Use of preparations for inhibiting or blocking the interaction between FAAH and NLRP3

Abstract

The present invention provides the use of a formulation that inhibits or blocks the interaction between FAAH and NLRP3. This formulation destabilizes the NLRP3 protein and degrades it, thereby treating inflammatory diseases associated with NLRP3 inflammasome activation. The formulation can also effectively treat inflammatory diseases associated with NLRP3 protein mutations.

Description

Technical Field

[0001] The present invention relates to the use of an agent that inhibits or blocks the interaction between FAAH and NLRP3. Background Art

[0002] Inflammasomes are multiprotein signaling platforms that drive inflammatory responses in response to exogenous or endogenous danger signals. Of the multiple distinct inflammasomes identified, the NLRP3 inflammasome has been the most studied. In addition to NLRP3 as a sensor, the NLRP3 inflammasome also comprises an adaptor (ASC; also known as PYCARD) and an effector (caspase 1). Following assembly of the NLRP3 inflammasome, caspase 1 is cleaved and activated, leading to the activation and release of the proinflammatory cytokines IL-1β and IL-18, as well as pyroptosis. Mutations within and around the NACHT domain of NLRP3 render NLRP3 constitutively active, causing cold pyrin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome (FCAS), Mueller-Weissler syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID, also known as chronic infantile neurological cutaneous and arthritic syndrome or CINCA).

[0003] Although a lot has been learned about the NLRP3 inflammasome, there are still many unanswered questions. One of the questions is whether there is a mechanism to maintain the stability of the NLRP3 protein before the inflammasome is activated. Since dysregulated NLRP3 inflammasome activity drives the development of many inflammatory, metabolic, neurodegenerative and autoimmune diseases, it is crucial to control the activity of the NLRP3 inflammasome. According to this principle, the NLRP3 protein in the cell is usually maintained at a very low level, which is not enough to activate. In a process called "priming", Toll-like receptors recognize danger-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPS), triggering the synthesis of large amounts of NLRP3 protein, but these proteins are still in an inactive, non-assembled state. The cell receives stimulation from a second signal, which leads to the activation and assembly of the NLRP3 inflammasome ( Figure 1), this delicate "two-signal" control mechanism ensures that NLRP3-induced inflammation is initiated only when absolutely necessary. One plausible hypothesis is that NLRP3 is inherently unstable and that cellular mechanisms maintain its stability, but this mechanism is currently poorly understood. Previous studies have reported that the assembled NLRP3 inflammasome complex undergoes autophagic degradation during activation, but how the unassembled NLRP3 protein maintains its stability prior to activation is less understood. Another question is the cellular localization of NLRP3. It is generally believed that NLRP3 is present on the membranes of mitochondria and the endoplasmic reticulum prior to inflammasome assembly, but the proteins that anchor NLRP3 to mitochondria and the endoplasmic reticulum remain a matter of debate. Published literature suggests that mitochondrial antiviral signaling (MAVS), cardiolipin, mitochondrion proteins, and STING can all anchor NLRP3 to mitochondria or the endoplasmic reticulum, but the anchoring effect of these molecules on NLRP3 may be limited to certain specific circumstances, such as during viral infection, or may only be effective for certain NLRP3 proteins.

[0004] Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a key regulatory role in the endocannabinoid system. It is primarily localized in the endoplasmic reticulum and mitochondria. It inactivates endogenous signaling lipids such as endocannabinoids (AEA) by hydrolyzing various fatty acid amides, thereby regulating various physiological processes such as pain, feeding, blood pressure, mood, and sleep. Whether FAAH affects NLRP3 protein stability or inflammasome activation has not yet been reported. Summary of the Invention

[0005] The present invention provides the use of an agent for inhibiting or blocking the interaction between FAAH and NLRP3.

[0006] To achieve the above objectives, the technical solution adopted is: use of a preparation that inhibits or blocks the interaction between FAAH and NLRP3 in screening drugs for treating diseases caused by NLRP3 inflammasome.

[0007] The present invention provides use of a preparation that inhibits or blocks the interaction between FAAH and NLRP3 in screening drugs for treating diseases caused by NLRP3 protein mutations.

[0008] The present invention provides use of a preparation for inhibiting or blocking the interaction between FAAH and NLRP3 in the preparation of a medicament for treating diseases caused by the NLRP3 inflammasome.

[0009] The present invention provides use of a preparation for inhibiting or blocking the interaction between FAAH and NLRP3 in preparing a drug for treating diseases caused by NLRP3 protein mutations.

[0010] Preferably, the disease caused by NLRP3 inflammasome is type II diabetes, gout, Alzheimer's disease, atherosclerosis, Parkinson's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, asthma, chronic obstructive pulmonary disease, nephritis, enteritis, or hepatitis.

[0011] Preferably, the disease caused by NLRP3 protein mutation is cryopyrin-associated periodic syndrome.

[0012] Preferably, the disease caused by NLRP3 protein mutation is familial cold autoinflammatory syndrome (FCAS), Muir-Wei syndrome (MWS) and chronic infantile neurological cutaneous and arthritic syndrome (CINCA).

