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Complement-coupled erythrocyte platform for rapid enrichment of candidate NLRP3 modulators
Lijia Shi1, Wenjing Liu1, Xueman Xie1
1State Key Laboratory of Bioreactor Engineering, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, East China University of Science and Technology, Shanghai, 200237, China.
Abstract:
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.
Insights
A novel erythrocyte-based assay screens for NLRP3 modulators, overcoming limitations of traditional methods. This cost-effective platform rapidly identifies potential drug candidates for inflammatory diseases.
Area of Science:
- Immunology
- Pharmacology
Background:
- NLRP3 inflammasome is a key target for inflammatory diseases.
- Conventional screening methods for NLRP3 modulators are resource-intensive and technically challenging.
Purpose of the Study:
- To develop and validate a high-throughput screening platform for NLRP3 modulators using spectosis.
- To identify novel small molecules that modulate NLRP3 signaling via an erythrocyte-based assay.
Main Methods:
- Phenotypic screening platform based on spectosis (complement-driven erythrocyte death).
- Monitoring complement-mediated hemolysis for compound evaluation.
- Screening of 1000 small molecules, followed by three rounds of confirmatory screening.
- Counter-screening against C9 deposition and validation in THP-1 macrophages.
Main Results:
- 365 initial active compounds identified from 1000 screened molecules.
- 11 validated hits consistently passed confirmatory screening.
- Ten of eleven hits did not suppress complement C9 deposition, suggesting non-direct complement inhibition.
- Five candidates showed efficacy in attenuating IL-1β maturation and LDH release in macrophages.
Conclusions:
- The erythrocyte-based spectosis assay is a rapid and cost-effective method for enriching NLRP3 modulator candidates.
- This platform overcomes the throughput limitations of conventional macrophage-based assays.
- Identified compounds warrant further investigation for treating NLRP3-mediated inflammatory disorders.
