A universal cell-based method for high-throughput analysis of viroporin function and inhibition.

Viroporins are transmembrane proteins encoded mainly by RNA viruses that oligomerize to form pores essential for multiple stages of the viral life cycle. Although they represent attractive antiviral drug targets, progress has been limited in part by the absence of high-throughput approaches to assess viroporin activity and inhibition. Here, we present a novel cell-based, fluorescence-driven method (VipoFluX) that enables high-throughput discovery of viroporins and their characterization, including inhibition in eukaryotic cells. We confirmed the robustness of the method by reproducing datasets from established assays and demonstrating applicability across diverse viroporins. Using this platform, we screened for viroporin inhibitors across four targets (M2 from influenza A virus, E from severe acute respiratory syndrome virus 2, 2B from coxsackievirus B3, vpu from human immunodeficiency virus 1) and validated hits using two-electrode voltage clamp electrophysiology. Overall, this method provides a scalable and versatile tool for advancing viroporin research and accelerating antiviral drug discovery.