Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: C172 inhibits NLRP3 activation via CFTR-independent manner. (A) C172 structure. (B) Immunoblot analysis of IL-1β and cleaved caspase-1 in culture supernatants (SN) of LPS-primed BMDMs treated with various doses (above lanes) of C172 and then stimulated with nigericin. (C and D) ELISA of IL-1β (C) and IL-18 (D) in supernatants from LPS-primed BMDMs treated with various doses (above lanes) of C172 and then stimulated with nigericin. (E) Immunoblot analysis of the indicated proteins in lysates from BMDMs treated with LPS for 3 h and stimulated with different doses of C172 for 30 min (C172 after LPS) or BMDMs treated with different doses of C172 for 30 min and then stimulated with LPS for 3 h (C172 before LPS). (F) ELISA of IL-1β in supernatants from LPS-primed WT or Cftr −/− BMDMs that were treated with nigericin with or without the presence of C172 (20 µM). Data are from three independent experiments with biological duplicates in each (C, D, and F; mean and SEM of n = 6) or are representative of three independent experiments (B and E). Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 blocks NLRP3 inflammasome activation. (A) CY-09 structure. (B) Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs treated with various doses (above lanes) of CY-09 and then stimulated with nigericin and immunoblot analysis of the precursors of IL-1β (pro–IL-1β) and caspase-1 (pro–caspase-1) in lysates of those cells (input). (C–G) ELISA of IL-1β in supernatants from LPS-primed BMDMs treated with various doses of CY-09 and then stimulated with nigericin, MSU (C), ATP (D), cytosolic LPS (E), cytosolic poly(dA:dT) (F), or Salmonella (G). Data are from three independent experiments with biological duplicates in each (C–G; mean and SEM of n = 6) or are representative of three independent experiments (B). Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 inhibits NLRP3 inflammasome assembly. (A) Immunoblot analysis of ASC oligomerization in lysates of BMDMs treated with various doses (above lanes) of CY-09 and then stimulated with nigericin. SN, supernatants. (B) Confocal microscopy analysis in LPS-primed BMDMs treated with CY-09 (10 µM) and then stimulated with nigericin, followed by staining with MitoSOX and MitoTracker red. Nuclei were stained with DAPI. Bars, 10 µm. (C) Qualification of potassium efflux in LPS-primed BMDMs treated with different doses of CY-09 and then stimulated with nigericin. (D and E) Qualification of potassium (D) and chloride (E) efflux in LPS-primed BMDMs treated with different doses of indicated inhibitors and then stimulated with nigericin. (F) IP and immunoblot analysis of the interaction of Flag–NLRP3 and VSV–NLRP3 in the lysates of HEK-293T cells. (G) Immunoblot analysis of NLRP3 by SDD-AGE or SDS-PAGE assay in WT or Nlrp3 −/− BMDMs treated with various doses of CY-09 and then stimulated with nigericin. (H) IP and immunoblot analysis of the interaction of endogenous NLRP3 and ASC in LPS-primed BMDMs treated with various doses (above lanes) of CY-09 and then stimulated with nigericin. Data are from three independent experiments with biological duplicates in each (C–E; mean and SEM of n = 6) or are representative of three independent experiments (A, B, and F–H). Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Western Blot, Confocal Microscopy, Staining, SDS Page

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 binds to the ATP-binding site of NLRP3 NACHT domain. (A) Cell lysates of LPS-primed BMDMs were incubated with different concentrations of biotin–CY-09, which were then pulled down with streptavidin beads. (B) Cell lysates of LPS-primed BMDMs or PMA-differentiated THP-1 cells were incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (C) Purified human NLRP3 protein was incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) Purified human NLRP3 protein was incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (E) MST assay for the affinity between CY-09 and purified GFP-NLRP3 protein. (F and G) Cell lysates from HEK-293T cells transfected with Flag-tagged NLRP3, NOD1, NOD2, AIM2, NLRC4, NLRP3–LRR, NLPR3–NACHT, or NLRP3–PYD constructs were incubated with indicated concentration of biotin–CY-09, which were then pulled down with streptavidin beads. Data are representative of three independent experiments.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Binding Assay, Incubation, Purification, Transfection, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 inhibits NLRP3 ATPase activity. (A) ATPase activity assay for purified human NLRP3 in the presence of different concentrations of CY-09. (B) ATPase activity assay for purified Flag–NLRP3, NLRC4, NLRP1, NOD2 or RIG-I with or without presence of CY-09 (1 µM). (C) Cell lysates from HEK-293T cells transfected with Flag-tagged WT NLRP3 or NLRP3 constructs with Walker A or Walker B motif mutation were incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) ATP-binding assay for purified Flag–NLRP3 in the presence of different concentrations of CY-09. (E) ATPase activity assay for purified Flag–NLRP3 or mutants with or without presence of CY-09 (1 µM). (F) Docking complex of NLRP3 with CY-09. CY-09 is shown in sticks and colored green, while NLRP3 is shown in cartoon and colored light blue. Data are from three independent experiments with biological duplicates in each (A, B, and E; mean and SEM of n = 6) or are representative of two or three independent experiments (C and D). Statistics were analyzed using an unpaired Student’s t test: ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activity Assay, Purification, Transfection, Construct, Mutagenesis, Incubation, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 inhibits NLRP3 activation in MSU-induced peritonitis and a mouse model of MWS. (A and B) ELISA of IL-1β in the serum (A) or peritoneal cavity (B) of C57BL/6J mice intraperitoneally injected with MSU (1 mg/mouse) with or without CY-09 (40 mg/kg) or MCC950 (40 mg/kg). Data are representative of two independent experiments (mean and SEM of n = 6). (C) FACS analysis of neutrophil numbers in the peritoneal cavity of C57BL/6J mice intraperitoneally injected with MSU (1 mg/mouse) with or without CY-09 (40 mg/kg) or MCC950 (40 mg/kg). Data are representative of two independent experiments (mean and SEM of n = 6). (D) ELISA of IL-1β in serum of Nlrp3 −/− mice intraperitoneally injected with L. monocytogenes ( L.mon ; 5 × 10 6 ) for 8 h with or without CY-09 (40 mg/kg). (E) Weight of WT or Nlrp3A 350VneoR crossed with LysM-Cre mice ( NLRP3 -mut) treated with CY-09 or MCC950 at day 9. Data are representative of two independent experiments (mean and SEM). WT vehicle, WT CY-09, and WT MCC950 ( n = 6), NLRP3 -mut vehicle ( n = 4), NLRP3 -mut CY-09 or MCC950 ( n = 5). (F) NLRP3 -mut mice treated with CY-09 or MCC950 on day 9. (G) Survival of NLRP3 -mut mice treated with vehicle or CY-09 up to day 49 (CY-09 withdrawn at day 25). Data are representative of two independent experiments. CY-09 group ( n = 8), MCC950 group ( n = 8), vehicle group ( n = 9). Statistics were analyzed using an unpaired Student’s t test (A–E) or a generalized Wilcoxon test (G): ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Injection

