 (2.8) completeness 98.2 (99.8)

Multiplicity 2.8 (2.8)

Refinement statistics

Resolution 65.51 - 3.2 (3.37 - 3.2)

no. of reflections used 26241

Rwork/Rfree 21.66/22.36

no. of protein atoms 6593

no. of ligand atoms 98

no. of metal ions 4

r.m.s. deviation from ideal bond 0.007

lengths (A)

rms deviation from ideal bond 1.129

angles (o)

Ramachandran statistics

no. of residues 817

most favored 92.1 %

allowed 7.6 %

generously allowed 0.3 %

disallowed 0.0 %

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of increasing IRE1 RNase activity comprising contacting IRE1 with an agent that binds to the Q site of an IRE1 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 IRE 1.

2. The method of claim 1, wherein the IRE1 is a S. Cerevisiae IREl .

3. The method of claim 1, wherein the IREl is a mammalian IREl .

4. The method of claim 3, wherein the IREl is a human IRE la.

5. The method of claim 1, wherein the agent interacts with one or more amino acid residues selected from the group consisting of S984, K985, E988, K992, P1077, LI 108 and Fl 112 of S. Cerevisiae IREl, or a homologous residue of a mammalian IREl .

6. The method of claim 1 , wherein the agent is capable of interacting with one or more amino acid residues selected from the group consisting of S984, K985, E988, K992, P1077, LI 108 and Fl 112 of S. Cerevisiae IREl, or a homologous residue of a mammalian IREl .

7. The method of claim 1, wherein the agent is a flavonoid.

8. The method of claim 7, wherein the agent is a flavonol.

9. The method of claim 7, wherein the agent is a flavone.

10. The method of claim 9, wherein the flavone is luteolin or apigenin.

11. The method of claim 8, wherein the flavonol is selected from the group consisting of quercetin, kampferol, isorhamentin, morin, staurosporine, fisetin, galangin, myrecetin, and taxifolin.

12. The method of claim 11, wherein the flavonol is quercetin.

13. The method of claim 8, wherein the flavonol is characterized by a 5-hydroxyl moiety on the 3 -hydroxy flavone core.

14. The method of claim 8, wherein the flavonol is characterized by a 4 '-substitution on the 3 -hydroxy flavone core wherein said 4 '-substitution is capable of hydrogen bonding with an amino acid residue of the Q site IRE1.

15. The method of claim 14, wherein the 4 '-substitution is a 4'-hydroxyl.

16. 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 IRE1 dimer, wherein binding of the agent to the Q site enhances the RNase activity of IRE 1, and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.

17. The method of claim 16, wherein the IRE1 is a S. Cerevisiae IREl .

18. The method of claim 16, wherein the IREl is a mammalian IREl .

19. The method of claim 18, wherein the IREl is a human IRE la.

20. The method of claim 17, wherein the agent interacts with one or more amino acid residues selected from the group consisting of S984, K985, E988, K992, P1077, LI 108 and Fl 112 of S. Cerevisiae IREl, or a homologous residue of a mammalian IREl .

21. The method of claim 16, wherein the agent is capable of interacting with one or more amino acid residues selected from the group consisting of S984, K985, E988, K992, P1077, LI 108 and Fl 112 of S. Cerevisiae IREl, or a homologous residue of a mammalian IREl .

22. The method of claim 16, wherein the agent is a flavonoid.

23. The method of claim 22, wherein the agent is a flavonol.

24. The method of claim 22, wherein the agent is a flavone.

25. The method of claim 24, wherein the flavone is luteolin or apigenin.

26. The method of claim 23, wherein the flavonol is selected from the group

consisting of quercetin, kampferol, isorhamentin, morin, staurosporine, fisetin, galangin, myrecetin, and taxifolin.

27. The method of claim 26, wherein the flavonol is quercetin.

28. The method of claim 23, wherein the flavonol is characterized by a 5-hydroxyl moiety on the 3 -hydroxy flavone core.

29. The method of claim 23, wherein the flavonol is characterized by a 4'- substitution on the 3 -hydroxy flavone core wherein said 4 '-substitute is capable of hydrogen bonding with an amino acid residue of the Q site IREl .

30. The method of claim 29, wherein the 4 '-substitution is a 4'-hydroxyl.

31. A method of activating IREl RNase activity comprising contacting said IREl with an agent that binds to the Q site of an IREl oligomer, wherein said binding stabilizes the oligomer or promotes oligomerization and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.

32. 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, and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.

33. 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 and the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.

34. The method of claim 33, wherein the IREl is a mammalian IREl .

35. The method of claim 34, wherein the IREl is a human IREla.

36. 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.

