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: 10 ³ -10 ⁴ ) 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 an entry inhibitor BSAD. CryoEM revealed that CA inhibits the V-ATPase rotary motor by occupying unique binding sites in the membrane-embedded V _o 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.