[0013] Preferably, the agent that inhibits or blocks the interaction between FAAH and NLRP3 includes at least one of 3'-carbamoylbiphenyl-3-ylcyclohexylcarbamate, biochanin A, N-benzylpalmitamide, N-(4-chloro-3-pyridinyl)-4-[(2,2-difluoro-1,3-benzodioxol-5-yl)methyl]-1-piperazinecarboxamide, 4-morpholinyl-1,2,5-thiadiazol-3-ylcyclooctyl(methyl)carbamate and N-3-pyridazinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methylene]-1-piperidinecarboxamide.

[0014] Preferably, the agent that inhibits or blocks the interaction between FAAH and NLRP3 comprises 3'-carbamoylbiphenyl-3-ylcyclohexylcarbamate.

[0015] Preferably, the agent for inhibiting or blocking the interaction between FAAH and NLRP3 comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.

[0016] Beneficial effects:

[0017] The present invention uses a preparation that blocks the interaction between FAAH and NLRP3, causing the NLRP3 protein to be in an unstable state and degraded, thereby treating NLRP3 inflammasome-related diseases. Specifically, the NLRP3 protein produced in the cell after the first signal stimulation is in an inherently unstable state, and its stability depends on the interaction with the membrane enzyme FAAH, thereby being anchored to the mitochondrial and endoplasmic reticulum membranes; blocking the interaction between NLRP3 and FAAH can cause the NLRP3 protein to dissociate from the mitochondrial and endoplasmic reticulum membranes, and then cause K48 ubiquitination of the NLRP3 protein through the action of E3 ubiquitin protein ligase (CHIP). Finally, it is pulled into the autophagosome for degradation through the action of E3 ubiquitin protein ligase (NBR1), resulting in a significant decrease in the level of NLRP3 protein in the cell. After receiving the second stimulation signal, the cell is unable to assemble into a fully active inflammasome, thereby achieving the effect of treating NLRP3 inflammasome-related diseases. In addition, unlike many currently known drug molecules that work by inhibiting the endogenous ATPase activity of the NLRP3 protein (which are only effective against diseases caused by non-mutated NLRP3 proteins), the preparations used in the present invention that inhibit or block the interaction between FAAH and NLRP3 are effective against diseases caused by both mutant and non-mutated NLRP3 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This study showed that NLRP3 expression in macrophages is essentially negative under non-stimulated conditions. Stimulation with the first signal leads to high expression but lacks inflammasome activation. Only after stimulation with the second signal does NLRP3 become activated to produce inflammatory cytokines. Immunoblot analysis of NLRP3 and GAPDH in primed (mock) BMDM treated with or without LPS was performed.

[0019] Figure 2 FAAH knockout results in reduced NLRP3 inflammasome activation in this animal model. ELISA was used to measure IL-1β and TNF-α levels in the peritoneal cavity of WT, FAAH+ / -, and FAAH- / - BMDM mice. Serum: serum.

[0020] Figure 3 FAAH knockdown results in NLRP3 protein degradation. Figure (a) Immunoblot analysis of NLRP3, FAAH, and GAPDH in WT, FAAH+ / -, and FAAH- / - BMDM. Figure (b) Immunoblot analysis of NLRP3 and GAPDH in LPS-primed (mock) WT and FAAH- / - BMDM with or without CHX co-treatment. Figure (c) Immunoblot analysis of NLRP3, FAAH, and GAPDH in LPS-primed (mock) WT and FAAH- / - BMDM with or without nigericin co-treatment.

[0021] Figure 4 To demonstrate that FAAH knockdown results in reduced NLRP3 inflammasome activation in cells, ELISA was used to measure IL-1β levels in the supernatants of WT, FAAH+ / -, and FAAH- / - BMDMs following LPS stimulation, LPS stimulation followed by nigericin stimulation, or LPS stimulation followed by 3-MA followed by nigericin stimulation (Nigericin+3MA). Mean ± SEM, n = 3.

[0022] Figure 5 Demonstrating that FAAH knockdown results in NLRP3 protein degradation dependent on autophagy rather than the proteasome pathway, (a) Immunoblotting analysis of NLRP3 and GAPDH in LPS-primed (mock) FAAH- / - BMDMs with or without co-treatment with 3MA, CQ, or wortmannin. (b) Immunoblotting analysis of NLRP3 and GAPDH in LPS-primed (mock) FAAH- / - BMDMs transfected with scrambled or ATG5-specific siRNA.

[0023] Figure 6 To show that FAAH knockdown leads to increased K48 ubiquitination of NLRP3 protein, which is required for NLRP3 protein degradation, cell lysates from untreated or LPS-primed WT and FAAH- / - BMDMs were immunoprecipitated with control (isotype) or NLRP3-specific antibodies and then immunoblotted with K48-Ub, K63-Ub, or NLRP3 antibodies.

[0024] Figure 7 Demonstrating that FAAH-induced NLRP3 knockdown requires the E3 ligase CHIP. Figure (a) Proteins extracted from BMDM and FAAH- / - BMDMs were immunoprecipitated with an NLRP3-specific antibody and then immunoblotted with NLRP3, NBR1, and CHIP antibodies. Figure (b) Immunoblotting analysis of NLRP3, CHIP, and GAPDH in LPS-primed FAAH- / - BMDMs transfected with CHIP-specific siRNA.

