An inhibitor of ASC-dependent inflammasomes for the treatment of inflammatory diseases

Abstract

The present application relates to compounds, e.g., compounds of Formula (I) and pharmaceutically acceptable salts thereof, and compositions comprising compounds of Formula (I) or Formula (II), or a pharmaceutically acceptable salt of either of the foregoing, for treating inflammatory diseases.

Description

[0001] AN INHIBITOR OF ASC-DEPENDENT INFLAMMASOMES FOR THE TREATMENT OF INFLAMMATORY DISEASES

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 783,166, filed on April 3, 2025, and U.S. Provisional Application Serial No. 63 / 655,530, filed on June 3, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant No. GM118112 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] FIELD

[0007] The present application relates to compounds, e.g., compounds of Formula (I) and pharmaceutically acceptable salts thereof, and compositions comprising compounds of Formula (I) or Formula (II), or a pharmaceutically acceptabsle salt of either of the foregoing, for treating inflammatory diseases.

[0008] BACKGROUND

[0009] Inflammasome activation is implicated in the pathogenesis of various inflammatory diseases, including autoimmune diseases (e.g., rheumatoid arthritis, lupus), metabolic disorders (e.g., type 2 diabetes, obesity), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease), and / or inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis). Most of the existing therapies in the clinic rely solely on monoclonal antibodies that target inflammatory cytokine Interleukin 1 (IL-1) or antagonizes its receptor. However, targeting only IL-1 interferes with part of the inflammasome response and given its protective role during infections, targeting IL-1 can potentially cause immunosuppression. Thus, there is an unmet clinical need to develop novel therapeutics to treat inflammatory diseases.

[0010] SUMMARY

[0011] Provided herein are compounds of Formula (I): or a pharmaceutically acceptable salt thereof, wherein

[0012] Ring A is phenyl or 5-6 membered heteroaryl; m is 0 or 1;

[0013] R1is halogen, C1-C6 alkyl, -NRARB, or phenyl optionally substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl;

[0014] RAand RBare independently hydrogen and C1-C6 alkyl; and

[0015] R2and R3are independently hydrogen or allyl; wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is not Compound 2 (C-2) or a pharmaceutically acceptable salt thereof

[0016] Also provided herein are methods for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein

[0017] Ring A is phenyl or 5-6 membered heteroaryl; m is 0 or 1;

[0018] R1is halogen, C1-C6 alkyl, -NRARB, or phenyl optionally substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl;

[0019] RAand RBare independently hydrogen and C1-C6 alkyl; and R2and R3are independently hydrogen or allyl.

[0020] Also provided herein are methods for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound 2 (C-2), which is 5-((5-methylfuran-2-yl)methylene)-2- thioxodihydropyrimidine-4, 6( 1 / , 5 / 7)-dione, having the structure:

[0021] Also provided herein are methods for treating an inflammatory disorder in a subject, the method including: (a) determining or having determined that the subject has an inflammatory disorder; and (b) administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof. Also provided herein are methods for reducing inflammation in a subject having an inflammatory disorder, the method including administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0022] Also provided herein are methods for treating an inflammatory disorder in a subject, the method including: (a) determining or having determined that the subject has an inflammatory disorder; and (b) administering to the subject a therapeutically effective amount of Compound 2 (C-2). Also provided herein are methods for reducing inflammation in a subject having an inflammatory disorder, the method including administering to the subject a therapeutically effective amount of Compound 2 (C-2).

[0023] In some embodiments, the inflammatory disorder is an autoimmune disease, a metabolic disease, a neurodegenerative disease, or an inflammatory bowel disease.

[0024] In some embodiments, the inflammatory disorder is an autoimmune disease. In some mbodiments, the autoimmune disease rheumatoid arthritis or lupus.

[0025] In some embodiments, the inflammatory disorder is a metabolic disorder. In some mbodiments, the metabolic disorder is type 2 diabetes or obesity.

[0026] In some embodiments, the inflammatory disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer’s disease or Parkinson’s disease. In some embodiments, the inflammatory disorder is an inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn’s disease or ulcerative colitis.

