Complementary target engagement and functional assays to probe NLRP3 inflammasome pathway antagonism

Abstract Inflammasomes are critical to the innate immune system and implicated in numerous chronic and acute inflammatory diseases. Having tools to screen inflammasome modulatory drugs is critical, especially for the NLRP3 inflammasome that is triggered by a wide array of stimuli. We have developed three cell-based methods for quantifying inflammasome function: a NanoBRET™ NLRP3-specific target engagement assay, a caspase-1 activity assay and an IL-1β release assay. For the NLRP3 target engagement assay, cells are transfected with a NLRP3-Nanoluc construct and incubated with a cell-permeable, fluorescent NLRP3 binder (tracer), generating a bioluminescent resonance energy transfer (BRET) signal. The homogeneous caspase-1 and IL-1β assays can be run directly with cells in culture or with transferred culture medium, enabling application of both luminescent assays to the same cell wells via split-sample analysis. Using all three assays, we tested a series of reported NLRP3 inhibitors, including MCC950, oridonin, NBC6, NBC19, CY09, OXSi2 and OLT1177™ (dapansutrile). MCC950 inhibition of caspase-1 activity and IL-1β release was demonstrated in human and mouse cell models. We used LPS-primed THP-1 cells treated with nigericin for testing the series of inhibitors. The NLRP3 target engagement assay used HEK293 cells in suspension in non-binding plates. All three assays demonstrated good inhibition for MCC950, oridonin, NBC6, NBC19 and CY09 with rank order potency consistent with published results. We did not detect inhibition with OxSi2 or OLT1177™. These three assays enable screening for inflammasome antagonists and provide a mechanistic platform for characterizing NLRP3 inflammasome inhibitors.