[0025] Figure 8 To demonstrate that FAAH knockdown-induced NLRP3 degradation requires the selective receptor protein NBR1, immunoblot analysis of NLRP3, NBR1, and GAPDH was performed in LPS-primed FAAH- / - BMDM transfected with NBR1-specific siRNA. Input refers to unprecipitated cell lysate.

[0026] Figure 9Figure 3: FAAH interacts with and stabilizes NLRP3. (a) Cell lysates from LPS-primed BMDMs were immunoprecipitated with isotype- or NLRP3-specific antibodies, followed by immunoblotting for NLRP3 and FAAH. (b) Cell lysates from untreated or LPS-primed (mock) BMDMs were immunoprecipitated with FAAH-specific antibodies, followed by immunoblotting for NLRP3 and FAAH. (c) Proximity ligation assay (PLA) of NLRP3-FAAH interaction in LPS-primed WT, FAAH+ / -, and FAAH- / - BMDMs; scale bar, 20 μm.

[0027] Figure 10 Domain mapping analysis showing the interaction between FAAH and NLRP3 led to the design of AS-1 peptides. Figures a and b show co-transfection of a Flag-tagged NLRP3 construct and an HA-tagged FAAH construct into HEK293T cells. Immunoblot analysis of HA and Flag proteins in cell lysates immunoprecipitated with an anti-Flag antibody.

[0028] Figure 11 Demonstrating the interaction between mutant NLRP3 and FAAH in cells. (a) HEK293T cells were cotransfected with Flag-tagged wild-type and three mutant NLRP3 constructs and an HA-tagged FAAH construct. Immunoblotting analysis of HA and Flag proteins in cell lysates immunoprecipitated with anti-Flag antibodies. (b) LPS-primed NLRP3-R258W knock-in BMDMs were immunoprecipitated with isotype- or NLRP3-specific antibodies, followed by immunoblotting analysis of NLRP3-R258W and FAAH. (c) PLA of the NLRP3-R258W-FAAH interaction in LPS-stimulated NLRP3-R258W knock-in BMDMs; scale bar, 20 μm.

[0029] Figure 12 Immunoblot analysis of NLRP3 and GAPDH in WT, FAAH+ / -R258W, and FAAH- / -R258W BMDMs showed that FAAH knockdown leads to mutant NLRP3 protein degradation.

[0030] Figure 13FAAH knockout results in reduced mutant NLRP3 inflammasome activation and ameliorated CAPS disease symptoms in an animal model. a, Photographs of WT, NLRP3-R258W, FAAH+ / -R258W, and FAAH- / -R258W BMDM mice at 12 weeks of age. b, Axillary lymph nodes, spleen, and liver of WT, NLRP3-R258W, FAAH+ / -R258W, and FAAH- / -R258W BMDM mice.

[0031] Figure 14 Figure 3. FAAH knockout results in reduced mutant NLRP3 inflammasome activation in cells. a, ELISA analysis of IL-1β in supernatants of WT, NLRP3-R258W, FAAH+ / -R258W, and FAAH- / -R258W BMDMs after 3 hours of LPS stimulation. Mean ± SEM, n = 3, **p < 0.01. b, Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and pro-caspase-1 and pro-IL-1β in cell lysates (lysates) of LPS-stimulated BMDMs from WT, NLRP3-R258W, FAAH+ / -R258W, and R258WFAAH- / -R258W mice. Lysis indicates precipitate.

[0032] Figure 15 Figure 1 shows that FAAH knockdown leads to the dissociation of NLRP3 protein from mitochondrial and endoplasmic reticulum membranes. (a) Immunoblotting analysis of NLRP3, CRT, TOM20, and GAPDH from cytosolic and membrane fractions of cell lysates from LPS-induced WT and FAAH- / - BMDMs. (b) Immunoprecipitation of cell lysates from WT and FAAH- / - BMDMs with anti-NLRP3 antibodies, followed by immunoblotting analysis of NLRP3, CRT, and TOM20. Cells were left unstimulated (control) or stimulated with LPS for 3 hours. (c and d) NLRP3-TOM20 interaction (PLA) (c) and NLRP3-CRT interaction (d) in LPS-induced WT and FAAH- / - BMDMs; scale bar, 20 μm.

[0033] Figure 16 Figure 3. Mutant NLRP3 interacts with mitochondrial and endoplasmic reticulum membranes in cells expressing normal FAAH. a and b, PLA (a) and NLRP3-TOM20 interaction (b) in LPS-primed WT, FAAH+ / -R258W, and FAAH- / -R258W BMDMs; scale bar, 20 μm.

[0034] Figure 17Effects of different FAAH inhibitors on NLRP3 protein degradation. Fourteen FAAH inhibitors were tested: JZL195, URB597, LY2183240, PF3845, Biochanin A, N-Benzyllinolenamide, Carprofen, BIA10-2474, FAAH-IN-2, N-Benzylpalmitamide, 1-monomyristin, JNJ-42165279, JZL-184, JZP-430, PF-04457845, and SA47. Six of these inhibitors, URB597, Biochanin A, N-Benzylpalmitamide, JNJ-42165279, JZP-430, and PF-04457845, showed significant activity, as defined by their ability to reduce NLRP3 protein levels by more than 30% at a concentration of 40 μM.