[0027] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0028] Also provided herein are methods for inhibiting inflammasome activity in a cell including an inflammasome, the method including contacting the cell with an effective amount of Compound 2 (C-2). In some embodiments, the inflammasome activity comprises IL-ip / IL-18 secretion, ASC oligomerization, caspase-1 activation, and / or pyroptosis.

[0029] In some embodiments, the subject has been previously diagnosed as having, or is suspected of having, an inflammatory disease.

[0030] In some embodiments, the cell is in a sample from a subject. In some embodiments, the cell is in a subject and the contacting comprises administering to the subject a therapeutically effective amount of Compound 2 (C-2).

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0032] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0033] DESCRIPTION OF DRAWINGS

[0034] FIGS. 1A-1J. Compound 2 (C-2) inhibited NLRP3-driven pyroptosis and IL-ip secretion. FIG. 1 A: A schematic depicting the chemical structure of C-2 chemical structure. FIG. IB: Dose response curve of C-2 was generated using a lactate dehydrogenase (LDH) assay to calculate the IC50 value in human CD14+ monocytes. FIG. 1C: LDH release was measured in cells treated with or without Nigericin. FIG. ID: IL-ip production was measured in cells treated with or without Nigericin. FIG. IE: Effect of C-2 on membrane permeabilization was tested by measuring cell death when treated with C-2 or DMSO vehicle control. FIG. IF : Western blot was performed to examine the gasderm D (GSDMD) processing and release of mature IL-ip in human CD 14+ monocytes that were primed for 3 hours with LPS, and treated with or without C-2 (20 M) before adding nigericin (20 pM) for 1 hour. FIG. 1G: Western blot showing ASC oligomerization and caspase- 1 activation of DSS cross-linked pellets and supernatant / lysate, respectively, from human CD14+ monocytes stimulated with LPS and nigericin, and followed by treatment with C- 2. FIG. 1H: LPS-primed bone-marrow-derived macrophages (BMDMs) treated with DMSO vehicle control or C-2 were stimulated with different inflammasome agonists before measuring the levels of LDH and IL-ip. FIGS. 1I-1J: Status of ASC oligomers formation was assessed in LPS-primed BMDMs pretreated with C-2 for 1 hour before adding indicated inflammasome stimuli.

[0035] FIGS. 2A-2H. C-2 targeted and stabilized mitochondrial phosphate carrier protein (MPCP). FIG. 2A: Left panel shows compound P-2, an allkyene-tagged C-2. Right panel shows Log2 fold change measured in LPS and nigericin stimulated human CD14+ monocytes after treatment with P-2 or the vehicle control (DMSO). FIG. 2B: Top 10 C-2 bound proteins was identified in an unbiased manner by Peptide Mapping Nano LC- MS / MS. FIG. 2C: Western blot of MPCP in streptavidin pulldown from clicked lysate. Human CD14+ monocytes were activated for 3 hours with LPS, treated the last 1 hour with indicated concentrations of P-2, and later stimulated with nigericin for 2 hour. Lysates were subjected to click chemistry and streptavi din-beads were used to pull down the P-2 interacting proteins. FIG. 2D: Immunoblot of MPCP in streptavidin pulldown from clicked lysates after competition experiment with C-2 (at increasing concentrations) and P-2 (at 20 pM). FIG. 2E: Lysate of HEK293T cells stably expressing MPCP were treated with P-2 or C-2 across a range of increasing temperatures. FIG. 2F: Levels of IL-ip and TNF-a were measured in supernatants harvested from wild type (WT) and Mpcp~'~ BMDM treated with or without C-2. FIG. 2G: LDH level was measured in the supernatant harvested from WT and Mpcp~ ~ BMDMs. WT BMDMs were stimulated with LPS, followed by treatment with or without C-2, and then nigericin was added. Mpcp~'~ BMDMs were treated with LPS+nigericin. FIG. 2H: Western blot showing the effects of C-2 on GSDMD processing and caspase- 1 activation after WT and Mpcp~'~ BMDMs were primed with LPS and stimulated with nigericin.

