Use of compound or composition comprising NLRP1 inflammasome activation inhibitor for treatment of human airway inflammation

Abstract

The present invention relates to a method of preventing or treating airway inflammation and / or related complications triggered by enterovirus 3C protease activated NLRP1 by using a compound or composition comprising an inhibitor of NLRP1 inflammasome activation. In one embodiment, the enterovirus 3C protease is from a viral species selected from the group consisting of human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C). In some embodiments, the compound is selected from the group consisting of MLN 4924, TAS4464, ZM223, MG132, and bortezomib.

Description

[0001] This invention application is a divisional application of the invention patent application filed on March 18, 2021, with application number 202180034911.4 (international application number PCT / SG2021 / 050142) entitled "Use of a compound or composition comprising an NLRP1 inflammasome activation inhibitor for the treatment of human airway inflammation". Technical Field

[0002] The present invention provides a method for preventing or treating airway inflammation and / or related complications triggered by NLRP1 activated by enterovirus 3C protease, a compound for the method or a composition comprising the compound, and the use of the compound in the preparation of a pharmaceutical agent for preventing or treating airway inflammation and / or related complications triggered by NLRP1 activated by enterovirus 3C protease. Background Technology

[0003] The human innate immune system employs a large number of germline-encoded sensor proteins to detect microbial infections and initiate first-line immune responses (Jones et al., Science 354 (2016)). Nod-like receptor (NLR) proteins are a family of innate immune sensors capable of detecting pathogen-associated molecular patterns (PAMPs) in the cytosol [Jones et al., Science 354 (2016); Shaw et al., Curr. Opin. Immunol. 20:377-382 (2008); Kaneganti et al., Immunity 27:549-559 (2007)]. Upon activation, NLR proteins nucleate inflammasome complex assemblages, leading to pyroptotic cell death and the secretion of pro-inflammatory cytokines such as IL-1β and IL-18 [Latz, Current Opinion in Immunology 22:28-33 (2010)]. Among all NLR sensors, NLRP1 remains one of the few whose homologous PAMP ligands have not yet been identified. Germline activation mutations in NLRP1 cause Mendelian syndrome, characterized by multiple self-healing keratoacanthomas of the skin and hyperkeratosis of the laryngeal and corneal epithelium [Grandemange et al., Annals of the Rheumatic Diseases 76:1191-1198 (2017); Zhong et al., Cell 67:187-202 (2016); Mamai et al., J. Invest. Dermatol. 135:304-308 (2015)]. Carriers of certain common NLRP1 single nucleotide polymorphisms (SNPs) have an increased risk of autoimmune diseases such as asthma and vitiligo [Sui et al., Arthritis Rheum. 64:647-654 (2012); Levandowski et al., PNAS 110:2952-2956 (2013)]. Human NLRP1 differs from its rodent homologs in terms of domain tissue, ligand specificity, and tissue distribution [Sand et al., Cell Death Dis 9:24 (2018)], and its exact role in the human immune response in vivo remains unclear.

[0004] Recent studies have described a novel pathway through which certain alleles of the rodent NLRP1 homolog Nlrp1b are activated by bacterial toxins, such as anthrax lethal factor (LF) [Boyden and Dietrich, Nat. Genet. 38:240-244 (2006); Newman et al., PLoS Pathog. 6:e1000906 (2010); Hellmich et al., PLoS One 7:e49741 (2012); Levinsohn et al., PLoS Pathog. 8:e1002638 (2012); Chavarria-Smith et al., PLoS Pathog. 9:e1003452 (2013)]. Anthrax LF cleaves directly at the N-terminus near Nlrp1b [Levinsohn et al., PLoS Pathog. 8:e1002638 (2012); Chavarria-Smith et al., PLoS Pathog. 9:e1003452 (2013)]. This cleavage leads to N-degradation determinant (N-degron)-mediated degradation of the self-inhibiting N-terminal fragment, thereby releasing the non-covalently bound FIIND. UPA -CARD(aa1213-1474) fragment to activate caspase-1 [Chui et al., Science 364:82-85 (2019); Sandstrom et al., Science 364 (2019); Xu et al., EMBO J. (2019)]( Figure 1 A). A common LF cleavage site is clearly absent in human NLRP1 (which contains an N-terminal PYRIN domain not found in rodents). Figure 1A). Therefore, LF does not cleave or activate human NLRP1. Despite their differences, both human NLRP1 and its rodent homolog can be activated by chemical inhibitors of dipeptidases DPP8 and DPP9 (but the underlying mechanisms remain to be fully elucidated) [Zhong et al., J. Biol. Chem. 293:18864-18878 (2018); Okondo et al., Cell Chem Biol. 25:262-267 (2018); de Vasconcelos et al., Life Sci Alliance 2 (2019)]. Rodent Nlrp1 can also be activated by Toxoplasma gondii infection in a process that clearly shows protease-mediated cleavage [Cirelli et al., PLoS Pathog. 10:e1003927 (2014); Ewald et al., Infect. Immun. 82:460-468 (2014)]. Whether any naturally occurring pathogens or danger-associated signals (DAMPs) can activate human NLRP1, and whether they do so via a “functional degradation” pathway similar to that recorded in mice, remains an open question. Identification of one or more possible homologous PAMP ligands that trigger the human NLRP1 inflammasome and assessment of their mechanisms of NLRP1 activation to improve airway inflammation in humans are needed. Summary of the Invention

[0005] This invention stems from the identification of PAMPs that can trigger inflammasomes activated by NLRP1 in the human airway. More specifically, the inventors have identified PAMPs involving cullin ZER1 / ZYG11B The inflammasome pathway activated by NLRP1 of NEDD8 activator enzyme (NAE) and potential inhibitors of said pathway.

[0006] In a first aspect of the invention, a compound or composition comprising the compound is provided for the prevention or treatment of airway inflammation and / or related complications triggered by NLRP1 cleaved by enterovirus 3C protease, wherein the compound or composition is an inhibitor of NLRP1 inflammasome activation.

[0007] The genus Enteroviruses encompasses 234 human pathogens belonging to the following seven species that circulate worldwide: human enteroviruses A through D (HEV-A, HEV-B, HEV-C, and HEV-D) and human rhinoviruses A through C (HRV-A, HRV-B, and HRV-C). Echoviruses and Coxsackievirus B (CV-B) are classified within the HEV-B species, and poliovirus (PV) is classified within HEV-C.

[0008] In some embodiments, the 3C protease is derived from a viral species selected from the following: human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

[0009] The cleavage of NLRP1 by the 3C protease produces a polypeptide fragment of NLRP1 with an N-terminal glycine residue (sensitive to the N-glycine degradation determinant pathway).

[0010] In some embodiments, the compound inhibits the degradation of N-glycine degradation determinant pathway ubiquitination and NLRP1 cleavage products.

[0011] In some implementations, the compound inhibits cullin ZER1 / ZYG11B .

[0012] In some embodiments, the compound is an inhibitor of NEDD8 activating enzyme (NAE).

[0013] In some embodiments, the compound or composition comprises a pharmaceutically acceptable salt or solvate of the inhibitory compound, or a pharmaceutically functional derivative thereof.

[0014] In some embodiments, the compound is selected from:

[0015] MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride salt;

[0016] TAS4464, IUPAC name 7H-pyrrolo[2,3-d]pyrimidin-4-amine, 7-[5-[(aminosulfonyl)amino]-5-deoxy-β-D-furanribosyl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]-or its hydrochloride salt;

[0017] ZM223, IUPAC name N-[6-[[2-(4-aminophenyl)thioalkylacetyl]amino]-1,3-benzothiazo-2-yl]-4-(trifluoromethyl)benzamide;

[0018] MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, and

[0019] Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

[0020] In some implementations, the 3C protease is derived from human rhinovirus.

[0021] In some embodiments, the composition comprises an inhibitory compound and a pharmaceutically acceptable adjuvant, diluent, or carrier.

[0022] In a second aspect of the invention, the use of compounds or compositions according to any aspect of the invention for manufacturing medicaments for the prevention or treatment of human airway inflammation and / or related complications triggered by NLRP1 activated by enterovirus 3C protease is provided.

[0023] In some embodiments, the agent reduces IL-1β and IL-18 secretion, ASC oligomerization, and / or lysogenic cell death.

[0024] In some embodiments, the 3C protease is derived from a viral species selected from the following: human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

[0025] In some embodiments, the compound inhibits the degradation of N-glycine degradation determinant pathway ubiquitination and NLRP1 cleavage products.

[0026] In some implementations, the compound inhibits cullin ZER1 / ZYG11B .

[0027] In some embodiments, the compound is an inhibitor of NEDD8 activating enzyme (NAE).

[0028] In some embodiments, the compound is selected from:

[0029] MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride salt;

[0030] TAS4464, IUPAC name 7H-pyrrolo[2,3-d]pyrimidin-4-amine, 7-[5-[(aminosulfonyl)amino]-5-deoxy-β-D-furanribosyl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]-or its hydrochloride salt;

[0031] ZM223, IUPAC name N-[6-[[2-(4-aminophenyl)thioalkylacetyl]amino]-1,3-benzothiazo-2-yl]-4-(trifluoromethyl)benzamide;

[0032] MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, and

[0033] Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

[0034] Determining the effective dose range for administration to subjects requiring prevention or treatment of airway inflammation and / or related complications triggered by enterovirus 3C protease activation (NLRP1) is within the capabilities of those skilled in the art. In some embodiments, the effective dose range may be between about 0.1 μM and 1 μM.

[0035] In a third aspect of the invention, a method is provided for preventing or treating airway inflammation and / or related complications triggered by NLRP1 activated by enterovirus 3C protease in a subject, the method comprising administering a therapeutically effective amount of a compound or composition according to any aspect of the invention.

[0036] In some embodiments, the 3C protease is derived from a viral species selected from the following: human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

[0037] In some embodiments, the compound inhibits the degradation of N-glycine degradation determinant pathway ubiquitination and NLRP1 cleavage products.

[0038] In some implementations, the compound inhibits cullin ZER1 / ZYG11B .

[0039] In some embodiments, the compound is an inhibitor of NEDD8 activating enzyme (NAE).