[0035] Figure 18 Figure 5 shows that URB597 causes the dissociation of NLRP3 protein from FAAH. PLA of NLRP3-FAAH interaction in LPS-primed WT BMDM with or without URB597 treatment. Scale bar, 20 μm.

[0036] Figure 19 URB597 reduces NLRP3 inflammasome activation in cells. a, Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and pro-caspase-1 and pro-IL-1β in cell lysates (lysates). BMDMs were stimulated with LPS for 3 hours, then treated with the indicated concentrations of URB597 for an additional 3 hours, and then challenged with nigericin for 30 minutes. b, ELISA analysis of IL-1β in supernatants of BMDMs stimulated with LPS for 3 hours, then treated with the indicated concentrations of URB597 for 3 hours, and then challenged with nigericin for 30 minutes. Mean ± SEM, n = 3, ***p < 0.001.

[0037] Figure 20 URB597-induced NLRP3 protein degradation is mediated by autophagy. Figure 1: Immunoblot analysis of NLRP3 and GAPDH in LPS-primed WT BMDMs with or without co-treatment with URB597, 3MA, CQ, or wortmannin.

[0038] Figure 21URB597 is shown to cause mutant NLRP3 protein degradation in mouse BMDM cells. Immunoblot analysis of NLRP3-R258W and GAPDH was performed in NLRP3-R258W BMDMs stimulated with LPS and then treated with the indicated doses of URB597 for an additional 3 hours.

[0039] Figure 22 URB597 induces dissociation of NLRP3 protein from FAAH in CAPS patient monocytes. Figure 1. PLA of the NLRP3-FAAH interaction in PBMCs from patient 1. Cells were stimulated with LPS for 3 hours and then treated with 40 μM URB597 for an additional 3 hours.

[0040] Figure 23 URB597 reduces NLRP3 inflammasome activation in monocytes from patients with CAPS. a, ELISA of IL-1β in supernatants of PBMCs isolated from patient 1 and her healthy mother. Cells were stimulated with LPS for 3 hours and then treated with 40 μM URB597 or 10 μM MCC950 for an additional 3 hours. Mean ± SEM, n = 3, ***p < 0.001. NS, not significant. b and c, PBMCs from patients 2 (b) and 3 (c), respectively, were stimulated with LPS for 3 hours and then treated with 40 μM URB597 for 3 hours. N2 is the healthy mother of patient 2, and N3 is an unrelated healthy boy. Mean ± SEM, n = 3, ***p < 0.001.

[0041] Figure 24 AS-1 peptides are shown to cause dissociation of NLRP3 protein from FAAH. Figure 1: PLA of the NLRP3-FAAH interaction in LPS-primed WT BMDM treated with or without AS-1 peptides.

[0042] Figure 25 AS-1 peptides induce NLRP3 protein degradation. Figure 1: Immunoblot analysis of NLRP3 and GAPDH in LPS-primed WT BMDMs treated with or without AS-1 peptides for different durations and concentrations.

[0043] Figure 26 AS-1 peptides were shown to reduce NLRP3 inflammasome activation in cells. Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and pro-caspase-1 and pro-IL-1β in cell lysates (lysates). BMDMs were stimulated with LPS for 3 hours, then treated with various concentrations of AS-1 peptides for an additional 3 hours, followed by nigericin stimulation for 30 minutes. DETAILED DESCRIPTION

[0044] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0045] The structural formula of 3'-carbamoylbiphenyl-3-ylcyclohexylcarbamate (URB597) is as follows (I):

[0046]

[0047] The structural formula of tert-butyl 4-nitrophenyl 4-(3-phenoxybenzyl)piperazine-1-carboxylate (JZL195) is as follows (II):

[0048]

[0049] The structural formula of 5-([1,1'-biphenyl]-4-methyl)-N,N-dimethyl-1H-tetrazolyl-1-carboxamide (LY2183240) is as follows (III):

[0050]

[0051] The structural formula of N-3-pyridine-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide (PF-3845) is as follows (IV):

[0052]

[0053] The structural formula of biochanin A is as follows (V):

[0054]

[0055] The structural formula of N-Benzyl-(9Z,12Z,15Z)-octadecatrieamide is as follows (VI):

[0056]

[0057] The structural formula of Carprofen is as follows (VII):

[0058]

[0059] The structural formula of 3-(1-(cyclohexyl(methylcarbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide (BIA10-2474) is as follows (VIII):

[0060]

[0061] The structural formula of 4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-ol (FAAH-IN-2) is as follows:

[0062]

[0063] The structural formula of N-Benzylpalmitamide is as follows:

[0064]

[0065]

[0066] The structural formula of 1-monomyristin is as follows:

[0067]

[0068] The structural formula of N-(4-chloro-3-pyridyl)-4-[(2,2-difluoro-1,3-benzodioxol-5-yl)methyl]-1-piperazinecarboxamide (JNJ-42165279) is as follows:

[0069]

[0070] The structural formula of 4-nitrophenyl 4-[bis(1,3-benzodioxazol-5-yl)hydroxymethyl]-1-piperidinic acid ester (JZL-184) is as follows (XIII):