[0036] FIGS. 3A-3J. C-2 mitigated IL-ip secretion in NLRP3 activated mouse models. Serum levels of IL-ip (FIGS. 3A, 3D, and 3H), IL-6 (FIGS. 3B, 3E, and 31) and TNF-a (FIG. 3C) from C57BL / 6 mice pretreated with two different doses of C-2 or vehicle control were measured by ELISA 2 hours after LPS injection (i.p.). FIGS. 3D-3G: After injecting the MSU crystal treated mice with or without C-2 pretreatment, the levels of IL-ip (FIG. 3D) and IL-6 (FIG. 3E) were measured using ELISA, and the levels of neutrophils (FIG. 3F) and monocytes (FIG. 3G) were measured by flow cytometry. FIGS. 3H-3J: LPS- challenged C57BL / 6 mice were intraperitoneally injected with vehicle or C-2, prior to ATP exposure. After 1 hour, the levels of IL-ip (FIG. 3H) and IL-6 (FIG. 31), and the number of free extracellular ASC oligomers (FIG. 3 J) were analyzed in cell-free supernatants of the peritoneal exudates.

[0037] FIG. 4. Structure activity relationship (SAR) for C-2.

[0038] FIGS. 5A-5G. Analogs -1, -2, -3, -7, -8, -9, -11, -12 and -13 from Table 1 showed similar biological activity to C-2. LPS-primed WT BMDMs treated with or without either C-2 (20 pM) or the indicated analogs (20 pM) were stimulated with nigericin (20 pM) for 1 hour to assess the LDH release (FIGS. 5A and 5B), IL-ip production (FIGS. 5C and 5D), and TNF-a production (FIGS. 5E and 5F). FIG. 5G: Western blot showing ASC oligomerization and caspase-1 activation of DSS cross-linked pellets and supernatant / lysate, respectively, from WT BMDMs that were stimulated with LPS and nigericin, and followed by treatments with C-2 or the indicated analogs.

[0039] DETAILED DESCRIPTION

[0040] The present application is based in part on the findings that Compound 2 (C-2), as described herein, prevented ASC oligomerization, caspase- 1 activation, IL-ip secretion and pyroptosis, all biomarkers of inflammasome activity, in human CD 14+ monocytes treated with LPS + nigericin, conditions which activate the NLRP3 inflammasome.

[0041] These inhibitory effects were not restricted to the NLRP3 inflammasome pathway. Compound 2 (C-2) also blocked AIM2, NLRC4 and PYRIN inflammasome-mediated IL- ip release and ASC oligomer formation but did not block responses to non-canonical inflammasome pathway. These findings suggest that Compound 2 (C-2) could be an inhibitor of ASC itself, because all these distinct inflammasome pathways require ASC for its activation. Rather than binding directly to ASC, the data indicates that Compound 2 (C- 2) targets and stabilizes MPCP, a mitochondrial phosphate carrier protein that mediates proton-coupled symport of phosphate ions necessary for mitochondrial oxidative phosphorylation of ADP to ATP. MPCP, in turn, plays a role in inflammasome biology. Cells lacking MPCP failed to produce IL-ip and do not die under NLRP3 inflammasome activation phenocopying the effects seen in cells treated with Compound 2 (C-2).

[0042] Moreover, no changes in TNF-a levels were observed, indicating that the inflammasome is the main pathway affected. Mpcp~'~ cells exhibited impaired GSDMD and caspase- 1 cleavage. Compound 2 (C-2) demonstrated an in vivo biological relevance in three different experimental models of inflammasome-triggered diseases which the induction of IL-ip by intraperitoneal injection of LPS, MSU or LPS+ATP is NLRP3 dependent. Pre-treatment with Compound 2 (C-2) reduced serum concentrations of IL-ip after LPS septic shock and MSU / ATP -induced peritonitis, suppressed the recruitment of inflammatory cells, neutrophils and monocytes, and decreased the amount of free extracellular ASC oligomers.