[0040] In some embodiments, the compound is selected from:

[0041] MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride salt;

[0042] TAS4464, IUPAC name 7H-pyrrolo[2,3-d]pyrimidin-4-amine, 7-[5-[(aminosulfonyl)amino]-5-deoxy-β-D-furanribosyl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]-or its hydrochloride salt;

[0043] ZM223, IUPAC name N-[6-[[2-(4-aminophenyl)thioalkylacetyl]amino]-1,3-benzothiazo-2-yl]-4-(trifluoromethyl)benzamide;

[0044] MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, and

[0045] Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

[0046] In some implementations, subjects given the prophylaxis or treatment will have reduced IL-1 secretion, ASC oligomerization, and / or lysogenic cell death in the airways compared to untreated subjects.

[0047] In some embodiments, the compound is MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride. Attached Figure Description

[0048] Figure 1The activation of the human NLRP1 inflammasome by enterovirus 3Cpro is shown. (A) Domain structures of human NLRP1 and rodent Nlrp1b. Mouse Nlrp1b is activated by cleavage of anthrax lethal factor (LF) toxin followed by proteasome degradation of the repressive N-terminal fragment. (B) Percentage of 293T-ASC-GFP-NLRP1-FLAG cells with ASC-GFP spots after overexpression of the Myc-tagged viral protease. Cells were fixed for 24 h after transfection with the indicated protease or empty vector control. Treatment with tapabostat (2 μM, 24 h) was used as a positive control. The number of cells with ASC-GFP spots was visually scored using a wide-field epifluorescence microscope at 20x magnification. ASC-GFP spot formation in more than 100 cells per condition was scored. n = 3 biological replicates. *P < 0.05; **P < 0.01 (two-way ANOVA); ns, not significant. (C) Blue non-denaturing PAGE (BN-PAGE) analysis of NLRP1 auto-oligomerization. 293T-NLRP1-FLAG cells were transfected with the indicated plasmid. One day after transfection, cells were treated with a mimic or with 2.5 μM MG132 for 24 h. Cells were lysed 48 h after transfection. 20 μg of total lysate was used for blue non-denaturing PAGE or SDS-PAGE, followed by Western blotting. (D) Effect of MG132 on NLRP1-dependent ASC-GFP spot formation. 293T-ASC-GFP or 293T-ASC-GFP-NLRP1-FLAG cells were transfected with HRV14-3Cpro and treated with 2.5 μM MG132 6 h after transfection. ***P<0.001 (two-way ANOVA). (E) HRV14-3Cpro induced mature IL-1B secretion and ASC oligomerization in immortalized human keratinocytes. Tet-ON HRV-3Cpro or 3Cpro was harvested 24 hours after administration of 1 μg / mL doxycycline (DOX). C146A N / TERT keratinocytes and conditioned medium. The conditioned medium was concentrated 10 times, followed by SDS-PAGE and IL-1β Western blotting. Endogenous ASC oligomers were extracted by 1% SDS after covalently cross-linking 1% NP40 insoluble precipitate with 1 mM DSS in PBS. (F) Live-cell imaging of ASC-GFP N / TERT cells transfected with HRV14-3Cpro. White arrows indicate pyroptosis cells with ASC-GFP spots and PI inclusions. (G) Stable transduction of Cas9 control and NLRP1 KO keratinocytes with Tet-ON HRV14-3Cpro. Cells were induced and harvested, as shown. Figure 1E. * = Non-specific bands observed by ASC protein blotting. (H) IL-1β ELISA of conditioned medium from NLRP1 KO and Cas9 control Tet-ON HRV14-3Cpro N / TERT keratinocytes 24 hours after DOX induction. ***P<0.001; ****P<0.0001 (two-way ANOVA). n = 3 independent cell seedings and inductions. (I) Trypan blue inclusion assay of NLRP1 KO and Cas9 control Tet-ON HRV14-3Cpro N / TERT keratinocytes 24 hours after DOX induction. **P<0.01 (two-way ANOVA), n = 3 independent cell seedings and inductions, as shown in E. Figure 1 As shown in H.

[0049] Figure 2 This demonstrates that HRV-3Cpro can act as an effective trigger for pyroptotic cell death in immortalized human keratinocytes. (A) Expression of Myc-tagged viral protease in 293T-ASC-GFP-NLRP1-FLAG cells. Two exposure times are shown. Note: Myc-HRV16-3Cpro expression is low but can activate ASC-GFP spots. (B) Representative wide-field fluorescence images of ASC-GFP spots, NLRP1-HA, and Myc-tagged protease. 293T-ASC-GFP cells were treated with NLRP1-HA and Myc-tagged gZiPro, HRV14-3Cpro, or HRV14-3Cpro. C146A Co-transfected and fixed 24 hours post-transfection. (C) Validation of ASC KO in N / TERT cells. ASC KO #2 was selected for further experiments. (D) Validation of CASP1 KO in N / TERT cells. #1 was selected for... Figure 2 Further experiments are shown in E. (E) N / TERT KO cells were transfected with the indicated 3Cpro variant and harvested 24 hours post-transfection. Conditioned medium was analyzed by IL-1β ELISA. **P < 0.01 (two-way ANOVA). n = 3 independent transfections. (F) Cell death patterns in taperstatin-treated or doxycycline-induced Tet-ON HRV14-3Cpro N / TERT cells were determined by annexin VI and PI content. Data represent one of two independent experiments.

[0050] Figure 3This illustrates how 3CPro activates NLRP1 through direct cleavage at a single site between p.Gln130 and Gly131. (A) HRV14-3Cpro cleaves near the N-terminus of NLRP1. Top inset: Antibody used to detect NLRP1 self-proteolytic fragments. The epitope of the N-terminal NLRP1 antibody is between NLRP1 aa130 and aa230. Bottom inset: Transfection of 293T cells with C-terminal HA-tagged NLRP1 and Myc-tagged HRV14-3Pro. Visualization of full-length NLRP1 and its cleavage products using N-terminal fragment-specific NLRP1 antibodies and antibodies against the C-terminal HA tag. Black arrows indicate the proposed proteolytic relationships between the observed NLRP1 fragments. Note the reduced expression of catalytically active 3CPro in all transfected plasmids (e.g., see also...). Figure 2 B. Figure 2 D). (B) HRV14-3Cpro cuts NLRP1 at a single site. F1212A 293T cells were treated with FLAG-tagged NLRP1. F1212A Transfection with gradually increasing doses of HRV14-3Cpro. Cell lysates were harvested 48 hours post-transfection. (C) Recombinant HRV14-3Cpro cleaves human NLRP1. Cell-free lysates (20 μg) from NLRP1-HA transfected 293T cells were incubated with recombinant HRV14-3Cpro (0.1, 0.3, 1 μg) for 90 min at 33°C and analyzed by SDS-PAGE. (D) Mapping of the 3Cpro cleavage site. Top: PYRIN domain (PYD) followed immediately by the NLRP1 linker region. Glutamine (Q) residues are underlined. Bottom: 293T cells were co-transfected with the indicated NLRP1 Q>A mutant and HRV14-3Cpro. 3Cpro C146A Used as a negative control. Total cell lysates were harvested 48 hours post-transfection. (E)Q130A eliminated HRV14-3Cpro cleavage. 293T-ASC-GFP-NLRP1 WT -FLAG and 293T-ASC-GFP-NLRP1 Q130A -FLAG cells were transfected with Myc-HRV14-3Cpro or treated with taperstat for 48 hours. Total cell lysates were analyzed by SDS-PAGE. *, possibly a nonspecific NLRP1 degradation product. Black arrow, 3Cpro-dependent cleavage product (aa131-1213). (F)Q130A eliminated 3CPro-dependent (but not taperstat-dependent) NLRP1 activation in 293T cells. Cells were then... Figure 1Cells were transfected as shown in E or treated with taperstatin and fixed 24 hours post-transfection. The number of cells with ASC-GFP spots was visually scored using a wide-field epifluorescence microscope at 20x magnification. *P<0.01; ****P<0.0001 (two-way ANOVA). n = 3 independent transfections / treatments. (G) Wild-type (instead of NLRP1) Q130A This restored 3Cpro-dependent IL-1β secretion in NLRP1 knockout human keratinocytes. First, NLRP1-knockout keratinocytes were treated with NLRP1... WT -FLAG or NLRP1 Q130A -Stable lentiviral expression of FLAG cells was rescued and further transduced using Tet-ON HRV14-3Cpro. NLRP1 KO or rescued cells were treated with doxycycline (1 μg / mL) or taperstatin (2 μM). Conditioned medium was harvested 24 hours after drug treatment and analyzed by IL-1B ELISA. ****P < 0.0001 (two-way ANOVA). n = 3 cell seedings and drug treatments. (H) and (I). Wild-type (instead of NLRP1) Q130A This restored 3Cpro-dependent pyroptosis in NLRP1 knockout human keratinocytes. (From...) Figure 1 Cells harvested from G were analyzed by Western blotting (H) and trypan blue exclusion (I).

[0051] Figure 4 The results show that HRV14-3CPro cannot activate mouse Nlrp1b. (A) 293T-ASC-GFP was treated with NLRP1. WT -HA or NLRP1 Q130A - HA was co-transfected with the indicated protease. Cell lysates were harvested 48 hours post-transfection. (B) Cells treated in S2A were fixed and imaged by wide-field GFP fluorescence targeting ASC-GFP spot formation. *P<0.05, **P<0.01 (two-way ANOVA). n = 3 independent transfections. (C) Representative morphology of N / TERT cells expressing HRV14-3Cpro or treated with taperxistat. Inset: Cells exhibiting “membrane bulging” (a typical feature of lytic cell death). (D) Percentage of trypan blue positive cells in Tet-ON-HRV14-3Cpro RAW264.7 cells after doxycycline induction with taperxistat (20 μM). (E) Percentage of trypan blue positive cells after anthrax LF treatment.

