Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases

Abstract

The ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.

Fig. 1. MM01 is an inhibitor of ASC-mediated pro-Casp-1 activation.

A Dose–response curve of compound MM01 (up to 40 µM) from an in vitro ASC-mediated pro-Casp-1 activation assay. B Recombinant caspase-1 activity was monitored in the presence of MM01 (10 µM) to exclude compounds affecting active caspase-1. An inhibitor of the caspase catalytic site, zVAD (20 nM), was used as a control. C Recombinant caspase-3 activity was measured in the presence of zVAD (20 nM) and MM01 (5 µM). D Recombinant caspase-9 activity was measured in the presence of zVAD (20 nM) and MM01 (5 µM). As an activation control, procaspase-9 incubated with Na-citrate buffer was used. E Apoptosome activity was monitored after reconstitution in vitro in the presence of zVAD (20 nM) and MM01 (5 µM). In all cases, data represent the mean ± SD of three independent experiments (***p < 0.001).

Fig. 2. MM01 inhibits ASC oligomerization in vitro.

A Electronic microscopic images of negatively stained preparations of human recombinant ASC filaments in the presence of MM01 (20 µM). B Western blot of lysates and crosslinked cytosolic pellets of ASC-YFP transfected HEK293 cells treated with MM01 (10 µM). GAPDH was used as a loading control for lysate samples. C Live-cell imaging of THP-1 ASC GFP cells treated with MM01 (10 µM) and stimulated with LPS (100 ng/ml) and nigericin (10 µM). The blue signal corresponds to DAPI staining and the red signal to the cytoplasmic marker WGA (wheat germ agglutinin). D Percentage of ASC specks in the THP-1 ASC GFP cells treated as in C and measured by flow cytometry. Data represent the mean ± SD of three independent experiments (***p < 0.001). E Western blot of crosslinked cytosolic pellets from PMA-differentiated THP-1 ASC GFP cells stimulated with LPS and nigericin and treated with the indicated compounds as in the above-described conditions.

Fig. 3. ASC-MM01-binding site characterization.

A Docking model of ASC with MM01. Green lines represent hydrogen bonds, red lines show van der Waals interactions, and blue lines depict π interactions. Western blot (B) and percentage (C) of ASC specks in HEK293 cells transfected with the different ASC mutants and measured by flow cytometry. Data represent the mean ± SD of three independent experiments (***p < 0.001). D Live-cell imaging of HEK293 cells transfected with ASC mutants. The blue signal corresponds to DAPI staining and the red signal to the cytoplasmic marker WGA.

Fig. 4. MM01 inhibits ASC-dependent inflammasome activity in vitro.

A IL-1β secretion was evaluated by ELISA following activation of the NLRP3 inflammasome with LPS (100 ng/ml) and nigericin (Nig; 10 µM). Cells were treated with MM01 at 10 µM. B Measurement of LDH release into the extracellular medium under the above-described conditions. C THP-1 cells were stimulated as described above, and supernatants (SN) and pellets were analyzed by immunoblotting for IL-1β and cleaved caspase-1. A representative blot is shown. IL-1β measured by ELISA (D) and LDH release (G) were evaluated upon activation of the NLRP1 inflammasome with LPS (100 ng/ml) and MDP (50 µg/ml) in THP-1 cells. Cells were treated with MM01 at 10 µM. IL-1β secretion (E) and LDH release (H) were analyzed upon stimulation of the AIM2 inflammasome with poly (dA:dT) (200 μg/ml) in PMA-differentiated THP-1 cells treated or not with MM01 at 10 µM. IL-1β (F) and LDH secretion (I) were evaluated upon NLRC4 activation mediated by Salmonella typhimurium in THP-1 cells. Cells were treated with MM01 at 10 µM. MM01 inhibits LPS/Nig stimulation of isolated human PBMCs. IL-1β (J) and LDH (K) release were evaluated upon activation of the NLRP3 inflammasome with LPS (100 ng/ml) and nigericin (10 µM) and +/−MM01 at 10 µM in human PBMCs. L Western blot of crosslinked cytosolic pellets from PBMCs stimulated as described above. In all cases, asterisks represent significant differences to the stimulated control as determined by a one-way ANOVA with Tukey’s multiple comparisons test **p < 0.05; ***p < 0.001. All data expressed as mean ± SD of three independent experiments.

Fig. 5. MM01 inhibits ASC-dependent inflammasome activity in stimulated murine peritoneal macrophages and in MSU-induced peritonitis mouse model.

IL-1β (A), IL-6 (B), and TNF-α (D) secretion was evaluated by ELISA upon activation of the NLRP3 inflammasome with LPS (100 ng/ml) and ATP (2.5 mM) +/− MM01 at 1, 5, or 10 µM in isolated peritoneal macrophages. C Percentage of LDH release under the above-described conditions. In all cases, asterisks represent significant differences to the stimulated control (LPS/ATP), as determined by a one-way ANOVA with Tukey’s multiple comparisons test **p < 0.05; ***p < 0.001. All data expressed as mean ± SD of three experiments. E IL-1β detection by ELISA in the peritoneal cavity of C57BL/6 mice injected with MSU crystals with or without MM01 (10 mg/kg) treatment. F Neutrophil numbers in the peritoneal cavity of C57BL/6 mice treated as in the above-described conditions. Data are representative of two independent experiments (mean ± SD of n = 12). Asterisks represent significant differences to the stimulated control as determined by a one-way ANOVA with Tukey’s multiple comparisons test ***p < 0.001.