Spirodalesol analog 8A inhibits NLRP3 inflammasome activation and attenuates inflammatory disease by directly targeting adaptor protein ASC

Abstract

Pharmacological inhibition of the Nod-like receptor family protein 3 (NLRP3) inflammasome contributes to the treatment of numerous inflammation-related diseases, making it a desirable drug target. Spirodalesol, derived from the ascomycete fungus Daldinia eschscholzii, has been reported to inhibit NLRP3 inflammasome activation. Based on the structure of spirodalesol, we synthesized and screened a series of analogs to find a more potent inhibitor. Analog compound 8A was identified as the most potent selective inhibitor for NLRP3 inflammasome assembly, but 8A did not inhibit the priming phase of the inflammasome. Specifically, while 8A did not reduce NLRP3 oligomerization, we found that it inhibited the oligomerization of adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), as ASC speck formation was significantly reduced. Also, 8A interrupted the assembly of the NLRP3 inflammasome complex and inhibited the activation of caspase-1. Subsequently, we used a cellular thermal shift assay and microscale thermophoresis assay to demonstrate that 8A interacts directly with ASC, both in vitro and ex vivo. Further, 8A alleviated lipopolysaccharide-induced endotoxemia, as well as monosodium urate-induced peritonitis and gouty arthritis in mice by suppressing NLRP3 inflammasome activation. Thus, 8A was identified as a promising ASC inhibitor to treat inflammasome-driven diseases. Pharmacological inhibition of the Nod-like receptor family protein 3 (NLRP3) inflammasome contributes to the treatment of numerous inflammation-related diseases, making it a desirable drug target. Spirodalesol, derived from the ascomycete fungus Daldinia eschscholzii, has been reported to inhibit NLRP3 inflammasome activation. Based on the structure of spirodalesol, we synthesized and screened a series of analogs to find a more potent inhibitor. Analog compound 8A was identified as the most potent selective inhibitor for NLRP3 inflammasome assembly, but 8A did not inhibit the priming phase of the inflammasome. Specifically, while 8A did not reduce NLRP3 oligomerization, we found that it inhibited the oligomerization of adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), as ASC speck formation was significantly reduced. Also, 8A interrupted the assembly of the NLRP3 inflammasome complex and inhibited the activation of caspase-1. Subsequently, we used a cellular thermal shift assay and microscale thermophoresis assay to demonstrate that 8A interacts directly with ASC, both in vitro and ex vivo. Further, 8A alleviated lipopolysaccharide-induced endotoxemia, as well as monosodium urate-induced peritonitis and gouty arthritis in mice by suppressing NLRP3 inflammasome activation. Thus, 8A was identified as a promising ASC inhibitor to treat inflammasome-driven diseases. Inflammasomes are multiprotein signaling complexes that control inflammatory responses by regulating the proteolytic enzyme caspase-1 (1Rathinam V.A. Fitzgerald K.A. Inflammasome complexes: emerging mechanisms and effector functions.Cell. 2016; 165: 792-800Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). Inflammasomes are assembled in the cytosol of host cells to detect exogenous pathogenic or endogenous warning signals that, in turn, activate inflammatory caspases that promote cytokine production and induce pyroptotic cell death (2Li Y. Huang H. Liu B. Zhang Y. Pan X. Yu X.Y. et al.Inflammasomes as therapeutic targets in human diseases.Signal Transduct. Target. Ther. 2021; 6: 247Crossref PubMed Scopus (90) Google Scholar). Based on the structural features and unique activators, the inflammasomes are divided and named NOD-like receptor (NLR), AIM2-like receptor family members, and pyrin (3Sharma D. Kanneganti T.D. The cell biology of inflammasomes: mechanisms of inflammasome activation and regulation.J. Cell Biol. 2016; 213: 617-629Crossref PubMed Scopus (486) Google Scholar). The canonical inflammasome complex consists of a cytosolic sensor (NLR or AIM2-like receptor