[0052] Figure 5 The 3Cpro-triggered NLRP1 activation in humans requires cullin. ZER1 / ZYG11BN-terminal glycine degradation determinant pathway mediated. (A) Overexpression of NLRP1 (aa131-1474) did not induce spontaneous ASC-GFP spot formation. 293T-ASC-GFP cells were transfected with wild-type NLRP1 or mutants and fixed 48 hours post-transfection. (B) Summary of different pathways regulating post-cleavage N-terminal fragment degradation in human NLRP1 and mouse Nlrp1b. The two types of N-degradation determinant pathways require different recognition receptors and exhibit different sensitivities to small molecule inhibitors. (C) MLN4924 (NEDD8 / cullin inhibitor) and proteasome inhibitors eliminated 3Cpro-induced ASC-GFP spots. 293T-ASC-GFP-NLRP1-FLAG cells were transfected with HRV14-3Cpro and treated for 24 hours with the following indicated drugs: MLN4924, 1 μM; MG132, 2.5 μM; and bortezomib 0.5 μM; phenylalanine, 1 mM. (D) MLN4924 inhibits NLRP1 auto-oligomerization. 293T-NLRP1-FLAG cells were transfected with HRV14-3Cpro and treated with the indicated drug for 24 hours after transfection (16 hours post-transfection). Whole-cell lysates were analyzed by BN-PAGE or SDS-PAGE followed by Western blotting. The right arrow indicates the HRV14-3Cpro cleavage product (aa131-1213). (E) MLN4924 inhibits endogenous 3Cpro-induced NLRP1 inflammasomes and pyroptosis. Tet-ONHRV14-3Cpro N / TERT keratinocytes were treated with doxycycline in combination with the indicated drug for 24 hours. Cell lysates, conditioned medium, and 1% NP40-insoluble complex were analyzed by Western blotting. (F) Figure 3 IL-1β ELISA in conditioned medium in E. ****P<0.0001. n=3 independent doxycycline inductions / drug treatments. (G) Genetically required Cullin for 3Cpro-induced NLRP1 activation. ZER1 / ZYG11B (Instead of UBR2). Indicated CRISPR KO 293T-ASC-GFP cells were treated with wild-type NLRP1 with HRV14-3Cpro or with NLRP1. M77T Transfection. Cells were fixed 24 hours post-transfection. ASC-GFP spots were scored using a 20x wide-field microscope. ****P<0.0001, n=3 transfections, >100 cells. (H)Cullin ZER1 / ZYG11B (Instead of UBR2) is responsible for the degradation of 3Cpro cleavage products. CRISPR KO 293T-ASC-GFP cells stably expressing NLRP1-FLAG were transfected with HRV14-3Cpro. Cell lysates were harvested 48 hours after transfection.

[0053] Figure 6 The gain-of-function MLRP1 mutant M77T is shown to destabilize the entire N-terminal fragment, disrupting the NLRP1 fold. (A) Expression of wild-type or NLRP1 mutant in 293T-ASC-GFP cells. Cells were transfected with the indicated construct and lysed 48 h post-transfection. (B) Tet-ON HRV14-3Cpro N / TERT cells were pretreated with the indicated inhibitor and induced with doxycycline (1 μg / mL) for 24 h. Conditioned medium was analyzed by IL-1β ELISA. ****P<0.0001 (two-way ANOVA). n=3 independent treatments. (C) Expression of wild-type or NLRP1 mutant cells. M77T Representative images of ASC-GFP spots in transfected ZZ-dKO 293T-ASC-GFP cells. Note that ZZ-dKO clone 8 has reduced ASC-GFP expression. Spot formation in ASC-GFP negative cells was not scored. (D) Cas9 control and UBR2 KO 293T-ASC-GFP-NLRP1-FLAG cells were transfected with HRV14-3Cpro and lysed 48 hours post-transfection.

[0054] Figure 7This study illustrates the requirement of NLRP1 for inflammasome assembly activation during live HRV infection. (A) NLRP1 vs. NLRP3 (RNAseq FKPM) expression levels in NHBEs. n = 21, independent cultures of NHBEs collected from healthy donors. (B) NLRP1 vs. NLRP3 expression in nasal biopsies from healthy individuals. Dot pattern: positive staining by RNAscope. Nuclear staining: hematoxylin. (C) Overview of cytokine characterization analysis of HRV16-infected and taperstat-treated NHBEs. (D) Cells were seeded with HRV16 at MOI = 5 and incubated at 33°C for 48 h. After cell debris removal, cells were subjected to Luminex arrays on conditioned medium. n = 3 independent infections and treatments. Cytokines / chemokines induced at least 5-fold (p < 0.05, Student's t-test, log-normal values) are indicated by square boxes. (E) MCC950 does not affect HRV16-triggered IL-1β secretion in NHBEs. NHBEs were pretreated with MCC950 (5 μM) or rupinxivir (10 nM) followed by HRV16 inoculation or taperidone (2 μM) treatment. (F) MCC950 did not affect HRV16-triggered IL-1β secretion in NHBEs. (G) Profile of HRV infection at the top of 3D human bronchial epithelial cultures. (H) Alcian blue (AB) / periodic acid-Schiff (PAS) staining of 3D human bronchial epithelial cultures infected with HRV16 (MOI = 3) and treated with taperidone (2 μM). (I) IL-18 levels in the top or base culture medium 48 hours after infection or taperidone treatment. **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). n = 3 independent infections / treatments of cultures. (J) Secretion of cleaved IL-1β and IL-18 and ASC oligomerization in HRV16-infected primary NHBE cells. Cells were seeded with HRV16 (MOI=5) or treated with taperstat (2 μM) for 48 h at 33 °C.

[0055] Figure 8 HRV16 infection leads to 3Cpro-dependent NLRP1 cleavage and activation of reconstructed human NLRP1 inflammasomes. (A) HRV16 infection leads to NLRP1 cleavage in HeLa-Ohio-NLRP1-HA cells. MOI = 1. (B) Q130A eliminates HRV16-induced NLRP1 cleavage and ASC-GFP oligomerization. HeLa-Ohio-ASC-GFP-NLRP1 cells were subjected to 33°C. WT or NLRP1 Q130ACells were infected with HRV16 at MOI=1. Cell pellet was harvested 48 hours post-infection. (C) HRV16 infection for cells expressing wild-type NLRP1 or NLRP1 Q130A The effect of ASC-GFP spot formation in HeLa-Ohio-ASC-GFP cells. ***P<0.001 (two-way ANOVA). n = 3 replicates from one of two independent infections, >100 cells per case. (D) Expression of wild-type NLRP1 or NLRP1 Q130A Representative image of ASC-GFP spots in HeLa-Ohio-ASC-GFP cells. Arrow: ASC-GFP spot. Note that the ASC-GFP spots appear less compact in this cell line, even in taperstatin-treated positive control cells. (E) Expression levels of selected inflammasome components and antiviral genes extracted from normal human airway epithelial cells in publicly available datasets (nose #1-GSE107898; nose #2-GSE55458; bronchus #1-GSE107971; bronchus #2-GSE107897; alveolar #1: GSE61220). In datasets reporting multiple treatment conditions, only the basal / simulated condition was selected. RPM values ​​for nose #2-GSE55458 and bronchus #1-GSE107971 were logarithmically converted for heatmap generation. Color mapping (shown on a grayscale) was performed using the "Dual Gradient" method in Graphpad Prism 8. (F) IL-18 secretion in HRV16-infected primary human nasal epithelial cells 48 hours after infection (MOI=5).

[0056] Figure 9 This indicates that HRV16 infection leads to NLRP1 activation via direct cleavage, and requires cul ZER1 / ZYG11B The N-glycine degradation determinant pathway mediated by NLRP1. (A) Endogenous NLRP1, ASC, and caspase-1 are essential for HRV16-induced IL-18 secretion in NHBEs. As before, Cas9 control, NLRP1, ASC, and CASP1 KO NHBEs were infected with HRV16. Conditioned medium was harvested 48 hours post-infection. (B) NLRP1 is genetically required for HRV16-induced IL-18 cleavage and ASC oligomerization. As before, Cas9 control, NLRP1, and ASC KO NHBEs were infected with HRV16. Cleavage IL-18 was marked with arrows. (C) Mutating the NLRP1 cleavage site eliminated HRV16-triggered IL-18 secretion in NHBEs. NLRP1 KO NHBEs were infected with FLAG-tagged NLRP1. WT or NLRP1 Q130ARescue of lentiviral constructs (carrying a silent mutation at the PAM site). Rescue cells were infected with HRV16 or treated with taperstatin. Conditioned medium was analyzed by IL-18 ELISA. **P<0.01, ***P<0.001, n = 3 replicates from one of two independent infections. (D)Q130A eliminated NLRP1 cleavage, IL-18 maturation, and ASC oligomerization in NHBE. NLRP1 KO cells or NLRP1 WT Saved cells or NLRP1 Q130A Rescued cells were infected with HRV16 or treated with taperstat. Cells were harvested 48 hours post-infection. Arrow: NLRP1 cleavage product in lane 8 HRV16. *: Nonspecific NLRP1 degradation product appearing just below the aa131-1213 cleavage fragment. See also Figure 3 E, Figure 5 E, Figure 5 D. (E) MLN4924 and bortezomib blocked HRV16-induced IL-18 secretion. NHBE was pretreated with MLN4924 (1 μM) or bortezomib (1 μM) before HRV16 infection or taperidone treatment. IL-18 levels were measured by ELISA. ****, p < 0.0001 (two-way ANOVA), n = 3 independent infections / treatments. (F) MLN4924 and bortezomib blocked HRV16-induced IL-1β secretion. (G) MLN4924 did not affect 3Cpro-dependent VP2 maturation during the course of HRV16 infection. Western blot analysis was performed 48 hours after HRV16 inoculation. Figure 9 E- Figure 9 Lysates from HRV16-infected NHBE in F.

[0057] Figure 10Further results show that NLRP1 is a major inflammasome sensor for HRV infection. (A) Caspase-1 activity in conditioned medium of HRV16-infected or taperxistat-treated NHBEs. ****P<0.0001 (two-way ANOVA), n=4 replicates from one of two independent infections. (B) Caspase-1 activity in conditioned medium of HRV16-infected or taperxistat-treated NHBEs. ****P<0.0001, **P<0.01 (two-way ANOVA). n=3 infections. (C) Representative morphology of HRV16-infected or taperxistat-treated NHBEs. Black arrows indicate membrane bulging typical of lytic cell death. The lower inset is intentionally focused on floating cells; therefore, attached monolayers appear blurred. (D) Western blot validation of ASC and CASP1 KOs in NHBEs. (E) Genomic sequencing validation of NLRP1 KOs in NHBEs. (F) and (G). LDH release and IL-8 secretion in KO NHBEs infected with HRV16 or treated with taperstatin. **P<0.01, ***P<0.001 (two-way ANOVA). n=3 infections / treatments. ****P<0.0001 (two-way ANOVA).

