Complement-coupled erythrocyte platform for rapid enrichment of candidate NLRP3 modulators.

Despite the validation of NLRP3 as a therapeutic target for inflammatory disorders, conventional cell-based screens for NLRP3 modulators remain resource-intensive and technically demanding. Here, we report a phenotypic screening platform based on spectosis, a complement-driven erythrocyte death pathway mechanistically coupled to NLRP3 signaling. By monitoring complement-mediated hemolysis, this system enables high-throughput phenotypic enrichment of compounds for subsequent evaluation. A diverse library of 1000 small molecules was screened using a permissive primary threshold of 20% inhibition to ensure comprehensive coverage, yielding 365 initial actives. These underwent three rounds of confirmatory screening under identical conditions, yielding 11 validated hits that consistently passed all rounds. Importantly, counter-screening against terminal complement component C9 deposition showed that ten of these eleven compounds did not reduce C9 deposition, indicating that the majority of hits do not act via direct complement suppression. Translational validation in THP-1 macrophages identified five candidates that effectively attenuated both IL-1β maturation and LDH release, consistent with NLRP3 pathway modulation. This erythrocyte-based platform thus enables rapid, cost-effective enrichment of candidate chemotypes with potential NLRP3-related activity from large chemical libraries while circumventing the throughput limitations of conventional macrophage-based assays.