[0071]

[0072] The structural formula of 4-Morpholino-1,2,5-thiadiazol-3-yl cyclooctyl (methyl) carbamate (JZP-430) is as follows (XIV):

[0073]

[0074] The structural formula of N-3-pyridazinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridyl]oxy]phenyl]methylene]-1-piperidinecarboxamide (PF-04457845) is as follows (XV):

[0075]

[0076] The structural formula of 2-(methylamino)-2-oxoethyl {2-[1-(6-methyl-2-pyridyl)-4-piperidinyl]ethyl}carbamate (SA47) is as follows (XVI):

[0077]

[0078] Example 1

[0079] 1. Loss of FAAH leads to NLRP3 degradation

[0080] To evaluate the potential role of FAAH in NLRP3 inflammasome activation, we knocked down FAAH in mouse bone marrow-derived macrophages (BMDMs) using short-chain RNA (shRNA). This resulted in a significant reduction in the secretion of nigericin-triggered cleaved caspase 1 and IL-1β following lipopolysaccharide (LPS) priming, suggesting that FAAH may influence NLRP3 inflammasome activation. To systematically investigate the effects of FAAH loss, we generated FAAH knockout mice (FAAH ko-1) by deleting exons 15 to 2 of the FAAH gene using CRISPR-Cas9 technology. We verified the knockout status by genomic PCR and genome sequencing. FAAH- / - mice exhibited reduced FAAH enzyme activity and exhibited enhanced hypothermia responses and reduced pain sensitivity in both the tail immersion and hot plate tests following AEA challenge. FAAH- / - mice also exhibited low reproductive capacity, whereas FAAH+ / - mice reproduced normally. Compared with BMDM derived from wild-type mice, nigericin-triggered IL-1β release after LPS priming was reduced by approximately 50% and 70% in BMDM carrying heterozygous (FAAH+ / -) and homozygous (FAAH- / -) loss of FAAH, respectively. A significant reduction in IL-1β secretion was also observed in FAAH- / - BMDM after ATP and MSU challenge. In addition, loss of FAAH also reduced IL-18 release and pyroptosis. Consistent with these results, we also observed that serum IL-1β levels were reduced by approximately 45% and 70% after systemic LPS administration in FAAH- / - and FAAH- / - mice, respectively. Figure 2 In contrast, FAAH knockout did not increase serum TNF-α levels after systemic LPS administration. Loss of FAAH had no effect on NLRP3 mRNA levels, but NLRP3 protein was reduced, as approximately 45% and 65% reductions in NLRP3 protein levels were observed in FAAH+ / - and FAAH- / -, respectively ( Figure 3a). Furthermore, in the presence of the protein synthesis inhibitor cycloheximide, FAAH depletion accelerated NLRP3 reduction (Figure 3b), indicating that enhanced protein degradation is the underlying cause of the reduced protein levels. Overall, these results suggest that FAAH depletion inhibits NLRP3 inflammasome activity by inducing NLRP3 degradation.

[0081] Notably, while we observed an approximately 50% reduction in FAAH protein in FAAH+ / - cells, in FAAH- / - bone marrow cells and bone marrow-derived macrophages (BMDM), a result that was expected, we consistently detected a single protein band (Protein X) from FAAH- / - mice at the same position as FAAH on Western blots using three different commercially available antibodies. Protein X was also detected in the heart of FAAH- / - mice but was absent from other tissues we examined, including brain and liver, which express high levels of FAAH in wild-type mice. A second FAAH knockout mouse (FAAH ko-2), created by another company using a similar but slightly different strategy, also expressed Protein X in BMDM but absent from major tissues, including brain, liver, and kidney. Kupffer cells isolated from FAAH- / - mice did not express Protein X and showed no detectable levels of NLRP3 protein after LPS priming. Figure 3 c), and IL-1β release was reduced by nearly 90% after nigericin challenge ( Figure 4 ), indicating that FAAH loss has a greater impact on NLRP3 in Kupffer cells than in BMDM. Notably, a second FAAH gene has been identified in humans and many other species, but not in mice and rats. The identity of Protein X and its possible role in NLRP3 stability and activation are still under investigation.