[0043] Additional Definitions

[0044] To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Each of the patents, applications, published applications, and other publications that are mentioned throughout the specification and the attached appendices are incorporated herein by reference in their entireties.

[0045] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation, for example, within experimental variability and / or statistical experimental error, and thus the number or numerical range may vary up to ± 10% of the stated number or numerical range.

[0046] The term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0047] As used herein, the “subject” refers to any animal, including mammals such as primates (e.g., humans), mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and / or exhibited at least one symptom of the disease to be treated.

[0048] As used herein, terms “treat”, or “treatment” refer to therapeutic or palliative measures. Beneficial or desired clinical results include, but are not limited to, alleviation, in whole or in part, of symptoms associated with a disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0049] The phrase “therapeutically effective amount” means an amount of compound that, when administered to a subject in need of such treatment, is sufficient to achieve a desired beneficial result, for example, a reduction of symptoms in a subject, prolonging the life of a subject, improving the quality of life of a subject, and the like.

[0050] The term “pharmaceutically acceptable excipient” means a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed , Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed. Rowe el al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed, Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed:, Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0051] In addition, atoms making up the compounds of the present embodiments are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C. The compound disclosed herein is intended to include all tautomeric forms. Thus, Compounds of Formula (T)

[0052] Provided herein are compounds of Formula (I): or a pharmaceutically acceptable salt thereof, wherein

[0053] Ring A is phenyl or 5-6 membered heteroaryl; m is 0 or 1;

[0054] R1is halogen, C1-C6 alkyl, -NRARB, or phenyl optionally substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl;

[0055] RAand RBare independently hydrogen and C1-C6 alkyl; and

[0056] R2and R3are independently hydrogen or allyl; wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is not Compound 2 (C-2) or a pharmaceutically acceptable salt thereof

[0057] In some embodiments, Ring A is phenyl. In some embodiments, Ring A is 5-6 membered heteroaryl. In some embodiments, Ring A is furanyl.

[0058] In some embodiments, m is 0. In some embodiments, m is 1.

[0059] In some embodiments, R1is halogen. In some embodiments, R1is iodo.

[0060] In some embodiments, R1is C1-C6 alkyl. In some embodiments, R1is methyl.

[0061] In some embodiments, R1is -NRARB.

[0062] In some embodiments, RAand RBare the same. In some embodiments, RAand RBare different. In some embodiments, RAand RBare each hydrogen. In some embodiments, RAand RBare each C1-C6 alkyl. In some embodiments, RAand RBare each methyl. In some embodiments, one of RAand RBis hydrogen and the other of RAand RBis C1-C6 alkyl.

[0063] In some embodiments, R1is phenyl optionally substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl.

[0064] In some embodiments, R1is unsubstituted phenyl.

[0065] In some embodiments, R1is phenyl substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl.

[0066] In some embodiments, R1is phenyl substituted with 1-2 substituents independently selected from fluoro, chloro, nitro, and methyl.

[0067] In some embodiments, R2is hydrogen.

[0068] In some embodiments, R2is allyl.

[0069] In some embodiments, R3is hydrogen.

[0070] In some embodiments, R3is allyl.

[0071] In some embodiments, R2and R3are each hydrogen.

[0072] In some embodiments, one of R2and R3is hydrogen and the other of R2and R3is allyl.

[0073] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the compounds in Table 1, or a pharmaceutically acceptable salt thereof.

[0074] Compound 2 (C-2)

[0075] Compound 2 (C-2), as used herein, refers to 5-((5-methylfuran-3-yl) methylene)-2- thioxodihydropyrimidine-4,6(lH,5H)-dione, having the structure:

[0076] Pharmaceutical Compositions

[0077] Some embodiments provide a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the compounds in Table 1, or a pharmaceutically acceptable salt thereof.

[0078] Some embodiments provide a pharmaceutical composition comprising a compound of Formula (II), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0079] Some embodiments provide a pharmaceutical composition comprising Compound 2 (C-2), and one or more pharmaceutically acceptable excipients.

