In Silico Discovery of a Protein-Protein Interaction Inhibitor for Influenza Viruses

Abstract

The Influenza virus poses a significant public health threat. Frequent mutations make the virus a difficult target for antiviral development. Amantadine and rimantadine are now ineffective against most circulating strains due to point mutations in the M2 channel, and neuraminidase inhibitors are also vulnerable to viral resistance. This highlights the need for the discovery of antivirals that inhibit infection by a new mechanism. We set out to design antivirals that would block influenza replication by inhibiting the interaction of Polymerase Basic 1 (PB1), a subunit of the viral polymerase complex, and Ran-Binding Protein 5 (Ran BP5), a host importin, with a small molecule that would bind to PB1. It has been shown that PB1 interacts with Ran BP5 for nuclear import, a necessary step in viral replication. Compounds that might block nuclear import of the polymerase complex were identified using high-throughput virtual screening coupled with in vitro validation assays. The Asinex protein-protein interactions library was docked with Glide and Autodock Vina to the PB1 subunit of 4WSB, the trimeric polymerase complex of bat influenza. The proposed binding site was chosen due to its proximity to the bipartite nuclear localization signal (NLS), and the top hits were rescreened using a higher exhaustiveness to confirm the results. Four compounds were chosen and tested in cell culture for inhibition of viral replication with an immunofluorescence assay and for cytotoxicity with a vital dye uptake assay. A lead compound was identified that has an EC50 of 13.89 µM against A/California/07/2009 and an EC50 of 16.19 µM against A/WS/33. This compound also showed some activity against B/USSR/69 (Brigit) with an EC50 of 37.52 µM. This lead compound is amenable to medicinal optimization.