[0058] Figure 11 This demonstrates that NLRP1 functions in parallel with other immune sensors, such as TLR and RLR. (A) Proposed model of HRV-triggered NLRP1 activation in human airway epithelium. (B) Sequence conservation of the 3Cpro cleavage site in primate species. The minimum cleavage site (QG) is shaded. Detailed Implementation

[0059] For convenience, bibliographical references mentioned in this specification are listed in the form of a reference list and added at the end of the embodiments. The entire contents of such bibliographical references are incorporated herein by reference. Any discussion of prior art does not acknowledge that the prior art is part of common general knowledge in the field of this invention. This invention relates to the identification of PAMPs that can trigger inflammasomes activated by NLRP1 in the human airway. More specifically, the inventors have identified PAMPs involving cullin ZER1 / ZYG11B The inflammasome pathway activated by NLRP1 of NEDD8 activator enzyme (NAE) and potential inhibitors of said pathway.

[0060] definition

[0061] For convenience, certain terms used in the specification, embodiments and appended claims are collected herein.

[0062] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a”, “an”, and “the” used herein and in the appended claims include plural indicators.

[0063] As used herein, the terms “comprising” or “including” should be interpreted as specifically referring to the presence of the stated features, integers, steps, or components mentioned, but do not exclude the presence or addition of one or more features, integers, steps, or components or groups thereof. However, in the context of this disclosure, the terms “comprising” or “including” also include “consisting of”. Variations of the word “comprising” (such as “comprise” and “comprises”) and variations of “including” (such as “include” and “includes”) have correspondingly changing meanings.

[0064] As used herein, the terms “nucleotide,” “nucleic acid,” or “nucleic acid sequence” mean oligonucleotide, polynucleotide, or any fragment thereof; DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent a sense or antisense strand); peptide nucleic acid (PNA); or any DNA-like or RNA-like material.

[0065] As used herein, the term “amino acid” or “amino acid sequence” means an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, as well as naturally occurring or synthetic molecules. Where “amino acid sequence” is described herein as referring to the amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and similar terms are not intended to limit the amino acid sequence to the complete naturally occurring amino acid sequence associated with the described protein molecule.

[0066] Salts that may be mentioned include acid addition salts and base addition salts. Such salts can be formed by conventional means, for example by reacting a compound of formula I in its free acid or free base form with one or more equivalents of a suitable acid or base, optionally in a solvent or medium in which the salt is insoluble, followed by removal of the solvent or medium using standard techniques, such as in a vacuum, by freeze-drying, or by filtration. Salts can also be prepared by exchanging the counterion of a compound of formula I in its salt form with another counterion, for example using a suitable ion exchange resin.

[0067] Examples of salts include acid addition salts derived from inorganic and organic acids, and salts derived from metals (such as sodium, magnesium, or preferably potassium and calcium).

[0068] Examples of acid addition salts include those formed with the following acids: acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acids (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetaminobenzoic acid, butyric acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactopyric acid, gentian acid, glucoheponic acid, gluconic acid (e.g., D-gluconic acid), glucuronic acid (… Examples include D-glucuronic acid, glutamic acid (e.g., L-glutamic acid), α-oxoglutarate, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanate, undecenoic acid, and valeric acid.

[0069] Specific examples of salts are those derived from mineral acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid); those derived from organic acids (such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acid); and those derived from metals (such as sodium, magnesium, or preferably potassium and calcium).

[0070] The solvate can be stoichiometric or non-stoichiometric. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates, and dihydrates.

[0071] For a more detailed discussion of solvates and the methods used to prepare and characterize them, see Bryn et al., Solid-State Chemistry of Drugs, 2nd ed., SSCI, Inc. of West Lafayette, Indiana, USA, 1999, ISBN 0-967-06710-3.

[0072] As used in the context of this invention, the term "treatment" refers to preventive, ameliorative, therapeutic, or curative treatment.

[0073] The term "subject" is defined herein as a vertebrate, particularly a mammal, and more particularly a human. For research purposes, the subject may, in particular, be at least one animal model, such as a mouse, rat, etc. For example, for the treatment of airway inflammation and related disorders, the subject may be a person suffering from an enterovirus infection.

[0074] For convenience, bibliographical references mentioned in this specification are listed in the form of a reference list and added at the end of the embodiments. The entire contents of such bibliographical references are incorporated herein by reference.

[0075] Those skilled in the art will understand that the invention can be practiced without excessive experimentation based on the methods given herein. The methods, techniques, and chemicals described are as set forth in the given references or in the embodiments found in standard biotechnology and molecular biology textbooks.

[0076] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the invention.

[0077] Example

[0078] Example 1

[0079] Materials and methods

[0080] Cell culture

[0081] 293T (ATCC#CRL-3216), HeLa-Ohio (ECACC General Cell Collection#84121901), and normal bronchial epithelial cells (Lonza#CC-2541) were obtained from commercial sources and cultured according to the vendor's protocol. Immortalized human keratinocytes (N / TERT-1 or N / TERT in this paper) were a gift from H. Reinwald (MTA) [Mihaylova et al., Cell Rep. 24:3000-3007.e3 (2018)]. All cell lines underwent routine mycoplasma testing using LonzaMycoAlert (Lonza#LT07-118).

[0082] Plasmid transfection and stable cell line generation using lentiviruses

[0083] Previously, 293T-ASC-GFP, N / TERT-ASC-GFP, and N / TERT NLRP1 KO cells were described [Zell et al., Arch. Virol. 163:299-317 (2018)]. All transient expression plasmids were cloned into the pCS2+ vector using standard restriction cloning with ClaI and XhoI. Polyclonal Cas9 / CRISPR knockout cell lines were generated using lentiCRISPR-v2 (Addgene #52961) and selected with puromycin. Knockout efficiency was tested by Western blotting 7–10 days after puromycin selection. Site-directed mutagenesis was performed using QuickChangeXL II (Agilent #200522). Constitutive lentiviral expression was performed using the pCDH vector (SystemBio). The doxycycline-induced Tet-ON lentiviral construct was based on the pTRIPZ backbone (ThermoFisher).

[0084] Antibody and cytokine analysis

[0085] The following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Adipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R&Dsystems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R&Dsystems, #AF6788), IL18 (Abcam ab207324), and VP2 (QED Bioscience, #18758). HRV16-3Cpro was detected using rabbit serum (a gift from Dr. Ann Palmenberg and Dr. James Gern (University of Wisconsin)). Cytokines and chemokines were measured using the Human IL-1B ELISA Kit (BD, #557953), the Human IL-18 ELISA Kit (MBL Bioscience, #7620), and the Immune Monitoring 65-Plex Human ProcartaPlex. TM The assembly was performed using the ThermoFisher EPX650-10065-901 kit.

[0086] HRV16 virus replication

[0087] The HRV used in this study was HRV-A16 (strain 11757; ATCC VR-283, Manassas, Virginia, USA), and it was propagated in the HeLa cell line (HeLa Ohio, ECACC 84121901, Porton Down, Salisbury, Wiltshire, UK). The HeLa cells were cultured in Eagle's Limit Essential Medium (EMEM). 30-2003 TM (Supplemented with 10% fetal bovine serum (FBS) (BioWest, Kansas City, Missouri, USA), 2% HEPES, and 1% antibiotic-antifungal agent (Anti-Anti) (Gibco)) and incubated in a humidified incubator at 37°C and 5% CO2. To propagate HRV16, HeLa cells were first seeded in 24-well plates overnight to achieve approximately 80%-90% confluence. Figure 3 A). Cells were washed with 1X Duchenne phosphate-buffered saline (dPBS) and infected with HRV16, followed by the addition of EMEM containing 2% FBS, 2% HEPES, and 1% Anti-Anti. Infected HeLa cells were incubated at 33°C for two to three days. Virus was harvested from the supernatant of infected HeLa cells when approximately 80% cytopathic effect (CPE) was observed. Figure 3 B). The HRV virus stock solution was centrifuged at 3500 rpm for 10 min at 4 °C to remove cell debris, and then aliquoted into cryovials for storage at -80 °C.

[0088] Human rhinovirus inoculation

[0089] HRV was diluted with the appropriate cell culture medium and seeded at multiples of infection (MOI) of 5.0 (NHBE) and 1.0 (HeLa), respectively. Infected cells were incubated at 33°C for 1 h. Conditioned uninfected cell culture medium from virus propagation was added as an uninfected control. HRV-infected cells and control cells were then incubated at 33°C for up to 48 hpi post-infection. Cell culture supernatant and cell lysates were collected between 24 and 72 hpi for relevant assays.

[0090] Virus quantification using rhinovirus plaque assay

[0091] HeLa cells (85%–95% confluence) in 24-well plates were mixed with 100 μL of hNEC-infected cells from 10... -1 Up to 10 -6Serially diluted samples were incubated together at 33°C for 1 h. The plate was shaken every 15 min to ensure equal distribution of virus. The inoculum was removed and each well was replaced with 1 mL of Avicel (FMC Biopolymer) covering material, and incubated at 33°C for 65–72 h. The covering material composition was optimized to obtain HRV plaques suitable for counting. Avicel powder was added to dual-strength MEM to prepare a 1.2% Avicel solution, with a final concentration containing 3% FBS, 2% HEPES, 1.5% NaHCO3, 3% MgCl2, and 1% Anti-Anti. After the incubation period, the Avicel covering material was removed, and the cells were fixed with 20% formalin in 1×PBS for 1 h. The formalin was removed, and the cells were washed with 1×PBS. The fixed cells were stained with 1% crystal violet for 15 min and then washed. Plaque forming units (PFU) are calculated as follows: Plaque number × dilution factor = number of PFU per 100 μL, and then expressed as PFU / mL.