protein), adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain], and effector caspase-1 (4Pandey A. Shen C. Feng S. Man S.M. Cell biology of inflammasome activation.Trends Cell Biol. 2021; 31: 924-939Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). As the central adapter that bridges the upstream sensors to procaspase-1, ASC is composed of pyrin domain (PYD) and caspase activation and recruitment domain, and both domains promote homotypic interactions (5Hoss F. Rodriguez-Alcazar J.F. Latz E. Assembly and regulation of ASC specks.Cell Mol. Life Sci. 2017; 74: 1211-1229Crossref PubMed Scopus (81) Google Scholar). Procaspase-1 is subsequently activated, leading to the cleavage of pro-IL-1β, pro-IL-18, and gasdermin-D in their mature forms, which drives pyroptosis (6Xue Y. Enosi Tuipulotu D. Tan W.H. Kay C. Man S.M. Emerging activators and regulators of inflammasomes and pyroptosis.Trends Immunol. 2019; 40: 1035-1052Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 7Place D. Kanneganti T. Recent advances in inflammasome biology.Curr. Opin. Immunol. 2017; 50: 32-38Crossref PubMed Scopus (244) Google Scholar). Nod-like receptor family protein 3 (NLRP3), a member of the NLR family, consists of an N-terminal PYD domain, a central NACHT domain that mediates ATP hydrolysis, and a C-terminal leucine-rich repeat domain. As an innate immune sensor, NLRP3 responds to many stimuli derived from exogenous pathogens or endogenous danger signals (8Chen J. Chen Z.J. PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation.Nature. 2018; 564: 71-76Crossref PubMed Scopus (360) Google Scholar). A recent study showed that inactive NLRP3 forms a double ring of 12 to 16 monomers that shield its pyrin domains from the cytosol. Before activation, NLRP3 interacts with the protein NEK7, which facilitates NLRP3 signaling. Upon activation, NLRP3 assembles an inflammasome complex via PYD-PYD interactions with ASC, forming a platform for caspase-1 dimerization and activation (9Andreeva L. David L. Rawson S. Shen C. Pasricha T. Pelegrin P. et al.NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.Cell. 2021; 184: 6299-6312.e22Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 10Sharif H. Wang L. Wang W.L. Magupalli V.G. Andreeva L. Qiao Q. et al.Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome.Nature. 2019; 570: 338-343Crossref PubMed Scopus (392) Google Scholar). On the one hand, its activation may protect against infectious pathogens. On the other hand, over- or long-lasting activation of the NLRP3 inflammasome has been implicated in the development of a range of human acute or chronic inflammatory disorders, including inflammatory bowel disease, gout, atherosclerosis, diabetes, neurodegenerative disease, depression, and cancer (11Yang Y. Wang H. Kouadir M. Song H. Shi F. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors.Cell Death Dis. 2019; 10: 128Crossref PubMed Scopus (743) Google Scholar, 12Swanson K.V. Deng M. Ting J.P. The NLRP3 inflammasome: molecular activation and regulation to therapeutics.Nat. Rev. Immunol. 2019; 19: 477-489Crossref PubMed Scopus (2190) Google Scholar, 13Gong T. Liu L. Jiang W. Zhou R. DAMP-sensing receptors in sterile inflammation and inflammatory diseases.Nat. Rev. Immunol. 2020; 20: 95-112Crossref PubMed Scopus (777) Google Scholar, 14Lv Q. Xing Y. Liu J. Dong D. Liu Y. Qiao H. et al.Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation.Acta Pharm. Sin. B. 2021; 11: 2880-2899Crossref PubMed Scopus (51) Google Scholar). Accordingly, NLRP3 is considered a highly desirable therapeutic target for these diseases (15Mangan M.S.J. Olhava E.J. Roush W.R. Seidel H.M. Glick G.D. Latz E. Targeting the NLRP3 inflammasome in inflammatory diseases.Nat. Rev. Drug Discov. 