The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.