[0082] 2. NLRP3 degradation through selective autophagy

[0083] To explore the molecular mechanism of NLRP3 degradation caused by FAAH deficiency, we evaluated the potential role of proteasome and autophagy, two cellular mechanisms responsible for protein degradation. Autophagy inhibitors 3-MA, wortmannin, and CQ, as well as ATG5 knockdown, increased NLRP3 protein levels in FAAH- / - BMDMs, suggesting a weakening effect of autophagy on NLRP3 degradation. Figure 5 a and 5b). In contrast, the proteasome inhibitors MG132, PS341, and b-AP15 all further reduced NLRP3 levels in these cells ( Figure 5c), indicating that autophagic degradation is enhanced after proteasome inhibition. These results indicate that loss of FAAH induces autophagic rather than proteasomal degradation of NLRP3. Since NLRP3 degradation in FAAH- / -BMDMs showed high specificity, while no reduction in the levels of NLRC4, AIM2, ASC, and NEK7 was observed, we speculate that this degradation is carried out through selective autophagy, which is usually accomplished by delivering ubiquitinated cargo to autophagosomes through specific autophagy receptors. Supporting this hypothesis, the degradation of NLRP3 in FAAH- / -BMDMs was reduced by the ubiquitination inhibitor PYR41, while increased by the deubiquitination inhibitor PR619. In addition, NLRP3 showed enhanced K48 ubiquitination, but no K63 ubiquitination, in FAAH- / -BMDMs compared with FAAH+ / +BMDMs ( Figure 6 Dopamine has been reported to induce ubiquitination-dependent autophagic degradation of NLRP3 in macrophages, and two of the many known E3 ligases, MARCH7 and CHIP, were associated with NLRP3 after dopamine treatment (but only MARCH7 was responsible for dopamine-induced NLRP3 degradation, a result we were able to replicate). We found that CHIP, but not MARCH7, bound to NLRP3 in FAAH- / - BMDMs ( Figure 7 a). In addition, CHIP knockdown reduced NLRP3 degradation induced by FAAH loss ( Figure 7 b). To identify the autophagy receptors involved, we evaluated p62, NBR1, and NDP52, three possible candidates. In FAAH- / - BMDM, NBR1 and NDP52, but not p62, were found to show increased interaction with NLRP3 ( Figure 7 a). However, only knockdown of NBR1, but not NDP52, reduced NLRP3 degradation ( Figure 8 Thus, loss of FAAH induces NLRP3 degradation through selective autophagy mediated by CHIP as an E3 ligase and NBR1 as an autophagy receptor.

[0084] 3. FAAH interacts with and stabilizes NLRP3 protein

[0085] To further understand how loss of FAAH leads to NLRP3 degradation, we searched for proteins that interact with NLRP3. Unexpectedly, we found that NLRP3 interacts with FAAH in BMDM primed with NLRP3, as shown by co-IP analysis ( Figure 9In support of this, FLAG-tagged NLRP3 colocalized with HA-tagged FAAH when coexpressed in HEK293T cells. Proximity ligation assay (PLA) further revealed that the NLRP3-FAAH interaction was strong in wild-type BMDMs, significantly weakened in FAAH+ / - BMDMs, and nearly absent in FAAH- / - BMDMs. Figure 9 c). This result indicates that protein X does not interact with NLRP3, or rather, that the FAAH antibody recognizes protein X in immunoblotting but not in PLA. NLRP3 was also found to associate with FAAH in the mouse macrophage cell line J774A.1. The interaction between NLRP3 and FAAH is highly selective, as FAAH does not interact with other inflammasome-associated proteins (including ASC, caspase 1, NEK7, NLRC4, and AIM2). Conversely, NLRP3 does not interact with monoacylglycerol lipase (MAGL), a hydrolase in the endocannabinoid system. To further understand the NLRP3-FAAH interaction, we performed mapping studies. The NACHT domain of NLRP3, spanning the region between aa 91 and aa 710, is both necessary and sufficient for interaction with FAAH. Further mapping revealed that the region between aa 494 and aa 710 is sufficient for the NLRP3-FAAH interaction. We also identified that the region between aa 150 and aa 265 of FAAH is critical for interaction with NLRP3, as the aa 1 to 265 fragment of FAAH interacted with full-length NLRP3, whereas the aa 1 to 150 fragment did not ( Figure 10 a, Figure 10 b). Interestingly, this region constitutes the majority of the amidase signature (AS) sequence (residues 134-257 of FAAH), a contiguous sequence of approximately 130 amino acids found in a large number of enzymes of the AS family, most of which are of bacterial and fungal origin.

[0086] These results strongly suggest that NLRP3 is intrinsically unstable and stabilized by its interaction with FAAH. To strengthen this conclusion, we reintroduced FAAH into FAAH- / - BMDM via a lentiviral expression system. Overexpression of FAAH in these cells rescued its ability to stabilize NLRP3, resulting in higher levels of NLRP3 after LPS priming and, correspondingly, higher IL-1β release after nigericin challenge than that observed in FAAH+ / + BMDM. In contrast, overexpression of FAAH-S241A, which harbors an inactivating mutation at residue 39 of the key catalytic nucleophile Ser241 and exhibits reduced NLRP3 binding when expressed in 293T cells, was ineffective in stabilizing NLRP3 in mouse BMDM or restoring nigericin-induced NLRP3 inflammasome activation. These results suggest that FAAH requires enzymatic activity to interact with and stabilize NLRP3. However, we cannot exclude the possibility that the protein conformational change affected by the S241A mutation, rather than the loss of enzymatic activity, is more critical for reducing NLRP3 interaction.