2018; 17: 588-606Crossref PubMed Scopus (921) Google Scholar). In this study, we identified a novel ASC inhibitor, 8A, that binds to and attenuates ASC in the nucleus and prevents its recruitment by NLRP3, thereby reducing caspase-1 activation and the maturation of IL-1β. This working model may improve the treatment of NLPR3-related diseases, including sepsis and gouty arthritis. Previously, spirodalesol derived from Daldinia eschscholzii was shown to inhibit NLRP3 inflammasome activation and improve sepsis (16Zhang A.H. Liu W. Jiang N. Xu Q. Tan R.X. Spirodalesol, an NLRP3 inflammasome activation inhibitor.Org. Lett. 2016; 18: 6496-6499Crossref PubMed Scopus (22) Google Scholar). Based on these observations, we synthesized and screened a series of spirodalesol derivates to identify more potent compounds with simple structures. The structures of these compounds and their inhibitory activities against IL-1β are shown in Fig. S1, A–C. Among these derivatives, compounds 7 and 8A showed almost the same potency. The most remarkable difference was that compound 8A inhibited IL-1β more efficiently than compound 7 at a concentration of 1 μM (Fig. S1, A–C). However, in our study, we found that the solubilities of these compounds were different. Precipitated solid was observed when the buffer solution of compound 7 was prepared at a concentration of 100 μM in the culture medium but was not found with compound 8A. Therefore, we selected compound 8A for further studies. As shown in Figure 1, Figure 8, B–D, 8A suppressed IL-1β secretion in bone marrow-derived macrophages (BMDMs) and THP1-derived macrophages triggered by lipopolysaccharide (LPS) + ATP without affecting cell viability or TNF-α secretion. Its potency was superior to the previously reported activity of andrographolide, an NLRP3 inflammasome inhibitor (17Guo W. Sun Y. Liu W. Wu X. Guo L. Cai P. et al.Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer.Autophagy. 2014; 10: 972-985Crossref PubMed Scopus (213) Google Scholar). Moreover, LPS + monosodium urate (MSU)–induced IL-1β secretion, although not that of TNF-α, was inhibited by 8A (Fig. 1, E and F). We subsequently sought to elucidate the role of 8A against inflammasome activation. We observed that LPS + ATP- or MSU-triggered elevation of caspase-1 p10 and IL-1β p17 levels in culture supernatants were significantly suppressed by 8A (Fig. 2, A–D). Moreover, the pro-IL-1β levels remained unaltered (Fig. 2, A–D). Activation of CASP1 was confirmed by FAM-FLICA staining (Fig. 2E). We also established that the cleavage of gasdermin-D and release of lactate dehydrogenase, downstream of caspase-1 activation induced by LPS + ATP, were downregulated by 8A (Fig. 2, F and G). These findings indicate the high potency of the anti-inflammatory action exhibited by 8A and that 8A might selectively inhibit the assembly phase, although not the priming phase, of NLRP3 inflammasome activation. To confirm this conjecture, we examined the phosphorylation of NF-κB and LPS-induced expression of NLRP3. As shown, 8A had no appreciable effect on LPS-induced phosphorylation of NF-κB or NLRP3 expression (Fig. S2). We examined the process of NLRP3 inflammasome formation using immunoprecipitation and immunofluorescence (IF). We observed that ATP stimulation-induced formation of the NLRP3/ASC/caspase-1 complex was interrupted by 8A in a dose-dependent manner (Fig. 3, A and B). Moreover, IF analysis revealed that ATP induced formation of ASC specks (indicated by white arrow), whereas 8A and MCC950 treatment reduced the number of specks and contributed to the retention of ASC within the nuclei (Fig. 3, C and D). This phenomenon was further confirmed by examining ASC levels in the nuclear and cytosolic fractions. As shown in Figure 3, Figure 8E, 8A treatment resulted in the retention of ASC in the nucleus. We transfected ASC-GFP plasmid into 293T cells with or with NLRP3 plasmid to further examine speck formation as well as the localization of ASC. As shown in Fig. S3, A and B, GFP diffused throughout the whole cell in the GFP vector-transfection group. We estimated that approximately 20% of cells spontaneously formed ASC specks without NLRP3-encoding plasmid transfection and that about 70% of cells produced ASC specks when co-transfected with NLRP3-encoding plasmid (indicated by white arrow). In addition, ASC was partially localized in the nuclei without NLRP3 induction (indicated by red arrow). Both 8A and MCC950 treatment reduced the formation of ASC speck in 293T cells and restrained part of the ASC in the nuclei (red arrow). These results further confirmed that 8A reduced ASC speck formation and inhibited the activation of caspase-1. It is well established that mitochondrial dysfunction, exemplified by the overproduction of reactive oxgen species (ROS), is for the assembly of the NLRP3 inflammasome L. control of the NLRP3 Immunol. PubMed Scopus Google Scholar, R. P. J. A role for in NLRP3 inflammasome activation.Nature. PubMed Scopus Google Scholar). As by we found that ATP treatment induced production was by 8A (Fig. A and thereby that the inhibitory activity of 8A is downstream of mitochondrial and signaling. 