[0092] 3D reconstruction of HRV16 infection in human bronchial epithelium

[0093] 3D cultures of bronchial epithelium were purchased from Mattek (AIR-1484AIR-100EpiAirway, 3DRespiratory Epithelial Human MicroTissues) and cultured using an extended culture protocol as recommended by the supplier.

[0094] CRISPR / Cas9 knockout

[0095] CRISPR / Cas9 editing was performed in 293T cells according to the method reported by the Doyon group [Agudelo et al., NatMethods.14:615-620(2017)] (incorporated hereby by reference), except that the guide RNA (sgRNA) was cloned into pSpCas9(BB)-2A-Puro(PX459)V2.0 (Addgene 62988). N / TERT and NHBE KO were performed using LentiCRISPR-V2 (Addgene 52961) and stable lentiviral transduction. The sgRNAs used are shown in Table 1.

[0096] Table 1: List of sgRNAs

[0097]

[0098]

[0099] Example 2

[0100] Enterovirus 3Cpro activates human NLRP1 inflammasome

[0101] In our investigation of common human pathogens, we considered human rhinovirus (HRV), the causative agent of the common cold. HRV is a member of the genus Enterovirus (family Picornaviridae, a family of single-stranded RNA viruses that cause a variety of human diseases, including hand-foot-mouth disease, pericarditis / myocarditis, and poliomyelitis) [Zell, Arch. Virol. 163:299-317 (2018)]. Importantly, HRV infection of primary human bronchial epithelial cells has been reported to induce caspase-1 activation and IL-1 secretion [Zell, Arch. Virol. 163:299-317 (2018); Piper et al., PLoS One]. [8:e63365(2013)], although the upstream sensing mechanism is unclear. Notably, all small RNA viruses (including HRV) encode two well-defined proteases, called 2Apro and 3Cpro, which are essential for cleaving viral genome precursor proteins into individual functional components [Palmenberg, Annu. Rev. Microbiol. 44:603-623(1990)]. These two proteases have also been reported to cleave host proteins to facilitate viral replication or immune evasion [Walker et al., PLoS One 8:e71316(2013); Croft et al., Sci. Rep. 8:1569(2018); de Breyne et al., Virology 378:118-122(2008); Mukherjee et al., PLoS Pathogens 7:e1001311(2011)].

[0102] To test whether HRV-3Cpro could activate human NLRP1, we expressed Myc-tagged HRV-3Cpro in a 293T reporter cell line stably expressing ASC-GFP and NLRP1-FLAG (designated 293T-ASC-GFP-NLRP1-FLAG). Compared to vector-transfected cells, 3Cpro derived from two HRV strains (HRV-14 serotype B and HRV16 serotype A) and a closely related enterovirus (Coxsackie B3) resulted in a significantly increased percentage of cells forming ASC-GFP spots. Figure 1 B, Figure 2 A- Figure 2B), similar to the DPP8 / 9 inhibitor taperxistat. In contrast, other viral proteases tested (including Zika NS2B-3 (gZiPro), norovirus (NV) 3CLpro, SARS-3CLpro, and smallpox (Variola) I7pro) did not have similar effects. Figure 1 B, Figure 2 A- Figure 2 B), although expression levels are similar or even higher. The ability of HRV14-3Cpro to activate human NLRP1 depends entirely on its enzymatic activity, as mutating its catalytic cysteine ​​residue (p.C146A) eliminates ASC-GFP spot formation. Figure 1 B, Figure 2 B). In orthogonal assays of NLRP1 activation, non-denaturing PAGE (Native-PAGE) showed that HRV14- and HRV16-3Cpro directly induced the formation of high molecular weight NLRP1 oligomers at the expense of monomeric NLRP1. Figure 1 C, lanes 2 and 3 compared to lane 1). We were unable to reliably determine the effect of HRV-2Apro on human NLRP1 because Myc-tagged 2Apro did not accumulate in 293T cells ( Figure 1 C, lane 5). These findings establish enterovirus 3Cpro (such as those encoded by HRV) as robust viral activators of the in vitro reconstructed human NLRP1 inflammasome. Furthermore, the action of 3Cpro requires continuous proteasome activity, as the proteasome inhibitor MG132 significantly blocked HRV-3Cpro-induced ASC-GFP spot formation in 293T-ASC-GFP-NLRP1 cells. Figure 1 D) and NLRP1 self-oligomerization ( Figure 1 C, lanes 1-3 compared to 8-10). Therefore, despite their very different pathogen origins and substrate specificities, 3Cpro and anthrax LT may trigger human NLRP1 and rodent Nlrp1b, respectively, via a common mechanism involving cleavage followed by “functional degradation.”

[0103] Recently, it has been demonstrated that primary and immortalized human keratinocytes endogenously express components of the NLRP1 inflammasome complex and therefore undergo rapid pyroptosis upon treatment with taperstat [Zhong et al., Cell 167:187-202.e17 (2016); Zhong et al., J. Biol. Chem. 293:18864-18878 (2018)]. This provides a robust cellular system for determining whether 3Cpro can activate the endogenous NLRP1 inflammasome. Immortalized human keratinocytes were stably transduced with a doxycycline-induced (Tet-ON) lentiviral construct encoding dsRed (vector control), HRV14-3Cpro, or its catalytically inactive mutant C146A (…). Figure 1 E). Following doxycycline treatment, cells expressing only active HRV14-3Cpro exhibited the basic characteristics of pyroptosis, including the secretion of cleaved mature IL-1β and the formation of detergent-insoluble ASC oligomers. Figure 1 E, lane 5 compared to lanes 1-4 and lane 6). This still occurs despite the lower expression of wild-type 3Cpro compared to its inactive mutant (p.C146A). Figure 1 E, lane 5 compared to 6). Pretreatment with MG132 completely eliminated IL-1β secretion and ASC oligomerization. Figure 1 E, lane 5 compared to lane 8). Live-cell imaging of human keratinocytes expressing GFP-tagged ASCs further confirmed that HRV14-3Cpro can induce ASC-GFP spot formation (E, lane 5 compared to lane 8). Figure 1 F). HRV14-3Cpro-induced pyroptosis is entirely dependent on NLRP1, as it eliminates doxycycline-induced IL-1 secretion and ASC oligomerization through CRISPR / Cas9 gene excision (NLRP1 KO Tet-ON HRV14-3Cpro). Figure 1 G, Lanes 2-3 compared to lanes 5-6 Figure 1 H) and lysis of cells (H) Figure 1 I). In experiments with transiently transfected HRV14- and HRV16-3Cpro, deletion of NLRP1, ASC, and CASP1 all showed similar inhibitory effects. Figure 2 C- Figure 2 Therefore, HRV-3Cpro can act as an effective trigger for pyroptosis in immortalized human keratinocytes by activating the endogenous NLRP1 inflammasome. This is similar to the NLRP1 reconstructed in 293T cells. Figure 1 C- Figure 1 D), this effect also requires intact proteasome activity ( Figure 1E, lane 7 compared to lane 8), suggests that a "functional degradation" mechanism may be at play. We also observed that a subset of keratinocytes expressing 3Cpro underwent apoptotic cell death when stained with annexin V (but not PI-containing cells). Figure 2 F). This observation is consistent with reported pro-apoptotic effects of 3Cpro in other cell types [Buenz and Howe, Trends in Microbiology 14:28-36 (2006); Croft et al., mBio 8 (2017)]. In contrast, taperstat only caused lysis-related cell death via PI-contained markers (F). Figure 2 F).

[0104] Example 3

[0105] 3Cpro activates NLRp1 through direct cleavage at a single site between Gln130 and Gly131.

[0106] Small RNA virus 3Cpro (including HRV14-3Cpro) is a cysteine ​​protease with well-defined catalytic activity and broad substrate preference [Palmberg, Annu. Rev. Microbiol. 44:603-623 (1990); Matthews et al., Cell 77:761-771 (1994)]. Just as anthrax LF directly cleaves rodent Nlrp1b (Chavarria-Smith and Vance, PLoS Pathog. 9:e1003452 (2013); Chavarria-Smith et al., PLoS Pathog. 12:e1006052 (2016)), we hypothesize that 3Cpro can activate human NLRP1 via direct cleavage. Overexpressed NLRP1 undergoes self-cleavage within its FIIND and therefore appears as two bands differing by approximately 20 kDa when visualized with an N-terminal specific antibody [Finger et al., J Biol. Chem. 287:25030-25037 (2012); D'Osualdo et al., PLoS One 6:e27396 (2011)]. Figure 3 Lane A, lane 2). In the presence of HRV14-3Cpro, two additional bands became visible using the same antibody ( Figure 3 A, lane 4), while C end FIIND UPA - The CARD fragment remains intact, indicating that HRV14-3Cpro is present at a single site near the N-terminus (rather than at the FIIND site). UPA - NLRP1 was used to cut within the CARD fragment. To more accurately visualize 3Cpro-specific cleavage, NLRP1 was used.F1212A The mutant was subjected to the same experiment, but it could not undergo self-cleavage within FIIND, and therefore SDS-PAGE still showed a single band. Figure 3 B, Lane 2). As the amount of HRV14-3Cpro increases, NLRP1 F1212A It was cleaved into individual proteolytic products that were approximately 20 kDa smaller than the full-length NLRP1. Figure 3 B). In both experiments, the proteolytic banding pattern could be interpreted by a single cleavage site approximately 20 kDa from the N-terminus of NLRP1. The same cleavage was observed when lysates from NLRP1-free cells were incubated with recombinant HRV14-3Cpro at 33°C (the preferred temperature for HRV infection), suggesting that the observed cleavage was most likely direct (B). Figure 3 C).

