GSK461364 Inhibits NLRP3 Inflammasome by Targeting NEK7 Phosphorylation

Abstract

NLRP3 inflammasome is a multiple protein complex sensing exogenous or endogenous stimuli, and aberrant activation of the NLRP3 inflammasome is implicated in various inflammatory disorders. While numerous small-molecule compounds targeting NLRP3 inflammasome activity have been developed, most have encountered limited success in clinical translation. Through screening of a kinase compound library, GSK461364 is identified as a potent and selective NLRP3 inflammasome inhibitor. Notably, GSK461364 confers significant protective effects in murine models of LPS-induced endotoxemia and DSS-induced colitis. Mechanistic study reveals that GSK461364 exerts its inhibitory effects via targeting Polo-like Kinase 1(PLK1). Specifically, that PLK1-mediated phosphorylation of NEK7, likely occurring at evolutionarily conserved serine residues (Ser221 and Ser260), is shown to enhance NEK7-NLRP3 binding, a critical step for NLRP3 inflammasome assembly. These findings not only establish GSK461364 as a novel therapeutic candidate for NLRP3-driven inflammatory diseases but also provide new insights into the regulatory mechanisms governing inflammasome activation through post-translational modification.

Keywords: GSK461364; NEK7; NLRP3 inflammasome; PLK1.

Figure 1

Identification of NLRP3 Inflammasome Inhibitor via Kinase Library Screening. A)Screening of 429 small‐molecule compounds obtained from Selleck Chemicals. Briefly, LPS‐primed macrophages were treated with inhibitors (10 µm) for 1 h, followed by Nigericin treatment. Then, IL‐1β secretion was quantified by ELISA. B)The secretion of IL‐1β by each compound and the chemical structures of the compounds GSK461364, AG‐1024, TDZD‐8, 10058‐F4, SP600125, and BI‐78D. C)ELISA analysis of IL‐1β and TNF‐α secretion in supernatants from LPS‐primed mouse peritoneal macrophages treated with GSK461364, 10058‐F4, TDZD‐8, AG‐1024, BI‐78D, or SP600125, followed by stimulation with nigericin. Results are represented as mean ± SD, and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two‐way ANOVA with the Bonferroni test for(C),^** p < 0.01.

Figure 2

GSK461364 is a specific inhibitor of the NLRP3 inflammasome. A–I)Mouse peritoneal macrophages were first primed with LPS (100 ng mL⁻¹, 3 h) and then treated with or without 10 µm GSK461364 for 1 h, followed by stimulation with ATP (5 mm, 1 h), nigericin (10 µm, 1 h), MSU (200 µg mL⁻¹, 6 h), flagellin transfection (2 µg mL⁻¹, 1 h), or poly(dA:dT) transfection (1 µg mL⁻¹, 16 h). A–C)ELISA analysis of IL‐1β and TNF‐α, and release of LDH in supernatants. D)Cell Lysates and supernatant were subjected to western blot analysis. E,F)ELISA analysis of IL‐1β and TNF‐α in supernatants treated with 1 µm, 5 µm, and 10 µm GSK461364. G)Immunoblot analysis of ASC oligomerization in cross‐linked cytosolic pellets treated with DMSO or 10 µm GSK461364. H)Representative immunofluorescence images of ASC specks (green) and nuclei (blue). Yellow arrows indicate ASC specks. Scale bars: 5µm. I)The percentage of cells containing ASC specks was quantified. Results are represented as mean ± SD, and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two‐way ANOVA with the Bonferroni test for(A‐C,E,I)^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001.

Figure 3

GSK461364 Attenuates LPS‐Induced Endotoxemia. A)chematic representation of the experimental design for LPS and GSK461364 administration in mice. Wild‐type C57BL/6J male mice were administered either DMSO or GSK461364 (10 or 25 mg kg⁻¹) intraperitoneally. After 0.5 h, the mice received an injection of either saline or LPS (25 mg kg⁻¹, i.p.) (n = 6 biologically independent mice) for 12 h. B)ELISA analysis of serum levels of IL‐1β, TNF‐α, and IL‐6 in mice. C)Representative images of hematoxylin and eosin (H&E)‐stained lung tissues of mice treated with GSK461364 (10 mg kg⁻¹). Scale bars,50µm. D)Western blot analysis of GSDMD and caspase‐1 in lung tissues of mice treated with GSK461364 (10 mg kg⁻¹). Results are represented as mean ± SD, and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two‐way ANOVA with the Bonferroni test for(B), ^*** p < 0.001.

Figure 4

GSK461364 Ameliorates DSS‐induced Colitis. A)Wild‐type mice were administered either 3% DSS in drinking water or normal drinking water for a duration of 7 days. Concurrently, GSK461364 was administered via intraperitoneal injection once daily from day 0 to day 7. B)Body weights of mice were recorded daily. C)DAI scores of mice were assessed and recorded daily. D) Colon lengths of mice. E)The average colon length of mice was calculated. F)HE staining of colon tissues. Scale bars,100µm. G)Immunoblot analysis of ASC oligomerization in colon tissues. Results are represented as mean ± SD, and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two‐way ANOVA with the Bonferroni test for(B,C,E), ^*** p < 0.001.

