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Small molecule inhibitor binds to NOD-like receptor family pyrin domain containing 3 and prevents inflammasome activation.

ABSTRACT: Despite recent advances in the mechanism of oxidized DNA activating NLRP3, the molecular mechanism and consequence of oxidized DNA associating with NLRP3 remains unknown. Cytosolic NLRP3 binds oxidized DNA which has been released from the mitochondria, which subsequently triggers inflammasome activation. Human glycosylase (hOGG1) repairs oxidized DNA damage which inhibits inflammasome activation. The fold of NLRP3 pyrin domain contains amino acids and a protein fold similar to hOGG1. Amino acids that enable hOGG1 to bind and cleave oxidized DNA are conserved in NLRP3. We found NLRP3 could bind and cleave oxidized guanine within mitochondrial DNA. The binding of oxidized DNA to NLRP3 was prevented by small molecule drugs which also inhibit hOGG1. These same drugs also inhibited inflammasome activation. Elucidating this mechanism will enable the design of drug memetics that treat inflammasome pathologies, illustrated herein by NLRP3 pyrin domain inhibitors which suppressed interleukin-1β (IL-1β) production in macrophages.

SUBMITTER: Lackner A

PROVIDER: S-EPMC11298654 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Small molecule inhibitor binds to NOD-like receptor family pyrin domain containing 3 and prevents inflammasome activation.

Lackner Angela A Cabral Julia Elise JE Qiu Yanfei Y Zhou Haitian H Leonidas Lemuel L Pham Minh Anh MA Macapagal Alijah A Lin Sophia S Armanus Emy E McNulty Reginald R

iScience 20240705 8

Despite recent advances in the mechanism of oxidized DNA activating NLRP3, the molecular mechanism and consequence of oxidized DNA associating with NLRP3 remains unknown. Cytosolic NLRP3 binds oxidized DNA which has been released from the mitochondria, which subsequently triggers inflammasome activation. Human glycosylase (hOGG1) repairs oxidized DNA damage which inhibits inflammasome activation. The fold of NLRP3 pyrin domain contains amino acids and a protein fold similar to hOGG1. Amino acids ...[more]

PMID: 39104412

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Project description:BackgroundCryopyrin-associated periodic syndrome (CAPS) is a group of rare, heterogeneous autoinflammatory disease characterized by interleukin (IL)-1β-mediated systemic inflammation and clinical symptoms involving skin, joints, central nervous system, and eyes. It encompasses a spectrum of three clinically overlapping autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease. CAPS is associated with gain-of-function missense mutations in NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), the gene encoding NLRP3. Moreover, most mutations leading to MWS occurred in exon 3 of NLRP3 gene. Here, we reported a novel mutation occurred in exon 1 of NLRP3 gene in an MWS patient and attempted to explore the pathogenic mechanism.MethodsGenetic sequence analysis of NLRP3 was performed in an MWS patient who presented with periodic fever, arthralgia, and multiform skin lesions. NLRP3 was also analyzed in this patient's parents and 50 healthy individuals. Clinical examinations including X-ray examination, skin biopsy, bone marrow aspiration smear, and blood test of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum levels of IL-1β, immunoglobulin E (IgE), antineutrophil cytoplasmic antibodies, antinuclear antibodies, and extractable nuclear antigen were also analyzed. The protein structure of mutant NLRP3 inflammasome was calculated by SWISS-MODEL software. Proteins of wild type and mutant components of NLRP3 inflammasome were expressed and purified, and the interaction abilities between these proteins were tested by surface plasmon resonance (SPR) assay.ResultsX-ray examination showed no abnormality in the patient's knees. Laboratory tests indicated an elevation of CRP (233.24 mg/L) and ESR (67 mm/h) when the patient had fever. Serum IL-1β increased to 24.37 pg/ml, and serum IgE was higher than 2500.00 IU/ml. Other blood tests were normal. Bone marrow aspiration smear was normal. A novel point mutation c.92A>T in exon 1 of NLRP3 gene was identified, which caused a p.D31V mutation in pyrin domain (PYD) of NLRP3. SPR assay showed that this point mutation may strengthen the interaction between the PYD of NLRP3 and the PYD of the apoptosis-associated speck-like protein. The mutation c.92A>T in exon 1 of the NLRP3 gene was not found in the patient's parents and 50 healthy individuals.ConclusionsThe mutation c.92A>T in exon 1 of the NLRP3 gene is a novel mutation associated with MWS. The p.D31V mutation might promote the activation of NLRP3 inflammasome and induce MWS in this patient.

Project description:The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC). Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.

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Small molecule inhibitor binds to NOD-like receptor family pyrin domain containing 3 and prevents inflammasome activation.

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Small molecule inhibitor binds to NOD-like receptor family pyrin domain containing 3 and prevents inflammasome activation.

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