[0080] Methods of Treatment

[0081] Also provided herein are methods for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as described herein. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the compounds in Table 1, or a pharmaceutically acceptable salt thereof.

[0082] Also provided herein are methods for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein Ring A is phenyl or 5-6 membered heteroaryl; m is 0 or 1;

[0083] R1is halogen, C1-C6 alkyl, -NRARB, or phenyl optionally substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl;

[0084] RAand RBare independently hydrogen and C1-C6 alkyl; and

[0085] R2and R3are independently hydrogen or allyl.

[0086] In some embodiments, Ring A is phenyl.

[0087] In some embodiments, Ring A is 5-6 membered heteroaryl. In some embodiments, Ring A is furanyl.

[0088] In some embodiments, m is 0. In some embodiments, m is 1.

[0089] In some embodiments, R1is halogen. In some embodiments, R1is iodo.

[0090] In some embodiments, R1is C1-C6 alkyl. In some embodiments, R1is methyl.

[0091] In some embodiments, R1is -NRARB.

[0092] In some embodiments, RAand RBare the same. In some embodiments, RAand RBare different. In some embodiments, RAand RBare each hydrogen. In some embodiments, RAand RBare each C1-C6 alkyl. In some embodiments, RAand RBare each methyl. In some embodiments, one of RAand RBis hydrogen and the other of RAand RBis C1-C6 alkyl.

[0093] In some embodiments, R1is phenyl optionally substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl.

[0094] In some embodiments, R1is unsubstituted phenyl.

[0095] In some embodiments, R1is phenyl substituted with 1-2 substituents independently selected from halogen, nitro, and C1-C6 alkyl.

[0096] In some embodiments, R1is phenyl substituted with 1-2 substituents independently selected from fluoro, chloro, nitro, and methyl.

[0097] In some embodiments, R2is hydrogen.

[0098] In some embodiments, R2is allyl.

[0099] In some embodiments, R3is hydrogen.

[0100] In some embodiments, R3is allyl.

[0101] In some embodiments, R2and R3are each hydrogen.

[0102] In some embodiments, one of R2and R3is hydrogen and the other of R2and R3is allyl.

[0103] Some embodiments provide a method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0104] Some embodiments provide a method for treating an inflammatory disorder in a subject, the method comprising:

[0105] (a) determining or having determined that the subject has an inflammatory disorder; and

[0106] (b) administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0107] Some embodiments provide a method for reducing inflammation in a subject having an inflammatory disorder, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0108] Some embodiments provide a method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0109] Some embodiments provide a method for treating an inflammatory disorder in a subject, the method comprising:

[0110] (a) determining or having determined that the subject has an inflammatory disorder; and

[0111] (b) administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0112] Some embodiments provide a method for reducing inflammation in a subject having an inflammatory disorder, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0113] Some embodiments provide a method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound 2 (C-2).

[0114] Some embodiments provide a method for treating an inflammatory disorder in a subject, the method comprising:

[0115] (a) determining or having determined that the subject has an inflammatory disorder; and (b) administering to the subject a therapeutically effective amount of Compound 2 (C-2).

[0116] Some embodiments provide a method for reducing inflammation in a subject having an inflammatory disorder, the method comprising administering to the subject a therapeutically effective amount of Compound 2 (C-2).

[0117] In some embodiments, the inflammatory disorder is an autoimmune disease, a metabolic disease, a neurodegenerative disease, or an inflammatory bowel disease.

[0118] In some embodiments, the inflammatory disorder is an autoimmune disease. In some embodiments, the autoimmune disease rheumatoid arthritis or lupus.

[0119] In some embodiments, the inflammatory disorder is a metabolic disorder. In some embodiments, the metabolic disorder is type 2 diabetes or obesity.

[0120] In some embodiments, the inflammatory disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer’s disease or Parkinson’s disease.

[0121] In some embodiments, the inflammatory disorder is an inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn’s disease or ulcerative colitis.

[0122] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a human.

