RESUMEN
Introduction: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many monoclonal antibody therapies illustrates the need for anti-viral treatments with low susceptibility to evolutionary escape. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly. This compound has minimal host toxicity, including once daily oral dosing in rats that achieves >200-fold of the 90% effective concentration (EC90) in blood. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low suceptability to evolutionary escape. We hypothesized that PAV-104 would be active against SARSCoV- 2 variants in human airway epithelial cells. Methods: Airway epithelial cells were differentiated from lung transplant tissue at air-liquid interface (ALI) for four weeks prior to challenge with Alpha (Pango lineage designation B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) SARS-CoV-2 variants. Viral replication was determined by quantitative PCR measurement of the SARS-CoV-2 nucleocapsid (N) gene. Dose-dependent virus inhibition and cytotoxicity of PAV-104 in the Calu-3 airway epithelial cell line was determined by PCR and MTT assay. Student's t-tests were used to evaluate statistical significance. Results: Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 showed comparable infectivity in human primary airway epithelial cells at ALI (N=3 donors), 47- to 550-fold higher than the parent (USA-WA1/2020) strain. PAV-104 reached 50% cytotoxicity in Calu-3 cells at 240 nM (Fig. 1A). Dose-response studies in Calu-3 cells demonstrated PAV-104 has a 6 nM 50% inhibitory concentration (IC50) for blocking replication of SARS-CoV-2 (USA-WA1/2020) (Fig.1B). In primary cells at ALI from 3 donors tested, there was >99% inhibition of infection by SARS-CoV-2 Gamma variant (N=3, MOI 0.1, P <0.01) with 100 nM PAV-104 (Fig. 1C). Addition of 100 nM PAV-104 2-hours post-infection, but not pre-infection, resulted in >99% suppression of viral replication, indicating a post-entry drug mechanism. PAV-104 bound a small subset of the known allosteric modulator 14-3-3, itself implicated in the interactome of SARS-CoV-2. Conclusion: PAV-104 is a host-targeted, orally bioavailable, pan-viral small molecule inhibitor with promising activity against SARS-CoV-2 variants in human primary airway epithelial cells. (Figure Presented).