[0107] Based on the dimensional difference between the cut and the full-length NLRP1, the 3Cpro cut site is mapped to the joint region immediately following the PYRIN structural domain (PYD). Figure 3 D, upper inset), the linker region is a non-conserved region in rodents. Since 3Cpro requires glutamine residues at the P'-1 substrate site [Matthews et al., Cell 77:761-771 (1994)], each of the 11 glutamine residues present in this linker region was changed to alanine via site-directed mutagenesis. Of all the alanine mutants tested, only the Q130A missense mutation eliminated the mutation via HRV14-3Cpro (… Figure 3 D, Lane 5 compared to other lanes) and other 3CPro ( Figure 4 A, lanes 1-5 compared to lanes 6-10) NLRP1 cleavage. Next, we generated stable expression of wild-type NLRP1 or the non-cleavable Q130A mutant (NLRP1). Q130A 293T-ASC-GFP reporter cells ( Figure 3 E, lane 2 compared to lane 5). 3Cpro-dependent cleavage bands were observed only in cells expressing wild-type NLRP1 ( Figure 3 E, lane 2 (black arrow), and therefore likely derived from aa131-1213. Compared to wild-type NLRP1, NLRP1 expression... Q130A The cells no longer nucleated ASC-GFP spots in response to HRV14-3Cpro expression; however, the cells' response to tepstatin remained intact relative to wild-type NLRP1. Figure 3 F). Using transfected NLRP1 Q130A Similar results were obtained with HRV16-3Cpro. Figure 4 B).

[0108] To study the endogenous inflammasome response, we used wild-type NLRP1 or the cleavage site mutant NLRP1. Q130A NLRP1 KO human keratinocytes were rescued. The "rescued" cells were further transduced using Tet-ON HRV14-3Cpro lentivirus. Only wild-type NLRP1 (not NLRP1) was detected. Q130A It restored HRV14-3Cpro-triggered IL-1β secretion and ASC oligomerization. Figure 3 G, Figure 3 H, lane 5 compared to lane 8) and lytic cell death ( Figure 3 I, Figure 4 C), while wild-type NLRP1 and NLRP1 Q130A Both re-enable NLRP1 KO cells to respond to taperstat ( Figure 3 G, Figure 3 H, lane 6 compared to lane 9; Figure 3 I, Figure 4 C). These results indicate that 3Cpro-triggered NLRP1 activation is a direct result of a discrete cleavage site between aaQ130 and aaG131 adjacent to the human-specific PYRIN domain. Since this site is not conserved in rodents, it was expected that HRV-3Cpro would not activate mouse Nlrp1b. This is consistent with the fact that HRV (similar to most pathogenic human enteroviruses) does not naturally infect rodents. To test this experimentally, wild-type and Nlrp1b-expressing KO mouse RAW264.7 cells were generated. Doxycycline induction failed to induce pyroptosis in either wild-type or Nlrp1b KO mouse cells. These results confirm that HRV14-3Cpro cannot activate mouse Nlrp1b ( Figure 4 D- Figure 4 E).

[0109] Example 4

[0110] 3Cpro triggers NLRP1 activation in humans, requiring cullin. ZER1 / ZYG11B N-terminal glycine degradation determinant pathway mediated

[0111] We attempted to further elucidate how 3Cpro cleavage of human NLRP1 triggers proteasome-dependent inflammasome activation. Compared to anthrax LF-mediated cleavage of mouse Nlrp1b, HRV-3Cpro removes most of the pathogenic gain-of-function germline mutations located in the entire human-specific NLRP1 PYRIN domain (PYD) (Grandmange et al., Annals of the Rheumatic Diseases 76:1191-1198 (2017); Zhong et al., Cell 167:187-202.e17 (2016); Drutman et al., PNAS USA 116:19055-19063 (2019); Herlin et al., Rheumatology (2019), doi:10.1093 / rheumatology / kez612). Several possible mechanisms may explain how 3Cpro cleavage activates NLRP1. For example, it could mitigate the inherent self-inhibitory effect of the PYD domain, or 2) the cleavage described could trigger instability of the post-cleavage maximal fragment (between the 3Cpro cleavage site and the FIIND self-hydrolysis site, aa131-1213). To differentiate between these two possibilities, we examined a truncated mutant of NLRP1 (aa131-1474), which mimics the major product produced by HRV14-3Cpro cleavage (the difference being the initiating methionine). Surprisingly, when expressed in 293T-ASC-GFP reporter cells, this mutant did not result in increased ASC-GFP spot formation relative to wild-type NLRP1. Furthermore, it remained completely sensitive to pabestat-mediated activation. Figure 5 A, Figure 6 A, lane 1 compared to lane 2). These results refute the first case and show that removing NLRP1 PYD alone is insufficient to explain HRV-3Cpro-triggered NLRP1 activation.

[0112] Recently, numerous reports have characterized the mechanism by which anthrax LF cleavage triggers Nlrp1b activation [Chui et al., Science 364:82-85 (2019); Sandstrom et al., Science 364 (2019), doi:10.1126 / sciencedotaau1330; Xu et al., EMBO J.38:e101996 (2019)]. LF cleavage produces an N-terminal degradation determinant recognized by type II N-degradation determinant receptors (such as UBR2), which degrades the N-terminal fragment of the proteasome-mediated repressive Nlrp1b (…). Figure 5(B) These findings address a long-standing question in the field of why Nlrp1b activation can be blocked by certain free amino acids (such as phenylalanine and leucine) that act as competitive inhibitors of UBR proteins [Wickliffe et al., Cell Microbiol. 10: 1352-1362 (2008)]. Inspired by these findings, we hypothesize that a similar pathway may explain the ability of HRV-3Cpro to activate human NLRP1. However, HRV-3Cpro cleavage of human NLRP1 produces a fragment with an N-terminal glycine (aa131-1273) that is not a canonical type II N-terminal degradation determinant recognized by the associated UBR protein [Wickliffe et al., Cell Microbiol. 10: 1352-1362 (2008); Varshavsky, PNAS USA 116: 358-366 (2019)]. Although this manuscript is under review, it describes a glycine-specific N-degradation determinant pathway [Timms et al., Science 365 (2019)]. The N-terminal glycine residue is targeted by receptors ZER1 and ZYG11B and their partner polymorphs cullin (CUL2 and CUL5) (referred to as cullin). ZER1 / ZYG11B ) recognition. cullin ZER1 / ZYG11B The mechanism involves ubiquitination of substrate proteins with N-terminal glycine residues, followed by degradation of these substrate proteins via the proteasome. Compared to the UBR system, the N-glycine degradation determinant pathway is insensitive to type II free amino acids, but can be inhibited by the NEDD8 / cullin inhibitor MLN4924 [Timms et al., Science 365 (2019)]. Figure 5 B). Using a series of chemical inhibitors, we tested whether the newly described N-glycine degradation determinant pathway contributes to HRV-3Cpro-triggered human NLRP1 activation. In 293T-ASC-GFP-NLRP1 reporter cells, MLN4924 blocked HRV14-3Cpro-induced ASC-GFP spot formation ( Figure 5 C), which is similar to the proteasome inhibitors MG132 and bortezomib. MLN4924 similarly blocked HRV14-3Cpro NLRP1 auto-oligomerization in 293T-NLRP1-FLAG cells. Figure 5 D, lane 5 compared to lane 3). Most notably, it leads to the stabilization of the NLRP1 fragment after cleavage (corresponding to aa131-1213, considering FIIND's self-proteolysis), to a degree similar to MG132 and bortezomib. Figure 5D, lanes 4-6 compared to lane 3, black arrow). The type II N-degradation determinant inhibitor phenylalanine has no effect on ASC-GFP spot formation or NLRP1 auto-oligomerization. Figure 5 C, Figure 5 D, lane 7). Similar effects were observed with endogenous NLRP1 inflammasomes in keratinocytes: MLN4924 and bortezomib completely blocked mature IL-1β secretion and ASC oligomerization upon HRV14-3Cpro induction. Figure 5 E, lanes 5 and 6 compared to lane 4, Figure 5 F). It also stabilized the cleaved NLRP1 fragment, which was below the detection limit in untreated cells (F). Figure 5 E, lanes 5 and 6 compared to lane 4 (black arrows). MLN4924 has a much smaller effect on taperstatin-induced IL-1β secretion in keratinocytes ( Figure 6 B) indicates that cullin ZER1 / ZYG11B The system cannot fully explain NLRP1 activation mediated by DPP8 / 9 inhibitors.

[0113] Given the known pleiotropic effects of MLN4924 on other cellular pathways, we used CRISPR / Cas9 to delete ZER1 and ZYG11B in 293T-ASC-GFP reporter cells (designated ZZ-dKO). Cloned ZZ-dKO 293T-ASC-GFP cells showed a significantly reduced percentage of ASC-GFP spots after co-expression of wild-type NLRP1 and HRV14-3Cpro, while UBR2 KO cells showed no difference compared to Cas9 control 293T-ASC-GFP cells. Figure 5 G, Figure 6 C). As an additional control, the patient-derived gain-of-function NLRP1 mutation p.M77T was examined. This mutation alone is sufficient to cause instability throughout the N-terminal segment ( Figure 6 A, lane 3 compared to lane 1), this is likely due to disruption of the NLRP1 fold [Zhong et al. Cell 167:187-202.e17(2016)], and with UBR or cullin ZER1 / ZYG11B The mediated N-degradation determinant pathway is irrelevant. In fact, it is caused by NLRP1. M77T The percentage of cells resulting in ASC-GFP spot formation was not different in ZZ-dKO or UBR2 KO compared to control cells. Figure 5 G and Figure 6 C). Therefore, cullin is particularly needed for HRV14-3Cpro-triggered human NLRP1 activation. ZER1 / ZYG11BThe NLRP1 fragment (aa131-1213) cleaved by 3Cpro was significantly more stable in ZZ-dKO cells compared to control cells. Figure 5 H, lanes 6-8 compared to lane 5), but unstable in UBR2KO cells ( Figure 6 D, lanes 5-6 compared to lane 4). These results further indicate that the NLRP1 self-hydrolyzed fragments cleaved by 3Cpro (corresponding to aa131-aa1212) are cullin ZER1 / ZYG11B Substrates for the N-terminal glycine degradation determinant pathway mediated by glycine.

[0114] In summary, these findings establish a detailed model for 3Cpro-triggered human NLRP1 activation, which is similar to but significantly different from the mouse model. Following 3Cpro cleavage between Q130 and G131, the cleaved NLRP1 fragment (aa131-1213) retains its self-inhibitory activity despite the loss of the PYD domain. Subsequently, the exposed N-terminal glycine is cullinized. ZER1 / ZYG11B The N-glycine-dependent degradation determinant system recognizes this. This constitutes an essential intermediate step for NLRP1 activation, as it delivers the cleaved, self-repressive N-terminal fragment of NLRP1 to the proteasome for "functional degradation," while simultaneously releasing FIIND. UPA -CARD oligomerizes itself and binds to ASC. This model is consistent with our initial observation that overexpression of the truncated mutant NLRP1 aa131-1474 does not lead to inflammasome activation. Figure 5 A). The most likely explanation is that the essential starting methionine derived from the Kozak sequence in our overexpression construct masks the second glycine residue, preventing it from being cullinized. ZER1 / ZYG11B The complex was fully recognized.