[0123] Methods of Inhibiting

[0124] Some embodiments provide a method for inhibiting inflammasome activity in a cell comprising an inflammasome, the method comprising contacting the cell with an effective amount of a compound of Formula (I).

[0125] In some embodiments, the cell is in a subject and the contacting comprises administering to the subject a therapeutically effective amount of a compound of Formula

[0126] (I), or a pharmaceutically acceptable salt thereof.

[0127] Some embodiments provide a method for inhibiting inflammasome activity in a cell comprising an inflammasome, the method comprising contacting the cell with an effective amount of a compound of Formula (II).

[0128] In some embodiments, the cell is in a subject and the contacting comprises administering to the subject a therapeutically effective amount of a compound of Formula

[0129] (II), or a pharmaceutically acceptable salt thereof. Some embodiments provide a method for inhibiting inflammasome activity in a cell comprising an inflammasome, the method comprising contacting the cell with an effective amount of Compound 2 (C-2).

[0130] In some embodiments, the inflammasome activity comprises IL-ip / IL-18 secretion, ASC oligomerization, caspase-1 activation, and / or pyroptosis. In some embodiments, the inflammasome activity is IL-ip / IL-18 secretion, ASC oligomerization, caspase-1 activation, and / or pyroptosis. In some cases, pyroptosis can be measured by a lactate dehydrogenase (LDH) assay.

[0131] In some embodiments, the cell is in a sample from a subject. In some embodiments, the subject has been previously diagnosed as having, or is suspected of having, an inflammatory disease.

[0132] In some embodiments, the cell is in a subject and the contacting comprises administering to the subject a therapeutically effective amount of Compound 2 (C-2).

[0133] The disclosure will be further described in the following examples, which do not limit the scope of the subject matter described in the claims.

[0134] EXAMPLES

[0135] Example 1: Identification of Compound 2 (C-2) from a Covalent Fragment Library

[0136] Compound 2 (C-2) was identified by screening 1300 compounds from a Covalent Fragment Library. This library strikes a balance between reactivity and selectivity. It was created by singling out compounds with specific structural fragments (e.g., functional groups) that forms covalent bonds with Lys, Cys, Ser, His and Tyr residues which are often found in ligand binding domains. In addition, molecules with highly reactive electrophilic and nucleophilic groups (non- selective binders) as well as compounds with non-drug-like cores were discarded, using appropriate PAINS filters. Several molecules were identified that blocked NLRP3 driven responses using LPS + nigericin as an activating stimulus. One of these molecules is now prioritized for further follow up - compound 2 (C-2).

[0137] Example 2; Compound 2 (C-2) inhibited ASC activities

[0138] To test the effect of C-2 on inflammasome activity, human CD 14+ monocytes from peripheral blood were primed with LPS and then C-2 was added 1 hour before activating the canonical NLRP3 inflammasome with nigericin. The results showed that C-2 prevented lacate dehydrogenase (LDH) release (pyroptosis) (FIG. 1C) and IL-ip secretion (FIG. ID), both outputs of NLRP3 inflammasome activation. C-2 also impaired membrane permeabilization (FIG. IE) and blocked the processing of GSDMD preventing the formation of the pore forming GSDMD NT fragment (FIG. IF). The release of mature IL- ip as well as oligomerization of adaptor protein apoptosis associated speck-like protein (ASC) and activation of caspase-1 were also compromised after C-2 treatment (FIG. 1G). To examine if C-2 can impair common events downstream of the inflammasome activation cascade independent of the receptor that is activated, stimuli were tested that trigger pyroptosis and IL-ip release but require activation of NLRC4, AIM2 and Pyrin inflammasomes instead of NLRP3. While Salmonella typhimurium and double-stranded DNA were used to trigger activation of NLRC4 and AIM2 inflammasomes, respectively, Pyrin inflammasome was activated by TcdB, a RhoA-inhibiting toxins from Clostridioides difficile. The C-2 inhibitory effects were not restricted to the NLRP3 inflammasome pathway. C-2 also blocked AIM2, NLRC4 and PYRIN inflammasome-mediated IL-ip release (FIG. 1H), and ASC oligomer formation in LPS-primed BMDM (FIGs. II and 1 J), but did not block responses to non-canonical inflammasome pathway. These findings suggest that C-2 can inhibit ASC, since all these distinct inflammasome pathways are ASC dependent.