Figure 5

GSK461364 targets PLK1 for the inhibition of NLRP3 inflammasome. A)Western blot analysis of Cellular Thermal Shift Assay (CETSA) for PLK1 binding to GSK461364 (10 µm) and the melting curve generated from CETSA was analyzed using ImageJ software. B)Surface plasmon resonance (SPR) analysis of the interaction between PLK1 and GSK461364. C)Western blot analysis and quantitative real‐time PCR analysis of PLK1 expression in mouse peritoneal macrophages transfected with si‐Ctrl or si‐PLK1 for 48 h. D–L)Mouse peritoneal macrophages were transfected with si‐Ctrl or si‐PLK1 for 48 h, primed with LPS (100 ng mL⁻¹, 3 h), and subsequently stimulated with ATP (5 mm, 1 h), nigericin (10 µm, 1 h), MSU (200 µg mL⁻¹, 6 h), flagellin (2 µg mL⁻¹, 1 h, transfection), or poly(dA:dT) (1 µg mL⁻¹, 16 h, transfection). D‐F)ELISA analysis of IL‐1 β and TNF‐α, and release of LDH in supernatants. G)Cell lysates and supernatants were analyzed by Western blot. H)Immunoblot analysis of ASC oligomerization in cross‐linked cytosolic pellets. I)Representative immunofluorescence images of ASC specks (green) and nuclei (blue). Yellow arrows indicate ASC specks. Scale bars: 5µm. J–L)ELISA analysis of IL‐1β and TNF‐α, and release of LDH in supernatants from mouse peritoneal macrophages treated with DMSO or GSK461364 (1 h) after si‐Ctrl or si‐PLK1 transfection for 48h. Results are represented as mean ± SD, and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two‐way ANOVA with the Bonferroni test for(C‐E), ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001.

Figure 6

PLK1 promotes the association between NLRP3 and NEK7. A) Co‐immunoprecipitation and immunoblot analysis of lysates from HEK293T cells transfected with Flag‐PLK1 and Myc‐NLRP3. Immunoprecipitation was performed using anti‐Flag and anti‐Myc antibodies. B) Co‐immunoprecipitation and immunoblot analysis of lysates from HEK293T cells transfected with Flag‐PLK1 and Myc‐NEK7. Immunoprecipitation was performed using anti‐Flag and anti‐Myc antibodies. C)Co‐immunoprecipitation and immunoblot analysis of lysates from mouse peritoneal macrophages treated with LPS (100 ng mL⁻¹, 3 h) followed by Nigericin (10 µm, 1 h) or untreated. D)The physical interaction between PLK1 and NEK7 was visualized as red puncta using proximity ligation assay (PLA) in mouse peritoneal macrophages primed with LPS (100 ng mL⁻¹, 3 h) and subsequently stimulated with nigericin (10 µm, 1 h) and ATP (100 µm, 1 h) for 1 h. Scale bars: 1 µm. E)Co‐immunoprecipitation and immunoblot analysis of lysates from mouse peritoneal macrophages treated with si‐Ctrl or si‐PLK1 followed by LPS (100 ng mL⁻¹, 3 h), or subsequently stimulated with nigericin (10 µm, 1 h). F)Co‐immunoprecipitation and immunoblot analysis of lysates from mouse peritoneal macrophages treated with LPS (100 ng mL⁻¹, 3 h) followed with or without GSK461364, or subsequently stimulated with nigericin (10 µm, 1 h). G)Co‐immunoprecipitation and immunoblot analysis of lysates from HEK293T cells transfected with Flag‐PLK1 and Myc‐NEK7 treated with or without 10 µm GSK461364 for 1 h. Immunoprecipitation was performed using anti‐Flag and anti‐Myc antibodies. H)Co‐immunoprecipitation and immunoblot analysis were performed on lysates from mouse peritoneal macrophages primed with LPS (100 ng mL⁻¹, 3 h), treated with or without 10 µm GSK461364 for 1 h, and subsequently stimulated with nigericin (10 µm, 1 h).

Figure 7

PLK1 mediates NEK7 phosphorylation, likely at Ser221 and Ser260. A)Immunoprecipitation and immunoblotting analysis of lysates from mouse peritoneal macrophages transfected with si‐Ctrl or si‐PLK1, primed with LPS (100 ng mL⁻¹, 3 h), and subsequently stimulated with or without nigericin (10 µm, 1 h), using anti‐NEK7 antibody and anti‐pan phospho‐serine/threonine antibody. B)Immunoprecipitation and immunoblotting analysis of lysates from mouse peritoneal macrophages primed with LPS (100 ng mL⁻¹, 3 h), treated with or without 10 µm GSK461364 for 1 h, and subsequently stimulated with or without nigericin (10 µm, 1 h), using anti‐NEK7 antibody and anti‐pan phospho‐serine/threonine antibody. C)GST pull‐down analysis of the interaction between GST‐NEK7 and His‐PLK1. D)Immunoblot analysis of NEK7 by SDS‐PAGE and Phos‐tag SDS‐PAGE. E)ADP‐Glo quantification of kinase activity at different concentrations of PLK1. F)ADP‐Glo quantification of kinase activity with DMSO or 10 µm GSK461364. G)The levels of IL‐1β released into the supernatant of 293T cells were measured following transfection with plasmids encoding IL‐1β, caspase‐1, ASC, NLRP3, PLK1, NEK7 WT, and NEK7 mutant, and subsequent stimulation with nigericin. H)Co‐immunoprecipitation and immunoblot analysis of lysates from HEK 293T cells transfected with Myc‐PLK1 and Flag‐NEK7 WT, Flag‐NEK7 Ser221, or Flag‐NEK7 Ser260. Immunoprecipitation was performed using anti‐Flag and anti‐Myc antibodies. Results are represented as mean ± SD, and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two‐way ANOVA with the Bonferroni test for(E), ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.