[0115] Example 5

[0116] NLRP1 is required for the activation of inflammasome assembly during HRV infection.

[0117] The role of live HRV infection in disease-associated human epithelial cell types was then examined. In HeLa-Ohio cells overexpressing NLRP1-HA, robust HRV16 viral replication was achieved 16 hours post-inoculation (MOI=1), as detected by accumulation of the viral capsid protein VP2. Figure 8 Lanes A, 4, 5, 10, and 11). This is accompanied by the presence of approximately 120 kDa NLRP1 cleavage product in infected cells ( Figure 8 Lanes A, 10 and 11). The small molecule pan-HRV-3Cpro inhibitor rupintrevir completely eliminated NLRP1 cleavage ( Figure 8 A, lane 12 compared to lanes 10 and 11). Using HeLa-Ohio cells that also expressed ASC-GFP, we next tested whether HRV16 infection led to the assembly of the NLRP1 inflammasome complex. Even though HeLa-Ohio cells showed less NLRP1 inflammasome activation than keratinocytes or HEK293T cells (even for the positive control taperstat), HRV16 resulted in significant ASC-GFP oligomerization ( Figure 8 B, Lane 2 compared to Lane 1) and spot formation ( Figure 8 C- Figure 8 D). These effects were observed only in HeLa-Ohio-ASC-GFP cells expressing wild-type NLRP1, but not in cells expressing cleavage-resistant NLRP1. Q130A No such observations were observed in the mutant cells. Figure 8 B, Lane 2 compared to Lane 5 Figure 8 C- Figure 8 D). Therefore, live HRV16 infection leads to 3Cpro-dependent NLRP1 cleavage and activates reconstructed human NLRP1 inflammasomes.

[0118] HRV is one of the most common human viral pathogens causing respiratory infections. Multiple dsRNA sensors (such as TLR3, MDA5, and RIG-I) are known to be endogenously expressed in human airway epithelial cells, all of which are involved in antiviral defense. However, the types of endogenous inflammasome components expressed in human airway epithelial cells have not been fully characterized. By examining publicly available RNA-seq datasets, we found that primary human airway epithelial cells [Landry et al., J. Infect. Dis. 217:897-905 (2018); Mihaylova et al., Cell Rep. 24:3000-3007.e3 (2018); Clark et al., PLoS One 10:e0115486 (2015); Hedstrom et al., Sci. Rep. 8:3502 (2018); Tian et al., BMC Genomics 16:529 (2015)] express a very limited number of NLR sensor species. Figure 8 E). Notably, other known inflammatory body sensory modalities (MEFV, NLRP3, NLRC5, and AIM2) were expressed at low levels or were completely absent. Figure 8 E). In 21 replicates, we confirmed that NLRP3 was not expressed in primary human bronchial epithelial cells (TPM < 1). Figure 7 A). NLRP3 was also undetectable in primary human nasal epithelium by in situ RNA staining. Figure 7B). Recently, an independent study confirmed the absence of the NLRP3 inflammasome in NHBE [Lee et al., Sci Immunol. 4 (2019), doi: 10.1126 / sciimmunoldotaau4643]. Although we cannot rule out the possibility that NLRP3 mRNA may be induced transcriptionally under specific conditions, these results suggest that NLRP1 (but not NLRP3) is the major inflammasome sensor constitutively expressed in human airway epithelial cells. In addition to NLRP1, all airway epithelial cell types endogenously express PYCARD (ASC), CASP1, and IL-1 cytokines (IL1B and IL18). Figure 8 E, Figure 7 A) indicates that they were “triggered” to undergo NLRP1-mediated inflammasome activation.

[0119] We analyzed the response of endogenous cytokines and chemokines in primary human bronchial epithelial cells (NHBE) to live HRV16 infection. Figure 7 C). Clearly, IL-18 (whose secretion is strictly dependent on inflammasome activation) was the most highly induced cytokine in both HRV16 infection and tapexat-treated NHBE. Figure 7 D). Subsequent ELISA experiments confirmed that IL-1β was also significantly secreted from HRV16-infected and taperstat-treated NHBEs, but its levels were lower than those of IL-18 and more variable. Figure 7 E- Figure 7 F) This may be because IL1B mRNA undergoes transcriptional regulation. Similar results were obtained using primary nasal epithelial cells. Figure 8 F). Consistent with the lack of endogenous NLRP3 in NHBE, IL-1β and IL-18 secretion were unaffected by the NLRP3 inhibitor MCC950 after HRV16 infection or taperdustat treatment, while the 3Cpro inhibitor ruminantril completely eliminated HRV16-induced (but not taperdustat-induced) IL-1β and IL-18 secretion. Figure 7 E- Figure 7 F). We performed apical HRV16 infection of 3D airlift cultures of human bronchial epithelium to more closely reflect early common cold infection in the human airway in vivo. Figure 7 G). Both HRV16 infection and taperidone resulted in significant epithelial cell destruction, increased mucin production, and goblet cell swelling. Figure 7 H). In the case of HRV16 infection, this is accompanied by the secretion of IL-18, particularly in the supernatant of the top culture where viral inoculation occurs (H). Figure 7I). In contrast, taperstat leads to the secretion of IL-18 in both the top and bottom media, likely due to its rapid diffusion across the culture. Figure 7 I).

[0120] HRV16-infected NHBE exhibits the basic characteristics of inflammasome activation, including 1) the hydrolysis of IL-18 and IL-1β proteins into their mature p17 form ( Figure 7 J, middle small figure), 2) endogenous ASC oligomerization ( Figure 7 J, lower small image), 3) Caspase-1 activation ( Figure 10 A), 4) Release of complete LDH activity ( Figure 10 B) and 5) characteristic membrane “bulging” ( Figure 10 (C, black arrow). In summary, these results indicate that HRV16 infection activates the NLRP1 (but not NLRP3) inflammasome pathway in primary human bronchial epithelial cells.

[0121] Example 6

[0122] HRV16 infection leads to NLRP1 activation via direct cleavage and requires cul ZER1 / ZYG11B N-glycine degradation determinant pathway mediated

[0123] We sought to further validate whether endogenous NLRP1 is a specific sensor for HRV-triggered inflammasome activation. The deletion of CRISPR / Cas9-mediated NLRP1, its downstream adaptor ASC, and caspase-1 precursor in primary NHBE completely eliminated IL-18 cleavage induced by HRV16 infection or taperidone treatment. Figure 9 A, Figure 10 D- Figure 10 E). Note that endogenous NLRP1 in NHBE is below the limit of detection of Western blot. Furthermore, NLRP1 knockout NHBE in HRV16 infection cannot cleave IL-18 into its mature p17 form (E). Figure 9 B, lanes 1-3 compared to lanes 7-12) or assembling ASC into high molecular weight oligomers ( Figure 9 B, lanes 1-3 compared to lanes 7-12). This is not caused by a general defect in viral bioogenesis, because the maturation of the capsid protein VP2 itself depends on 3Cpro activity [Cordingley et al., J. Biol. Chem. 265:9062-9065 (1990)], and is not affected by NLRP1 or ASC KO ( Figure 9B, upper inset). These results further support the view that NLRP1 is the primary inflammasome sensor for HRV infection in human NHBE. HRV-triggered inflammasome activation may occur in parallel with other immune sensing pathways, as NLRP1, ASC, and CASP1 KO NHBE are still able to produce other cytokines (such as IL-8) and undergo cell death after HRV16 infection. Figure 10 F- Figure 10 G).

[0124] To confirm that HRV-induced NLRP1 activation requires cleavage at aaQ130-G131, we used FLAG-tagged wild-type NLRP1 or NLRP1 Q130A Infection was performed using lentiviral constructs to rescue NLRP1 KO NHBE. As previously observed with keratinocytes ( Figure 3 E- Figure 3 I) In NLRP1 KO NHBE, wild-type NLRP1 and NLRP1 Q130A Both rescued taperstatin-dependent IL-18 secretion and ASC oligomerization. Figure 9 C, Figure 9 D, lanes 9 and 12 compared to lanes 3 and 6). However, despite similar VP2 maturation levels, only wild-type NLRP1 (rather than the non-cleavable mutant Q130A) rescued HRV16-induced IL-18 secretion and ASC oligomerization. Figure 9 C, Figure 9 D, lane 8 compared to lane 11). Furthermore, the 3Cpro cleavage fragment can only be found in cells expressing wild-type NLRP1 (not in cells expressing NLRP1). Q130A Detected in cells) Figure 9 D, lane 8 compared to lane 11, upper inset, black arrow. Note that this band appears near the degradation band in all samples (asterisk). In summary, these findings confirm that direct cleavage of 3Cpro is a necessary and sufficient step for HRV-induced NLRP1 activation in NHBE. To the best of our knowledge, these findings identify 3Cpro as the first PAMP activator sensed by human NLRP1.

[0125] This study investigated whether HRV-dependent NLRP1 activation requires the N-terminal glycine degradation determinant pathway. MLN4924 was used to investigate cullin ZER1 / ZYG11B The inhibition of IL-1β and IL-18 secretion in NHBE was completely blocked by the inhibition of the proteasome by bortezomib. Figure 9 EF). However, MLN4924 does not affect taperstatin-dependent IL-18 secretion and has only a moderate effect on taperstatin-dependent IL-1β secretion (EF). Figure 9E- Figure 9 F). These findings confirm cullin ZER1 / ZYG11B Both the proteasome and the proteasome are essential for HRV-triggered NLRP1 inflammasome activation in NHBE. As an important control, MLN4924 (unlike ruminantril and bortezomib) does not affect the accumulation of the mature capsid protein VP2, which is itself dependent on 3Cpro cleavage. Figure 9 G). Therefore, cullin is particularly needed for HRV-induced NLRP1 activation. ZER1 / ZYG11B This does not affect virus replication or 3Cpro activity.