[0139] Example 3: Compound 2 (C-2) stabilized MPCP

[0140] A cell permeable C-2 alkyne probe (P-2) was synthesized and a chemoproteomic assay was used to identify C-2-bound targets using click chemistry in an unbiased manner. P-2 like C-2 inhibits NLRP3 inflammasome activation in human cells. Primary human CD14+ monocytes were activated for 3 hours with LPS, treated the last 1 hour with P-2, and later stimulated with nigericin for 2 hours. Lysates were harvested and “clicked” with biotin-Nsf in the presence of CuSC>4 and TBTA. The clicked lysates were then isolated on streptavidin agarose beads followed by on-bead trypsin digestion and subsequently subjected to LC / LC-MS / MS to assess P-2-interacting proteins. It was found that instead of binding directly to ASC, C-2 targeted and stabilized a mitochondrial phosphate carrier protein (MPCP) (FIG. 2A and 2B) that mediates proton-coupled symport of phosphate ions necessary for mitochondrial oxidative phosphorylation of ADP to ATP. To validate MPCP as a top hit, endogenous MPCP was pulled down from LPS+Nigericin-stimulated human cells incubated with different concentrations of P-2 and checked P-2 ability to label MPCP in cell-based system. Notably, labeling MPCP by P-2 was dose dependent even when used at concentration as low as 5uM (FIG. 2C). Competition experiment with P-2 and C-2 demonstrated that P-2 and C-2 competed for the same target (FIG. 2D). The reduction of P-2 signals by the treatment with unlabeled C-2 indicated that C-2 occupied the same site as P-2 on the target protein. Direct binding of P-2 and C-2 to MPCP was assessed by thermal shift assay in the lysates from HEK293T cells overexpressing Flag-tagged human MPCP. It was found that either P-2 or C-2 increased the melting temperature of MPCP indicating that both compounds stabilized MPCP (FIG. 2E). MPCP, in turn, plays a role in inflammasome biology. Cells lacking MPCP failed to produce IL-ip and did not die under NLRP3 inflammasome activation, phenocopying the effects seen in the C-2-treated cells (FIGS. 2F-2G). No changes were observed in the TNF-a levels, indicating that inflammasome is the main pathway affected (FIG. 2F). Moreover, Mpcp~'~ cells exhibited impaired GSDMD and caspase- 1 cleavage expressions (FIG. 2H).

[0141] Example 4: In vivo effects of Compound 2 (C-2)

[0142] To test the potential therapeutic capability of this novel inhibitor, different mouse models of inflammasome-driven diseases were used. Mice were intraperitoneal administered vehicle or C-2 at either 25 or 50mg / kg, followed by theadministration of LPS (FIGS. 3A-3C), MSU (FIGS. 3D-3G), or LPS+ATP (FIGS. 3H-3J). Intraperitoneal injection of those NLRP3 inflammasome activators induces peritoneal inflammation, marked by an increase in the cytokines levels as well an infiltration of neutrophils and monocytes in mice intraperitoneal fluid. To evaluate whether C-2 can suppress this inflammatory response, cytokine concentration and neutrophil / monocyte content were measured by ELISA and flow cytometry, respectively, after 2 hours (LPS), 6 hours (MSU) or 1 hour (LPS+ATP). The results showed that pre-treatment with C-2 reduced the serum concentration of IL-ip after LPS septic shock (FIG. 3 A) and MSU / ATP-induced peritonitis (FIG. 3B). Additionally, pre-treatment with C-2 also suppressed the recruitment of inflammatory cells, neutrophils and monocytes (FIGS. 3F and 3G), and decreased the amount of free extracellular ASC oligomers (FIG. 3 J).