8A or MCC950 the of NLRP3 in (Fig. However, oligomerization of NLRP3 was significantly inhibited by MCC950 but not 8A. Moreover, ASC oligomerization was significantly reduced by treatment with 8A (Fig. These results indicate that ASC recruitment by NLRP3 is interrupted by 8A. Based on our that 8A reduced the cytosolic and oligomerization of ASC, we examined the 8A and ASC. We the cellular thermal shift assay a that facilitates the and simple of the target of in a cellular In the of ASC at and was at whereas in ASC was at (Fig. we found that 8A the levels of ASC at an of ASC in to 8A treatment (Fig. We also macrophages from mice to to confirm that 8A also to ASC in Accordingly, we an 8A and ASC (Fig. the microscale thermophoresis assay was used to the ASC and 8A. showed that 8A with the ASC with a of (Fig. As ASC is also in the we 8A IL-1β Therefore, we examined the effect of 8A on LPS + IL-1β Both 8A and IL-1β induced by LPS + ATP, while 8A inhibited LPS + IL-1β as a inhibitor did (Fig. A and M. Yu Y. R. S. L. L. et NLRP3 and inflammasome 2016; PubMed Scopus (307) Google Scholar). with the on the we that 8A is an ASC inhibitor. To confirm the therapeutic effect of 8A on NLRP3 diseases, we used an LPS-induced model and found that 8A significantly the of mice with sepsis (Fig. The LPS-induced in was also by 8A treatment (Fig. of inflammatory is to an to the development of We found that the of 8A inhibited the secretion of as IL-1β and in the (Fig. Moreover, we established that both and by LPS were alleviated in mice A and by reduced inflammatory cell in the (Fig. and reduced levels of and in the (Fig. staining of the confirmed caspase-1 activation 8A treatment (Fig. and in mice with LPS-induced the LPS for mice were mouse were with B, mouse were with levels of in the were by and mouse were to staining for in and E. in C and were as the of in and E were as the of mouse in group. LPS group. Figure LPS treatment also in by (Fig. A and of cells (Fig. and and expression of inflammatory (Fig. of which were 8A Moreover, 8A suppressed CASP1 activation in the mice (Fig. The in Figure Figure Figure 7 indicate that 8A treatment significantly alleviated LPS-induced in mice by the activation of the NLRP3 inflammasome. We further examined the of 8A on peritonitis and arthritis in the of in mice a inflammatory by of immune cells (Fig. and levels of caspase-1 cleavage in by (Fig. These immune responses were found to significantly suppressed by 8A and caspase-1 activation triggered by into the of mice were inhibited by 8A (Fig. 8, These findings indicate that 8A treatment reduce inflammation in mice by NLRP3 inflammasome activation. To the of our this is the study to a molecular compound that target ASC for NLRP3 inflammasome ASC, 8A suppressed the assembly of the NLRP3 thereby inflammatory diseases, including and gouty arthritis in Moreover, the unique mechanism by which 8A inflammasomes its in a of inflammatory A compounds been reported to or directly inhibit the activation of NLRP3. our study found that the andrographolide, and the compounds and inhibited NLRP3 inflammasome activation and colitis and LPS-induced sepsis in mice (17Guo W. Sun Y. Liu W. Wu X. Guo L. Cai P. et al.Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer.Autophagy. 2014; 10: 972-985Crossref PubMed Scopus (213) Google Scholar, W. Guo W. Y. S. W. 