Non‑nucleoside inhibitors of SARS‑CoV‑2 RdRp identified by high‑throughput screening and an approach to their mechanism of action by comparative studies

Abstract The RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pivotal antiviral target due to its essential role in viral replication and high conservation. Non-nucleoside analog inhibitors (NNIs) are a promising class of antiviral compounds that target SARS-CoV-2 RdRp, potentially circumventing resistance profiles associated with nucleoside analogues. Although multiple NNIs against SARS-CoV-2 RdRp have been reported, the knowledge regarding the modes of action remains fragmented, and the lack of a standardized cell-free assay has hindered cross-comparison of various NNIs. Notably, whether clinically approved drugs or the NNIs targeting hepatitis C virus (HCV) RdRp (NS5B) can be used to inhibit SARS-CoV-2 RdRp remains largely unexplored, and the chemical and mechanistic diversity of the NNIs are poorly defined. Here, we conducted a high-throughput screening of an FDA-approved drug library using a fluorescence-based RNA polymerase assay to identify novel NNIs targeting SARS-CoV-2 RdRp. Twenty-nine primary hits, along with sixteen previously reported SARS-CoV-2 NNIs and ten HCV RdRp NNIs, were systematically evaluated using a gel-based RdRp inhibition assay, resulting in seventeen positive compounds which include five novel inhibitors (verteporfin, benzbromarone, epigallocatechin gallate, mitoxantrone, and trifarotene) and two NNIs targeting HCV NS5B (JTK-109 and BILB-1941). Parallel dose-response analysis enabled direct efficacy comparison of the NNIs, showing suramin, C646, and baicalein as the most potent inhibitors. Gel mobility shift assay further revealed varied kinetics of the NNIs in disrupting the RdRp-RNA interaction, reflecting their diverse inhibitory mechanisms. Together, our findings expand the repertoire of NNIs against SARS-CoV-2 RdRp, establish a unified framework for comparative evaluation of RdRp inhibitors, and provide insights into the differential impacts of the NNIs on RdRp-RNA complex formation, thereby supporting future development of allosteric antiviral therapeutics.