Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.
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ABSTRACT:
SUBMITTER: Planes R
PROVIDER: S-EPMC9108100 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.
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Project description:The NLRP1 inflammasome is a multiprotein complex that is a potent activator of inflammation. Mouse NLRP1B can be activated through proteolytic cleavage by the bacterial Lethal Toxin (LeTx) protease, resulting in degradation of the N-terminal domains of NLRP1B and liberation of the bioactive C-terminal domain, which includes the caspase activation and recruitment domain (CARD). However, natural pathogen-derived effectors that can activate human NLRP1 have remained unknown. Here, we use an evolutionary model to identify several proteases from diverse picornaviruses that cleave human NLRP1 within a rapidly evolving region of the protein, leading to host-specific and virus-specific activation of the NLRP1 inflammasome. Our work demonstrates that NLRP1 acts as a 'tripwire' to recognize the enzymatic function of a wide range of viral proteases and suggests that host mimicry of viral polyprotein cleavage sites can be an evolutionary strategy to activate a robust inflammatory immune response.
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Project description:Cancer cells gain drug resistance through a complex mechanism, in which nuclear factor-?B (NF-?B) and interleukin-1? (IL-1?) are critical contributors. Because NACHT, LRR and PYD domains-containing protein (NLRP) inflammasomes mediate IL-1? maturation and NF-?B activation, we investigated the role of inflammasome sensor NLRP1 in acquired drug resistance to temozolomide (TMZ) in melanoma. The sensitivity of melanoma cells to TMZ was negatively correlated with the expression levels of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme to repair TMZ-induced DNA lesions. When MGMT-low human melanoma cells (1205Lu and HS294T) were treated with TMZ for over two months, MGMT was upregulated, and cells became resistant. However, the resistance mechanism was independent of MGMT, and the cells that acquired TMZ resistance showed increased NLRP1 expression, NLRP inflammasome activation, IL-1? secretion, and NF-?B activity, which contributed to the acquired resistance to TMZ. Finally, blocking IL-1 receptor (IL-1R) signaling with IL-1R antagonist decreased TMZ-resistant 1205Lu tumor growth in vivo. Although inflammation has been associated with drug resistance in various cancers, our paper is the first to demonstrate the involvement of NLRP in the development of acquired drug resistance. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, NLRP1 might be a suitable therapeutic target in drug-resistant melanoma, as well as in other cancers.
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Project description:The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.
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Project description:The obligate intracellular parasite Toxoplasma gondii is able to infect nearly all nucleated cell types of warm-blooded animals. This is achieved through the injection of hundreds of parasite effectors into the host cell cytosol, allowing the parasite to establish a vacuolar niche for growth, replication, and persistence. Here we show that Toxoplasma infection actives an inflammasome response in mice and rats, an innate immune sensing system designed to survey the host cytosol for foreign components leading to inflammation and cell death. Oral infection with Toxoplasma triggers an inflammasome response that is protective to the host, limiting parasite load and dissemination. Toxoplasma infection is sufficient to generate an inflammasome response in germfree animals. Interleukin 1? (IL-1?) secretion by macrophage requires the effector caspases 1 and 11, the adapter ASC, and NLRP1, the sensor previously described to initiate the inflammasome response to Bacillus anthracis lethal factor. The allele of NLRP1b derived from 129 mice is sufficient to enhance the B6 bone marrow-derived macrophage (BMDM) inflammasome response to Toxoplasma independent of the lethal factor proteolysis site. Moreover, N-terminal processing of NLRP1b, the only mechanism of activation known to date, is not observed in response to Toxoplasma infection. Cumulatively, these data indicate that NLRP1 is an innate immune sensor for Toxoplasma infection, activated via a novel mechanism that corresponds to a host-protective innate immune response to the parasite.
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Project description:Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1?/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.
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Project description:BackgroundPathogenic coronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. These viruses have induced outbreaks worldwide, and there are currently no effective medications against them. Therefore, there is an urgent need to develop potential drugs against coronaviruses.MethodsHigh-throughput technology is widely used to explore differences in messenger (m)RNA and micro (mi)RNA expression profiles, especially to investigate protein-protein interactions and search for new therapeutic compounds. We integrated miRNA and mRNA expression profiles in MERS-CoV-infected cells and compared them to mock-infected controls from public databases.ResultsThrough the bioinformatics analysis, there were 251 upregulated genes and eight highly differentiated miRNAs that overlapped in the two datasets. External validation verified that these genes had high expression in MERS-CoV-infected cells, including RC3H1, NF-κB, CD69, TNFAIP3, LEAP-2, DUSP10, CREB5, CXCL2, etc. We revealed that immune, olfactory or sensory system-related, and signal-transduction networks were discovered from upregulated mRNAs in MERS-CoV-infected cells. In total, 115 genes were predicted to be related to miRNAs, with the intersection of upregulated mRNAs and miRNA-targeting prediction genes such as TCF4, NR3C1, and POU2F2. Through the Connectivity Map (CMap) platform, we suggested potential compounds to use against MERS-CoV infection, including diethylcarbamazine, harpagoside, bumetanide, enalapril, and valproic acid.ConclusionsThe present study illustrates the crucial roles of miRNA-mRNA interacting networks in MERS-CoV-infected cells. The genes we identified are potential targets for treating MERS-CoV infection; however, these could possibly be extended to other coronavirus infections.
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