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Lett. 2016; 18: 6496-6499Crossref PubMed Scopus (22) Google Scholar). with the 8A has including a and of production and In the findings of this study indicate that spirodalesol 8A sepsis and gouty with the mechanism the of ASC to induce inflammasome findings that ASC as a target in therapeutic for sepsis or other inflammasome-driven inflammatory diseases. Therefore, 8A considered a compound of further to mice were from and were in with the for the and of of and by the and of were to and to reduce the number of The structures of spirodalesol and its analogs are in Fig. were at a concentration of in as a solution at and with a medium when The concentration of did not throughout the study control LPS from and ATP were from and activity assay were from the and + were from Life was from and were from Cell CASP1 was from was from for TNF-α, and human IL-1β were from The FAM-FLICA CASP1 assay was from MCC950 was from other were from and 293T cell were from the of Cell and in with 100 of 100 of and in a at were to the cells were from mice and with with and was to culture medium 3 these macrophages were within 7 to The cells were and on for without the cells were used for the as cells were with 8A at the was for 3 by to the The cell viability was using a at cells were with 100 LPS for 3 by 1 with 8A at the and with ATP or The cells were with and using a mice were LPS at with or without 8A treatment and and was for and from the and were LPS and 8A for the peritonitis were with the of 8A and for 3 a of in sterile the mice were and the were with 3 The number of cells was using a Life and caspase-1 was examined by the gouty arthritis mice were with 8A 3 a the in sterile was by was used to at the The levels of and TNF-α in the and cell culture were using IF of and on were as reported W. Liu W. Chen Y. S. Shen L. et NLRP3 inflammasome activation via mitochondrial 2017; PubMed Scopus Google Scholar, M. Guo W. Wu Y. C. L. Deng et inhibition and with Pharm. Sin. B. 2019; PubMed Scopus Google Scholar). cells or were in in in or and in a solution containing were with at by with were in an solution containing and using a or were with for at with for using and with were using of was in a manner as T. J. Liu W. J. M. Guo W. et al.NLRP3 mice from and via 2021; PubMed Scopus Google no of 1, 2, 3, high high of the cell and of and of and of The of was using and at levels high 3, 2, and 1, The are as a of of was as previously reported W. Guo W. Y. S. W. Zhang C. et 1 by a NLRP3 inflammasome activation and attenuates lipopolysaccharide-induced sepsis in Immunol. 2018; PubMed Scopus Google Scholar). were using by and The were with at and with a was using the cells were with or 8A for and to a D. R. M. T. C. et drug target in cells and using the cellular thermal shift PubMed Scopus Google Scholar). cells were divided into and part was for 3 at and The cells were at for 12 to for and this was one more Cell were by at for ASC expression was by the in thermal shift mice were 8A a for 3 macrophages were and to were using a The cell containing was used in this were from the cells transfected with the was using or and the protein was to an concentration for The compounds concentration were with the protein and by analysis was used for the ASC oligomerization was as previously W. Liu W. Chen Y. S. Shen L. et NLRP3 inflammasome activation via mitochondrial 2017; PubMed Scopus Google Scholar). were with and with at for The were at for at to with 1 of the were in of was to the which was at with for The was at and for buffer was used to the and was NLRP3 oligomerization assay was as previously reported J. Zhang Wang Y. Y. Zhang X.Y. et of NLRP3 for treatment of inflammatory Immunol. 2021; Scopus Google Scholar). the cells were in buffer containing and were by using a The nuclei and cells were by at for The supernatants were and at for The were in of buffer and for 1 at with The were in the and was to detect NLRP3 The results are as the of and were by using the control and The of was at a of are within the This The no of with the of the A. and W. W. C. S. and T. W. W. C. S. and T. W. W. W. J. and J. W. J. and J. R. Q. and W. W. and T. W. and This was by of of for the and from

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