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: Treatment of metabolic disorders in HFD-induced diabetic mice with CY-09. (A and B) Food intake and body weight change of WT or Nlrp3 −/− mice that were first fed with an HFD for 14 wk and then treated with CY-09 for 6 wk. n = 6–8 per group. (C and D) Fasting (C) or fed (D) blood glucose concentrations at week 6 in the mice described in A. n = 6–8. (E) Fasting blood insulin concentration at week 6 in the mice described in A. n = 6–8 per group. (F–I) Glucose tolerance test (F and H) and insulin tolerance test (G and I) performed at week 6 in the mice described in A. n = 6–8 per group. (J) Representative H&E staining of liver sections of WT or Nlrp3 −/− mice that were first fed with an HFD for 14 wk and then treated with CY-09 for 6 wk. Bar, 50 µm; n = 6. Data are shown as mean and SEM and are representative of two independent experiments. Statistics were analyzed using an unpaired Student’s t test (A–E) or two-way ANOVA for (F–I): *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Concentration Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 suppresses NLRP3-dependent metainflammation in diabetic mice. (A–H) WT or Nlrp3 −/− mice were first fed with an HFD for 14 wk and then treated with CY-09 for 6 wk. Plasma IL-1β (A) was assessed by ELISA. Liver (B, E, and G) and white adipose tissue (WAT; C, F, and H) were isolated and cultured for 24 h, and supernatants were analyzed by ELISA for IL-1β (B and C), TNF-α (E and F), or monocyte chemoattractant protein 1 (G and H). Caspase-1 activation in WAT was analyzed by immunoblot as indicated (D). n = 6–8 per group. Data are shown as mean and SEM and are representative of two independent experiments. Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture, Activation Assay, Western Blot

Journal: Pharmaceuticals

Article Title: In Silico and In Vitro Evaluation of Quercetin Metabolites Binding to Inflammatory Target Proteins

doi: 10.3390/ph19050655

Figure Lengend Snippet: Molecular docking analysis. ( A ) The tested compounds (quercetin, quercetin 3- O -glucuronide, quercetin 7- O -glucuronide, isorhamnetin, isorhamnetin 3- O -glucuronide, and the positive control (MCC950) were inside the binding site of the NLRP3 (pdb:6npy) protein. ( B ) Quercetin, isorhamnetin, and MCC950 with the interacting amino acids.

Article Snippet: Recombinant NLRP3 (Origene, TP750176) was labeled with the Monolith Protein Labeling Kit RED-NHS 2nd Generation (Cat. No. M0-L011, NanoTemper Technologies, Munich, Germany).

Techniques: Positive Control, Binding Assay

Journal: Pharmaceuticals

Article Title: In Silico and In Vitro Evaluation of Quercetin Metabolites Binding to Inflammatory Target Proteins

doi: 10.3390/ph19050655

Figure Lengend Snippet: Binding of quercetin, isorhamnetin, quercetin 7- O -glucuronide, quercetin 3- O -glucuronide, isorhamnetin 3- O -glucuronide, tamarixetin, hippuric acid, and 3,4-dihydroxytoluene to NLRP3 as determined by microscale thermophoresis.

Article Snippet: Recombinant NLRP3 (Origene, TP750176) was labeled with the Monolith Protein Labeling Kit RED-NHS 2nd Generation (Cat. No. M0-L011, NanoTemper Technologies, Munich, Germany).

Techniques: Binding Assay, Microscale Thermophoresis