A reversible broad-spectrum antiviral targets the human V-ATPase VO domain

Increasing threats of viral disease underscore the urgent need for broad-spectrum antiviral drugs (BSADs). Host proteins utilized by human pathogenic viruses are key BSAD targets. The vacuolar-type H⁺-ATPase (V-ATPase) has been identified as a proviral factor for most pH-dependent enveloped viruses classified as pandemic threats. We report here the discovery of cladoniamide A (CA)—a V-ATPase inhibitor with single-digit nanomolar antiviral activity and a high selectivity index (SI: 103-104) against human enveloped viruses [e.g., SARS-CoV-2 variants, influenza A viruses (H1N1, H5N1), respiratory syncytial virus, dengue serotypes 1–4, and Zika virus]. Transcriptome profiling, pH estimation assays, and V-ATPase bioassays indicate that CA interferes with V-ATPase-dependent acidification of the host endolysosomal network thus preventing viral entry. Using pseudoviruses derived from five pathogenic virus families, we confirmed that CA is a BSAD acting as an entry inhibitor. CryoEM revealed that CA inhibits the V-ATPase rotary motor by occupying unique binding sites in the membrane-embedded Vo motor. Importantly, intranasal CA treatment in mice infected with influenza A H1N1 significantly reduced viral load in the lung by four log orders. Together, these findings pave the way for developing next-generation BSADs targeted at unique druggable pockets that enable the reversible pharmacological modulation of the human V-ATPase.