Abstract

The formyl peptide receptor (FPR), a class A GPCR, plays a pivotal role in the pathogenesis of various diseases, including lung injury. In this study, a phenotype-based Epic® label-free screening was integrated with FPR-mediated calcium flux assays to identify dual antagonists of FPR1 and FPR2 from a compound library. Through comprehensive functional characterization, which included NanoBiT-based assays for G protein dissociation and β-arrestin2 recruitment as well as FlAsH-NanoBRET-based conformation detection, Kobe2602 was identified as a dual antagonist of FPR1 and FPR2. Kobe2602 effectively inhibited multiple FPR-mediated responses, including G protein dissociation, β-arrestin2 recruitment, calcium mobilization, superoxide production, degranulation, cell migration, and receptor internalization. Competition binding and conformational biosensor assays further demonstrated that Kobe2602 directly bound to FPR1 and FPR2 and induced conformational changes in both receptors. In a mouse model of LPS-induced acute lung injury, pathological damage was attenuated by Kobe2602. Our findings suggest that Kobe2602 is a dual FPR1/FPR2 antagonist with therapeutic potential in lung injury.