[0087] 4. FAAH also interacts with and stabilizes NLRP3-R258W

[0088] To assess whether FAAH also interacts with CAPS-associated mutant NLRP3, we expressed HA-tagged FAAH in 293T cells along with one of three FLAG-tagged murine NLRP3 mutants (NLRP3-R258W, NLRP3-T346M, and NLRP3-F521L). These correspond to the human NLRP3-R260W, NLRP3-T348M, and NLRP3-F523L mutations found in patients with FCAS, MWS, and NOMID, respectively. Co-IP analysis revealed that FAAH bound to all three NLRP3 mutant proteins ( Figure 11 a), suggesting that interaction with FAAH may be a common feature of CAPS-associated NLRP3 proteins. In addition, we found that NLRP3-R258W strongly interacted with FAAH in BMDM derived from NLRP3-R258W knock-in mice ( Figure 11 b and 11c). In LPS-primed BMDM, the levels of NLRP3-R258W were comparable to wild-type NLRP3, indicating that FAAH has similar ability to stabilize wild-type and mutant NLRP3 in these cells.

[0089] To evaluate the effects of FAAH loss on mutant NLRP3, we generated R258W FAAH+ / - mice by crossing NLRP3-R258W mice with FAAH- / - mice. In LPS-primed BMDM isolated from R258W FAAH+ / - mice, heterozygous loss of FAAH reduced NLRP3-R258W protein levels by 40% ( Figure 12 ), IL-1β release induced by nigericin was reduced by 50%. Notably, heterozygous loss of FAAH was sufficient to significantly reduce the inflammatory phenotype exhibited by the NLRP3-R258W mutation. Consistent with the reported results, NLRP3-R258W mice showed wrinkled coats, hair loss, smaller body weight and body mass, and severe skin inflammation, but FAAH+ / -R258W mice showed significant improvement in all these aspects (representative photos as shown in Figure 2). Figure 13 NLRP3-R258W mice also showed enlargement of axillary lymph nodes, spleen, and liver, whereas these organs removed from FAAH+ / -R258W mice were significantly smaller (but still significantly larger than those in WT mice; Figure 13 b) Histological studies of NLRP3-R258W mice showed thickening of the epidermis and dermis in affected skin and prominent portal inflammation in the liver, whereas these features were almost absent in FAAH+ / -R258W mice. We are currently obtaining FAAH- / -NLRP3-R258W mice. Furthermore, FAAH knockout resulted in reduced activation of the mutant NLRP3 inflammasome in cells ( Figure 14 a, Figure 14 b). In summary, the above results demonstrate that FAAH plays a key role in stabilizing CAPS-associated NLRP3-R258W protein.

[0090] 5. FAAH anchors NLRP3 to mitochondrial and ER membranes

[0091] The interaction between NLRP3 and FAAH prompted us to evaluate how FAAH, an intracellular membrane protein known to associate with the ER and mitochondria, affects the cellular localization of NLRP3. Using a simple differential centrifugation protocol, we separated cell lysates from LPS-primed BMDMs into cytosolic and membrane fractions after removal of large cellular debris. The majority of NLRP3 protein was present in the membrane fraction, which contained mitochondria but not the ER, suggesting that mitochondria may be more involved in the cellular localization of NLRP3 than the ER. Importantly, depletion of FAAH resulted in an increase in the cytosolic fraction of NLRP3 protein, whereas depletion of FAAH resulted in a decrease in the membrane fraction ( Figure 15a). Consistent with this result, a decreased NLRP3-TOM20 interaction was observed in FAAH- / -BMDM compared with FAAH+ / +BMDM, as shown by co-IP and PLA analysis ( Figure 15 b, 15c and 15d), indicating that NLRP3 is associated with mitochondria after FAAH loss. We also found that NLRP3-ER interaction was similarly reduced in FAAH- / - BMDMs using the ER marker CRT ( Figure 15 b and 15d). FAAH appears to be essential for inflammasome activation, as NLRP3, which is "protected" from degradation by the autophagy inhibitor 3-MA in FAAH- / - BMDM, exhibits considerable ability to elicit IL-1β release in response to nigericin challenge. This result also strongly suggests that the inhibitory effect of FAAH loss on NLRP3 inflammasome activation is due to NLRP3 protein degradation rather than increased levels of AEA and other FAAs hydrolyzed by FAAH, as the increase in these FAAs is caused by FAAH loss and is unlikely to be affected by 3-MA treatment. Similarly, the NLRP3 degradation effect caused by FAAH loss is unlikely due to increased FAAs. In support, AEA is unable to trigger NLRP3 degradation. Consistent with the finding that FAAH interacts with CAPS-mutated NLRP3, FAAH also plays a role in anchoring NLRP3-R258W to mitochondrial and ER membranes ( Figure 16 a,16b).

[0092] 6. Selective FAAH inhibitors induce NLRP3 degradation

[0093] The fact that the inactivating S241A mutation inactivates FAAH in stabilizing NLRP3 prompted us to evaluate the potential NLRP3 degradation activity of FAAH inhibitors. Fourteen FAAH inhibitors were tested, including JZL195, URB597, LY2183240, PF3845, Biochanin A, N-Benzyllinolenamide, Carprofen, BIA10-2474, FAAH-IN-2, N-Benzylpalmitamide, 1-monomyristin, JNJ-42165279, JZL-184, JZP-430, PF-04457845, and SA47. Six of them, including URB597, Biochanin A, N-Benzylpalmitamide, JNJ-42165279, JZP-430, and PF-04457845, showed significant activity, as defined by the ability to reduce NLRP3 protein levels by more than 30% at a concentration of 40 uM ( Figure 17However, no clear correlation was observed between the reported IC50 values for FAAH inhibition and their activity in inducing NLRP3 degradation. Whether the ability to inhibit FAAH enzymatic activity affects NLRP3 degradation requires further investigation. JZL195, URB597, LY2183240, PF3845, Biochanin A, N-Benzyllinolenamid, Carprofen, BIA10-2474, FAAH-IN-2, N-Benzylpalmitamide, 1-monomyristin, JNJ-42165279, JZL-184, JZP-430, PF-04457845, and SA47 were all purchased from MCE.