[0126] Summarize

[0127] Enterovirus 3C proteases (such as HRV-3Cpro) activate the human inflammasome sensor NLRP1 via direct cleavage at a single site between aaQ130 and G131. Figure 11 A). This discovery not only provides a unified mechanism for the NLRP1 inflammasome in humans and rodents [Chui et al., Science 364:82-85 (2019); Sandstrom et al., Science 364 (2019) 10.1126 / sciencedotaau1330; Xu et al., EMBO J.38: (2019)], but it also reveals the unexpected role of the recently described “N-terminal glycine degradation determinant” pathway in human innate immunity. Mechanically, 3Cpro cleaves the NLRP1 fragment, leaving a glycine residue (p.G131), which becomes cullin. ZER1 / ZYG11B The substrate of the "N-glycine degradation determinant" mechanism is subsequently degraded by the proteasome. Figure 11 A). This constitutes the release of NLRP1FIIND. UPA - The CARD card fragment is a key step in the complete assembly of the inflammasome complex composed of ASC and caspase-1. Notably, even though a single cleavage site mutation (Q130A) can completely block 3Cpro activation of the NLRP1 inflammasome, this mutation does not affect NLRP1 activation triggered by the DPP8 / 9 inhibitor taperstat.

[0128] The identification of enterovirus 3Cpro as a PAMP trigger for human NLRP1 challenges the widely held view that viral proteases largely disable host immune sensing. It also reveals the evolutionary trajectory of NLRP1. The 3Cpro cleavage site appears in the common ancestor of primates (i.e., apes, New World and Old World monkeys), but is absent in prosimians such as tarsiers and lemurs. Figure 11(B) It is conceivable that the recently evolved 3Cpro-responsive NLRP1 allele provided a selective survival advantage during the evolution of primates (including humans), and it is inferred that it can sense and initiate appropriate immune responses against certain simian-tropical enterovirus pathogens. This does not preclude the possibility that human NLRP1 can detect other pathogens or danger-derived signals besides 3Cpro.

[0129] The findings of this paper also establish human NLRP1 as a prominent viral sensor in the airway epithelium, which may function in parallel with other immune sensors such as TLR and RLR. Figure 11 (B) Because the inflammasome pathway does not require de novo protein synthesis, it is particularly well-suited as a "fail-safe" defense mechanism when host transcription and translation have been shut down by viral virulence factors and the host is no longer able to produce other antiviral molecules [Carrasco and Smith, Nature 264:807-809 (1976)]. Using HRV as a model enterovirus, we demonstrated that NLRP1 and its cofactors are essential for HRV-triggered inflammasome activation in primary human bronchial epithelial cells. Based on these results, we propose that the NLRP1 inflammasome plays a crucial role in both antiviral defense and pathological inflammation of the human airway epithelium. Respiratory viral infections (including HRV) are well-known risk factors for exacerbations of asthma and chronic obstructive pulmonary disease (COPD) [Jacobs et al., Clin. Microbiol. Rev. 26:135-162 (2013)]. Therefore, the NLRP1 inflammasome pathway is a potential therapeutic target for these diseases and their complications.

[0130] Specifically, the present invention includes, but is not limited to, the following:

[0131] 1. A compound or a composition comprising said compound, said compound or composition for the prevention or treatment of airway inflammation and / or related complications triggered by NLRP1 cleaved by enterovirus 3C protease, said compound or composition being an inhibitor of NLRP1 inflammasome activation.

[0132] 2. The compound according to claim 1 or a composition comprising the compound, wherein the 3C protease is derived from a viral species selected from the group consisting of human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

[0133] 3. The compound according to claim 1 or 2, or a composition comprising the compound, wherein the compound inhibits the degradation of N-glycine degradation determinant pathway ubiquitination and NLRP1 cleavage products.

[0134] 4. The compound according to claim 3 or a composition comprising the compound, wherein the compound inhibits culin ZER1 / ZYG11B .

[0135] 5. The compound according to claim 4 or a composition comprising the compound, wherein the compound is an inhibitor of NEDD8 activating enzyme (NAE).

[0136] 6. The compound according to any one of claims 1 to 5, or a composition comprising said compound, wherein said compound is selected from:

[0137] MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride salt;

[0138] TAS4464, IUPAC name 7H-pyrrolo[2,3-d]pyrimidin-4-amine, 7-[5-[(aminosulfonyl)amino]-5-deoxy-β-D-furanribosyl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]-or its hydrochloride salt;

[0139] ZM223, IUPAC name N-[6-[[2-(4-aminophenyl)thioalkylacetyl]amino]-1,3-benzothiazo-2-yl]-4-(trifluoromethyl)benzamide;

[0140] MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, and

[0141] Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

[0142] 7. The compound or composition comprising any one of claims 1 to 6, wherein the 3C protease is derived from human rhinovirus.

[0143] 8. The composition according to any one of claims 1 to 7, wherein the composition comprises a pharmaceutically acceptable salt or solvate or a pharmaceutically functional derivative of the inhibitory compound.

[0144] 9. The composition according to any one of claims 1 to 8, wherein the composition comprises an inhibitory compound and a pharmaceutically acceptable adjuvant, diluent or carrier.

[0145] 10. Use of any compound or composition according to any one of items 1 to 9 in the manufacture of a medicament for the prevention or treatment of human airway inflammation and / or related complications triggered by enterovirus 3C protease-activated NLRP1.

[0146] 11. The use according to item 10, wherein the agent reduces IL-1β and IL-18 secretion, ASC oligomerization, and / or lysogenic cell death.

[0147] 12. The use according to item 10 or 11, wherein the 3C protease is derived from a viral species selected from the following: human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

[0148] 13. The use according to any one of items 10 to 12, wherein the compound inhibits the degradation of N-glycine degradation determinant pathway ubiquitination and NLRP1 cleavage products.

[0149] 14. The use according to any one of claims 10 to 13, wherein said compound inhibits cullin ZER1 / ZYG11B .

[0150] 15. The use according to any one of items 10 to 14, wherein said compound is an inhibitor of NEDD8 activating enzyme (NAE).

[0151] 16. The use according to any one of items 10 to 15, wherein said compound is selected from:

[0152] MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride salt;

[0153] TAS4464, IUPAC name 7H-pyrrolo[2,3-d]pyrimidin-4-amine, 7-[5-[(aminosulfonyl)amino]-5-deoxy-β-D-furanribosyl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]-or its hydrochloride salt;

[0154] ZM223, IUPAC name N-[6-[[2-(4-aminophenyl)thioalkylacetyl]amino]-1,3-benzothiazo-2-yl]-4-(trifluoromethyl)benzamide;

[0155] MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, and

[0156] Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

[0157] 17. A method for preventing or treating airway inflammation and / or related complications triggered by NLRP1 activated by enterovirus 3C protease in a subject, the method comprising administering a therapeutically effective amount of a compound or composition according to any one of claims 1 to 9.

[0158] 18. The method according to claim 17, wherein the 3C protease is derived from a viral species selected from the following: human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

[0159] 19. The method according to item 17 or 18, wherein the compound inhibits the degradation of N-glycine degradation determinant pathway ubiquitination and NLRP1 cleavage products.

[0160] 20. The method according to any one of claims 17 to 19, wherein the compound inhibits cullin ZER1 / ZYG11B .

[0161] 21. The method according to any one of claims 17 to 20, wherein said compound is an inhibitor of NEDD8 activating enzyme (NAE).

[0162] 22. The method according to any one of claims 17 to 21, wherein the compound is selected from:

[0163] MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride salt;

[0164] TAS4464, IUPAC name 7H-pyrrolo[2,3-d]pyrimidin-4-amine, 7-[5-[(aminosulfonyl)amino]-5-deoxy-β-D-furanribosyl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]-or its hydrochloride salt;

[0165] ZM223, IUPAC name N-[6-[[2-(4-aminophenyl)thioalkylacetyl]amino]-1,3-benzothiazo-2-yl]-4-(trifluoromethyl)benzamide;

[0166] MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, and

[0167] Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

[0168] 23. The method according to any one of items 17 to 22, wherein, compared with untreated subjects, subjects given the prophylaxis or treatment will have reduced IL-1 secretion, ASC oligomerization, and / or lysogenic cell death in the airways.

[0169] 24. The method according to any one of items 17 to 23, wherein said compound is MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride.

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Claims

1. Use of a compound or composition in the manufacture of a medicament for the prevention or treatment of human airway inflammation triggered by enteroviruses, wherein the compound or composition is an inhibitor of NLRP1 inflammasome activation activated by enterovirus 3C protease by inhibiting ubiquitination and degradation of NLRP1 cleavage products via the N-glycine degradation determinant pathway, wherein the compound is: MLN4924, IUPAC name ((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-indene-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylaminosulfonate or its hydrochloride, or MG132, IUPAC name: N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopent-2-yl]amino]-1-oxopent-2-yl]amino]-1-oxopent-2-yl]carbamate, or Bortezomib, IUPAC name: [(1R)-3-methyl-1-[[(2S)-3-phenyl-2-(pyrazine-2-carbonylamino)propionyl]amino]butyl]boronic acid.

2. The use according to claim 1, wherein the enterovirus is selected from: human enterovirus A (HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), human enterovirus D (HEV-D), human rhinovirus A (HRV-A), human rhinovirus B (HRV-B), and human rhinovirus C (HRV-C).

3. The use according to claim 1, wherein the 3C protease is a human rhinovirus.

4. The use according to any one of claims 1 to 3, wherein the compound inhibits cullin ZER1 / ZYG11B .

5. The use according to any one of claims 1 to 4, wherein the compound is an inhibitor of NEDD8 activating enzyme (NAE).

6. The use according to any one of claims 1 to 5, wherein the pharmaceutical agent comprises a pharmaceutically acceptable salt or solvate or a pharmaceutically functional derivative of the inhibitory compound.

7. The use according to any one of claims 1 to 6, wherein the pharmaceutical preparation comprises the inhibitory compound and a pharmaceutically acceptable adjuvant, diluent, or carrier.

8. The use according to any one of claims 1 to 7, wherein the agent reduces IL-1β and IL-18 secretion, ASC oligomerization, and / or lysogenic cell death.

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