A novel NLRP3 inhibitor as a therapeutic agent against monosodium urate-induced gout

Abstract

Background: Since NEK7 is critical for NLRP3 inflammasome activation, NEK7 inhibitors could be employed as therapeutic agents against gout, a representative disease caused by NLRP3 inflammasome.

Methods: We designed NEK7 inhibitors based on biochemical kinome profiling of 2,7-substituted thieno[3,2-d]pyrimidine derivatives (SLC3031~3035 and SLC3037). Inflammasome activation was assessed by ELISA of IL-1b and immunoblotting of IL-1b maturation after treatment of bone marrow-derived macrophages with LPS+monosodium urate (MSU). NLPR3 binding to NEK7 and oligomerization were examined using immunoprecipitation and Blue Native gel electrophoresis, respectively. In vivo effect was investigated by studying gross and histopathological changes of food pad tissue of MSU-injected mice, together with assays of maturation of IL-1b and ASC speck in the tissue.

Results: SLC3037 inhibited inflammasome by MSU and other inflammasome activators through blockade of NLRP3 binding to NEK7 or oligomerization, and subsequent ASC oligomerization/phosphorylation. SLC3037 significantly reduced foot pad thickness and inflammation by MSU, which was superior to the effects of colchicine. SLC3037 significantly reduced content or maturation of IL-1b and ASC speck in the food pad. The number and height of intestinal villi were decreased by colchicine but not by SLC3037.

Conclusion: SLC3037, a NLRP3 inhibitor blocking NEK7 binding to NLRP3, could be a novel agent against diseases associated with NLRP3 inflammasome activation such as gout, cardiovascular diseases, metabolic syndrome or neurodegenerative diseases.

Keywords: NEK7; crystal; gout; inflammasome; monosodium urate.

Figure 1

Synthesis of the NEK7 inhibitor. SLC3031~3035 and SLC3037 were synthesized as described in “Materials and methods”. Reagent and condition for these reactions are as follows: (A) K₂CO₃, 1-ethylpiperazine, DMSO, 100°C, 3 h, 85%–89%; (B) Pd/C, H₂, EtOAc, RT, 12 h, 91%–95%; (C) (3-(N-methylsulfonamido)phenyl)boronic acid, 2 N Na₂CO₃, Pd(PPh₃)₂Cl₂, t-BuXPhos, 1,4-dioxane, 100°C, 3 h, 73%; (D) various amines, K₂CO₃, Pd₂dba₃, XPhos, 2-BuOH, 100°C, 1 h, 76%–78%; (E) 4-(4-ethylpiperazin-1-yl)aniline, TFA, 2-BuOH, 120°C, 24 h, 73%; (F) various boronic acid, 2 N Na₂CO₃, Pd(PPh₃)₂Cl₂, t-BuXPhos, 1,4-dioxane, 100°C, 3 h, 19-71% (G) 3-boronobenzoic acid, 2 N Na₂CO₃, Pd(PPh₃)₂Cl₂, t-BuXPhos, 1,4-dioxane, 100°C, 3 h; (H) N,N-dimethylethylenediamine, HATU, DIPEA, DMF, RT, 1 h, 54% over 2 steps; (I) 4-(4-ethylpiperazin-1-yl)aniline, K₂CO₃, Pd₂dba3, XPhos, 2-BuOH, 100°C, 1 h, 69%. (RT, room temperature).

Figure 2

Inhibition of the inflammasome by SLC3037. (A) BMDMs were primed with 100 ng/ml LPS for 4 h and then treated with 500 μg/ml MSU for 5 h, in the presence or absence of NEK7 inhibitors. The IL-1β content in the culture supernatant was determined by ELISA (n = 3 each). (B) BMDMs were treated with LPS+MSU with varying concentrations of SLC3037. The IL-1β content in the culture supernatant was determined by ELISA (n = 4 each). (C) After treatment of BMDMs as in (B), the cell extract or resuspended acetone precipitate of culture supernatant (SN) was subjected to immunoblot analysis using indicated antibodies. (D) BMDMs were primed with 100 ng/ml LPS for 3 h and then treated with LLOMe for 45 min, ATP for 45 min, nigericin for 45 min, or PA for 16 h in the presence or absence of SLC3037, and IL-1β content in the culture supernatant was determined by ELISA (n=4 each). (E) BMDMs pretreated with 100 ng/ml LPS for 3 h were transfected with poly(dA:dT) or flagellin for 3 h in the presence or absence of SLC3037, and the IL-1β content in the culture supernatant was determined by ELISA (n = 4 each). All data in this figure are the means ± SEM from more than three independent experiments. Red arrowheads indicate bands representing mature IL-1β. ***p < 0.001 by one-way ANOVA with Tukey’s test.

Figure 3

Inhibition of NLRP3 and ASC oligomerization by SLC3037. (A) BMDMs were treated with LPS+MSU in the presence or absence of SLC3037. The cell lysate was immunoprecipitated using the anti-NEK7 antibody, which was subjected to immunoblot analysis using the indicated antibodies as described in “Materials and methods” (red arrowhead, NLRP3 band) (B) After treating BMDMs as in (A), the cell lysate was separated on a Blue native gel, which was subjected to immunoblot analysis using anti-NLRP3 antibody. (C) BMDMs were treated with LPS+MSU in the presence or absence of SLC3037. The cell extract was subjected to immunoblot analysis using anti-ASC antibody after DSS crosslinking. (D) Cells were treated as in (C), and the cell extract was subjected to immunoblot analysis using anti-p-ASC (Tyr144) antibody.

Figure 4

In vivo effect of SLC3037 on MSU-induced gout. (A) Schematic drawing of the time of MSU, SLC3037, or colchicine administration. (SC, subcutaneous) (B) MSU was subcutaneously injected into the foot pad of mice, and foot pad thickness, %swelling, and Δfoot pad thickness were determined using a digimatic caliper (n = 16 each). (Con, control) (C) H&E staining of foot pad sections from mice of (B) (arrows, foci of inflammation) (scale bar: 100 μm). All data in this figure are the means ± SEM from more than three independent experiments. Rectangles were magnified. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s test.

Figure 5

Inhibition of MSU-induced inflammasome by SLC3037 in vivo. (A) Two days after subcutaneous injection of MSU to the foot pad (i.e., one day after the last administration of colchicine or SLC3037), IL-1β content in tissue extract of the foot pad was determined by ELISA (n=10 each). (B) Solubilized tissue extract of (A) was subjected to immunoblot analysis using indicated antibodies (left). Densitometric analysis of immunoblot bands was conducted (right) (n=3 each). (C) Paraffin sections of foot pad tissue from mice of (A) were subjected to immunofluorescence using anti-ASC antibody, and the number of ASC specks (arrows) was counted (n=3 each) (right). Representative immunofluorescence images are shown (left panel). (scale bar: 10 μm) All data in this figure are the means ± SEM from more than three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s test.

Figure 6

Biopsy of major organs after in vivo administration of SLC3037. (A) Major organs were obtained from C57BL/6 mice injected with MSU and then treated with SLC3037 or colchicine, which were subjected to histological analysis after H&E staining. (scale bar, 500 μm) (B) Height and number of intestinal villi were calculated from the pictures of (A) labelled as “intestine” (n = 4 each). All data in this figure are the means ± SEM from more than three independent experiments. *p < 0.05, by one-way ANOVA with Tukey’s test. ns, not significant.