RESUMEN
Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. 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 with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.
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).
RESUMEN
Background: Galectin-9 (Gal-9) is a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis. Multiple recent reports demonstrate that plasma levels of Gal-9 are elevated in the setting of severe COVID-19 disease. However, a causal role of Gal-9 in SARS-CoV-2 pathology remains to be elucidated. Here, we determined the impact of Gal-9 on SARS-CoV-2 replication and pro-inflammatory signaling in immortalized and primary human airway epithelial cells (AECs). Methods: Dose-dependent cytotoxicity of recombinant human Gal-9 in the Calu-3 AEC line was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 lineage P.1 (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the nucleocapsid (N) gene, immunofluorescence assay (IFA) of N protein, and titration of supernatant (TCID50). Viral entry was measured using luciferase activity of VSV-SARS-CoV-2 S-ΔG-Luciferase reporter pseudovirus. ACE2 and TMPRSS2 cell-surface expression were measured by flow cytometry. Pro-inflammatory factors (IL-6, IL-8, and TNFα) were detected by RT-PCR. Total RNA-seq was used to evaluate Gal-9 effects on the host transcriptome. Groups were compared by Student's t-test, and differential expression analyses were performed using DESeq2. Results: Gal-9 reached 50% cytotoxicity in Calu-3 cells at 597 nM. Gal-9 significantly increased SARS-CoV-2 expression (8.1 to 25.5 fold;p<0.0001) and infectious virus release (1.9 to 17.8 fold;p<0.038) in a dose-dependent manner in Calu-3 cells. Pseudovirus entry into Calu-3 cells was enhanced by Gal-9 (2.4 to 5.6 fold;p<0.0016), and the enhanced entry was inhibited by anti-ACE2 antibody (p<0.0027). Cell surface ACE2 and TMPRSS2 expression were unaffected by Gal-9. Gal-9 treatment accelerated virus-induced expression of IL-6, IL-8, and TNFα (p<0.018) in Calu-3 cells. Gal-9 increased SARS-CoV-2 production (p=0.03) and pro-inflammatory factor expression (p<0.05) in primary AECs (N=5 donors). RNA-seq data revealed that Gal-9 significantly induced IL-17, EIF2, IL-8 and IL-6 signaling pathways in the setting of SARS-CoV-2 infection. Conclusion: Gal-9 facilitates SARS-CoV-2 entry, replication, and virus-induced pro-inflammatory signaling in AECs ex vivo. Our data suggest that pharmacologic manipulation of Gal-9 should be explored as a SARS-CoV-2 therapeutic strategy.