Orally efficacious lead of the AVG inhibitor series targeting a dynamic interface in the respiratory syncytial virus polymerase

Abstract

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infections in infants and the immunocompromised, yet no efficient therapeutic exists. We have identified the AVG class of allosteric inhibitors of RSV RNA synthesis. Here, we demonstrate through biolayer interferometry and in vitro RNA-dependent RNA polymerase (RdRP) assays that AVG compounds bind to the viral polymerase, stalling the polymerase in initiation conformation. Resistance profiling revealed a unique escape pattern, suggesting a discrete docking pose. Affinity mapping using photoreactive AVG analogs identified the interface of polymerase core, capping, and connector domains as a molecular target site. A first-generation lead showed nanomolar potency against RSV in human airway epithelium organoids but lacked in vivo efficacy. Docking pose-informed synthetic optimization generated orally efficacious AVG-388, which showed potent efficacy in the RSV mouse model when administered therapeutically. This study maps a druggable target in the RSV RdRP and establishes clinical potential of the AVG chemotype against RSV disease.

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases

“…1c ) that was functional in initiation of RNA synthesis, but defective in elongation (Fig. 1f, g ), reminiscent of an RSV L construct truncated at the C-terminal end of the CD 34 . L trunc -P was readily amenable to addition of the MRK-1 ligand and a co-complex structure was solved at a resolution of 2.39 Å (Table 1 , Supplementary Fig.…”

“…YM-53403 and its derivatives, AZ-27 and PC-786, elicit resistance in a linker region between the CD and MTase domains 29 – 32 , with AZ-27 inhibiting initiation of RNA synthesis at the promoter 30 . An AVG series of compounds elicit resistance in the same linker region and in the CD 30 , 33 , 34 . These compounds bind to an interface between the PRNTase, CD, and MTase domains, as demonstrated by photoaffinity cross-linking, and inhibit the polymerase from elongating the RNA 30 , 33 , 34 .…”

“…An AVG series of compounds elicit resistance in the same linker region and in the CD 30 , 33 , 34 . These compounds bind to an interface between the PRNTase, CD, and MTase domains, as demonstrated by photoaffinity cross-linking, and inhibit the polymerase from elongating the RNA 30 , 33 , 34 . Finally, the BI compound series elicits resistance in the PRNTase domain, and compound BI-D inhibits cap addition and causes dysregulated elongation 35 , 36 .…”

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor

“…However, during manuscript preparation, the availability of Alphafold2 46 provided a glimpse of a model of full-length RSV L, which will require validation using experimental cryo-EM structures in the future. The binding sites of YM-53403 32 , AZ-27 33 , 34 , PC786 35 , AVG-233 36 , and AstraZeneca inhibitor cpd 1 37 had been mapped to the connecting domain of an alphafold2 predicted model. Escape mutations in residues L 1502 , Y 1631 , and H 1632 are clustered at the N-terminal boundary of the connecting domain of an alphafold2 predicted model (Supplementary Fig.…”

Biochemistry of the Respiratory Syncytial Virus L Protein Embedding RNA Polymerase and Capping Activities

“…AVG-388 is a nucleoside analog from a novel AVG class of allosteric inhibitors of RSV that has been shown to effectively block the activity of the RSV polymerase in vitro by disturbing the initiation of viral RNA synthesis at the promoter. AVG-388 is orally available and showed potency in the RSV mouse model when administered therapeutically [ 173 , 174 ].…”