37. The method of claim 36, wherein the IREl is a mammalian IREl .

38. The method of claim 37, wherein the IREl is human IREla.

39. A method activating the unfolded protein response (UPR) in a patient in need thereof comprising administering to said patient a therapeutically effective amount of an agent that binds to the Q site of an IREl dimer, wherein binding of the ligand to the Q site 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.

40. A method of treating a patient suffering from a condition associated with the accumulation of unfolded proteins in the endoplasmic reticulum comprising administering to said patient a therapeutically effective amount of an agent that binds to the Q site of an IRE1 oligomer and wherein said binding enhances the RNase activity of IRE 1, and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.

41. The method of claim 40, wherein the protein is selected from the group

consisting of 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 β-glucosidase, acid β-galactosidase, iduronate 2-sulfatase, a-L- iduronidase, galactocerebrosidase, acid a -mannosidase, acid β -mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β -galactosidase, N-acetylglucosamine-1 -phosphotransferase, acid

sphingmyelinase, NPC-1, acid a-glucosidase, β-hexosamine B, heparin N- sulfatase, a -N-acetylglucosaminidase, a -glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, a -N-acetylgalactosaminidase, a - neuramidase, β -glucuronidase, β-hexosamine A and acid lipase, polyglutamine, a -synuclein, Ab peptide, tau protein transthyretin and insulin.

42. A method for treating a protein conformation disease or disorder comprising administering 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.

43. The method of claim 42, wherein the protein is selected from the group

consisting of 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 β-glucosidase, acid β-galactosidase, iduronate 2-sulfatase, a-L- iduronidase, galactocerebrosidase, acid a -mannosidase, acid β -mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β -galactosidase, N-acetylglucosamine-1 -phosphotransferase, acid

sphingmyelinase, NPC-1, acid a-glucosidase, β-hexosamine B, heparin N- sulfatase, a -N-acetylglucosaminidase, a -glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, a -N-acetylgalactosaminidase, a - neuramidase, β -glucuronidase, β-hexosamine A and acid lipase, polyglutamine, a -synuclein, Ab peptide, tau protein, transthyretin and insulin.

44. The method of claim 42, wherein the protein conformational disease or disorder is a gain of function disease.

45. The method of claim 42, wherein the protein conformational disease or disorder is a loss of function disease.

46. A method of treating a patient suffering from cancer comprising administering to said patient a therapeutically effective amount of an agent that inhibits the binding of a ligand to the Q site of an IRE1 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.

47. A method of treating a condition mediated by plasma cells 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 IRE1 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.

48. The method of claim 47, wherein the condition is selected from the group

consisting of 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.

49. The method of claim 47, wherein the condition is cancer mediated by abnormal proliferation of plasma cells including, for example, multiple myeloma and plasma cell dyscrasia.

50. A method of treating 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 IRE1 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.

51. An isolated or purified polypeptide-ligand complex comprising an IRE1

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.

52. The complex of claim 51, wherein the oligomer is a dimer.

53. The complex of claim 51 , wherein two molecules of the agent are bound to the Q-site.

54. The complex of claim 51, wherein the IRE1 is S. Cerevisiae IRE1.

55. The complex of claim 51, wherein the IRE1 is a mammalian IRE1.

56. The complex of claim 51, wherein the agent is a flavonoid.

57. A method of identifying an agent that enhances RNase activity of IRE 1

comprising screening one or more candidate agents for ability to bind to the Q site of IRE 1.

58. The method of claim 57, further comprising measuring the RNase activity of IRE1.

59. The method of claim 57, wherein binding of the agent is determined in the presence of ADP.

60. The method of claim 58, wherein cleavage of an RNA substrate is measured, wherein said substrate comprises an IREl recognition loop.

61. The method of claim 60, wherein the substrate comprises an IREl recognition loop from XBP1 and a nucleotide sequence that dissociates upon cleavage.

62. The method of claim 60, wherein the RNA substrate is labeled.

63. The method of claim 62, wherein the label is a fluorescent label.

64. The method of claim 62, wherein RNA substrate further comprises a moiety that quenches the fluorescent label.

65. The method of claim 64, wherein cleavage of the substrate results in

fluorescence.

66. The method of claim 57, wherein binding is determined by identifying the

formation of a complex comprising the IREl oligomer and the agent bound to the Q site of the oligomer.

67. A method of activating RNase activity of an unphosphorylated IREl comprising administering an agent that binds to the Q site of an IREl dimer, wherein said binding stabilizes the oligomer or promotes oligomerization and wherein the Q site is a ligand binding domain in the dimer interface of kinase extension (KEN) domains.