[0143] Example 5: Structure activity relationships (SARs) for compound 2 (C-2)

[0144] To investigate the salient features of C-2, a small library of commercially available analogs of C-2 (see Table 1) was sourced from AA BLOCKS INC (9823 Pacific Heights Blvd., Suite F, San Diego, CA 92121 and Enamine Ltd 1 Distribution Way, Monmouth Jet., NJ 08852). The designs were based on (i) replacing the furan part of C-2 with aryl or diaryl groups (Ri) and (ii) having allylic group at R2 position (FIG. 4). Cell- based assays can be used to determine which part of the C-2 molecule can be modified to improve its potency, proteome wide selectivity, and pharmacokinetic (PK) properties.

[0145] Example 6: Analogs -I, -2, -3, -7, -8, -9, -11, -12 and -13 showed similar biological activity to C-2.

[0146] 5 To compare the biological activity of analogs shown in Table 1 with C-2, nigericin stimulated LPS-primed WT BMDMs treated with or without C-2 or the indicated analog were tested for LHD release and the production of IL-ip and TNFa. The LDH assay performed in mouse BMDM cells was used to calculate the IC50 values for the analogs shown in Table 1. Additionally, ASC oligomerization and caspase- 1 activation

[0147] 10 were also tested in LPS and nigericin stimulated WT BMDMs that were treated with C-2 or the indicated analog. It was found that analogs 1, 2, 3, 7, 8, 9, 11, 12, and 13 demonstrated similar biological activity to C-2 (FIGS. 5A-5G).

[0148] Table 1. Structures and ICso of Selected Compounds of Formula (I) and Formula

[0149] HD

[0150] 15 OTHER EMBODIMENTS

[0151] It is to be understood that while the subject matter of the present application has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the subject matter, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound 2 (C-2), which is 5-((5-methylfuran-3-yl) methylene)-2-thioxodihydropyrimidine-4,6(lH,5H)- dione, having the structure:

2. A method for treating an inflammatory disorder in a subject, the method comprising:(a) determining or having determined that the subject has an inflammatory disorder; and(b) administering to the subject a therapeutically effective amount of Compound 2 (C-2).

3. A method for reducing inflammation in a subject having an inflammatory disorder, the method comprising administering to the subject a therapeutically effective amount of Compound 2 (C-2).

4. The method of any one of claims 1-3, wherein the inflammatory disorder is an autoimmune disease, a metabolic disease, a neurodegenerative disease, or an inflammatory bowel disease.

5. The method of claim 4, wherein the inflammatory disorder is an autoimmune disease.

6. The method of claim 4 or 5, wherein the autoimmune disease rheumatoid arthritis or lupus.

7. The method of claim 4, wherein the inflammatory disorder is a metabolic disorder.

8. The method of claim 4 or 7, wherein the metabolic disorder is type 2 diabetes or obesity.

9. The method of claim 4, wherein the inflammatory disorder is a neurodegenerative disease.

10. The method of claim 4 or 9, wherein the neurodegenerative disease is Alzheimer’s disease or Parkinson’s disease.

11. The method of claim 4, wherein the inflammatory disorder is an inflammatory bowel disease.

12. The method of claim 4 or 11, wherein the inflammatory bowel disease is Crohn’s disease or ulcerative colitis.

13. The method of any one of claims 1-12, wherein the subject is a mammal.

14. The method of any one of claims 1-13, wherein the subject is a human.

15. A method for inhibiting inflammasome activity in a cell comprising an inflammasome, the method comprising contacting the cell with an effective amount of Compound 2 (C-2).

16. The method of claim 15, wherein the inflammasome activity comprises IL-ip / IL- 18 secretion, ASC oligomerization, caspase-1 activation, and / or pyroptosis.

17. The method of claim 15 or 16, wherein the cell is in a sample from a subject.

18. The method of claim 17, wherein the subject has been previously diagnosed as having, or is suspected of having, an inflammatory disease.

19. The method of claim 15, wherein the cell is in a subject and the contacting comprises administering to the subject a therapeutically effective amount of Compound 2 (C-2).

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