P20 Establishing a high-throughput drug screening platform for NLRP1 inflammasome modulators

Abstract Introduction and aims Ingenanes are naturally occurring plant-derived compounds of significant interest for their antimicrobial and anti-inflammatory properties, especially within dermatology and cancer research. Notably, ingenol mebutate (IM) is a topical treatment for actinic keratosis (AK), a precancerous skin condition caused by prolonged sun exposure. Although not fully understood, IM appears to work through a dual mechanism by promoting an inflammatory response and neutrophil-mediated cellular cytotoxicity. Nod-like receptor protein 1 (NLRP1) has recently emerged as a key regulator of inflammation and innate immunity in the skin. When activated, NLRP1 binds to apoptosis-associated speck-like protein containing a CARD (ASC), which oligomerizes and aggregates to form specks. Caspase-1 facilitates the activation and secretion of highly potent inflammatory interleukin (IL)-1 cytokines, leading to cell death. We have established a screening methodology to test a panel of ingenanes as potential NLRP1 modulators. Methods An NLRP1 fluorescent N/TERT ASC KO, ASC GFP reporter cell line was used to validate a high-throughput imaging workflow. Initially, known activators of NLRP1 were imaged over 12 h with ASC speck formation and propidium iodide (PI) inclusion used as a measure of NLRP1 activation. Subsequently, imaging results were validated by enzyme-linked immunosorbent assay (IL-1β) and Western blot (pro IL-1β, cleaved IL-1β, ASC and GAPDH). Results As expected, known activators of NLRP1 led to ASC speck formation and PI inclusion that resulted in IL-1β release. These results validated that the imaging system could correctly identify known NLRP1 activators and be utilized as a screening tool for unknown biologics. Next, we screened a 96-well plate library of ingenols to identify potential NLRP1 activators. Preliminary findings identified two Ingenanes as strong NLRP1 activators with high levels of ASC speck formation for further analysis. Conclusions Through this assay, we have identified a subset of ingenols that strongly activate the NLRP1 inflammasome, potentially contributing to developing new therapeutic ingenols for AK.