[0094] The most active compound tested, URB597, was selected for further investigation. In addition to mouse BMDMs, URB597 dose-dependently reduced NLRP3 protein levels in mouse peritoneal macrophages and two other macrophage cell lines we had studied. Furthermore, URB597 dose-dependently increased autophagy in mouse BMDMs, and the NLRP3-reducing effect of URB597 was significantly abolished by an autophagy inhibitor, but not a proteasome inhibitor. Figure 5 a, 5b, and 5c). Similar to the case of FAAH loss, URB597 treatment resulted in enhanced K48 ubiquitination of NLRP3 and promoted the binding of CHIP and NBR1 to NLRP3, indicating the same selective autophagy mechanism. Not surprisingly, URB597 disrupted the endogenous NLRP3-FAAH interaction in mouse BMDM ( Figure 18 On the other hand, dopamine did not disrupt the NLRP3-FAAH interaction.

[0095] 7. URB597 inhibits wild-type and CAPS-associated mutant NLRP3 inflammasome activation

[0096] As expected from its superior ability to trigger NLRP3 degradation, URB597 dose-dependently inhibited the secretion of IL-1β and cleaved caspase 1 triggered by NLRP3-specific stimuli, including niger, ATP, MSU, and alum, in LPS-primed mouse BMDM ( Figure 19 a and Figure 19 b). URB597 also inhibited apoptosis induced by nigericin. Importantly, 3-MA abolished the NLRP3 degradation effect of URB597 ( Figure 20), was able to “rescue” NLRP3 inflammasome activation that was inhibited by URB597. This provides additional evidence supporting the view that NLRP3 protein degradation, rather than increased AEA and other FAAs, is responsible for the observed NLRP3 inflammasome inhibition following NLFA3 degradation. In contrast to IL-1β release, URB597 had minimal effect on TNF-α release triggered by LPS and nigericin treatment. Furthermore, in BMDMs derived from NLRP3-R258W knock-in mice, URB597 also induced NLRP3-R258W protein degradation and reduced inflammasome activation after LPS challenge ( Figure 21 ).

[0097] To provide preliminary evidence on whether URB597 could be used to treat CAPS patients, we isolated PBMCs from three patients, whose profiles are shown in Table 1 .

[0098] Table 1 Blood biochemical indicators of three patients

[0099]

[0100]

[0101] The interaction of CAPS-mutant NLRP3 with FAAH and the dissociation of NLRP3 from FAAH after URB597 treatment were confirmed in PBMCs isolated from a 2-year-old girl, patient 1 ( Figure 22 ), the girl was diagnosed with FCAS, in which the NLRP3 gene has an A354T mutation. PBMCs from patient 1 showed increased spontaneous and LPS-triggered release of IL-1β compared with PBMCs obtained from her mother without CAPS ( Figure 23 a). Importantly, URB597 was able to reduce both spontaneous and LPS-triggered IL-1β release, with approximately 50% inhibition observed in LPS-triggered IL-1β release. Notably, MCC950 was ineffective in this patient. Enhanced spontaneous and LPS-triggered IL-1β release was also observed in PBMCs isolated from two other patients, a 3-year-old girl diagnosed with FCAS with a T350M mutation and a 7-month-old boy diagnosed with CINCA with an M408T mutation in the NLRP3 gene. URB597 again effectively inhibited both spontaneous and LPS-triggered IL-1β release, with 40% and 60% inhibition of LPS-triggered IL-1β release in patients 2 and 3, respectively ( Figure 23 b and 23c).

[0102] A peptide with 20 amino acids upstream and downstream of FAAH serine 241 was constructed (TAT-AS1: YGRKKRRQRRRGGSPLGLGTDIGGSIRFPSAFCG), and the AS-1 short peptide was tested in BMDM cells to block the interaction between FAAH and NLRP3 ( Figure 24 ), which leads to the degradation of NLRP3 ( Figure 25 ), further affecting the activation of NLRP3 inflammasome ( Figure 26 ).

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention. Sequence Listing <110> South China University of Technology <120> Use of preparations for inhibiting or blocking the interaction between FAAH and NLRP3 <130> WK21-YSY-CN1-0269 <141> 2021-07-15 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 34 <212> PRT <213> Artificial <400> 1 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Ser Pro Leu 1 5 10 15 Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe Pro Ser Ala Phe 20 25 30 Cys Gly

Claims

1. Use of agents that inhibit or block the interaction between FAAH and NLRP3 in screening for drugs to treat diseases caused by NLRP3 protein mutations; in, The preparation for inhibiting or blocking the interaction between FAAH and NLRP3 is URB597, and the disease caused by NLRP3 protein mutation is cryopyrin-associated periodic syndrome.

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