Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament

Abstract

The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z'-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro, the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens.

Tyrosine kinase inhibitors can activate the NLRP3 inflammasome in myeloid cells through lysosomal damage and cell lysis

“…Efforts to discover small-molecule modulators of the inflammasome have primarily focused on the identification of inhibitors (19)(20)(21), although some have also identified new activators (22). Smallmolecule activators of NLRP1, pyrin, and NLRP3 would simplify the study of these inflammasomes and, particularly in the case of NLRP3, might yield fundamental insight into activation mechanisms.…”

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity

“…Currently described NLRP3 inhibitors such as CY-09 or MCC950 directly bind to the ATP-binding motif of the NLRP3 NACHT domain [21,30]. Although several groups have investigated various molecular inhibitors interacting with different components of inflammasome complexes [16][17][18][19][20][21][22][23][24][25][31][32][33], to the best of our knowledge, there exists no specific inhibitor of NLRP3 PYD homo-interactions.…”

Tyrosine kinase inhibitors trigger lysosomal damage-associated cell lysis to activate the NLRP3 inflammasome

“…Efforts to discover small molecule modulators of the inflammasome have primarily focused on identification of inhibitors (21)(22)(23), though some have also identified new activators (24). Small molecule activators of NLRP1, pyrin and NLRP3 would simplify the study of these inflammasomes and, particularly in the case of NLRP3, might yield fundamental insight into activation mechanisms.…”