Protein-Structure Assisted Optimization of 4,5-Dihydroxypyrimidine-6-Carboxamide Inhibitors of Influenza Virus Endonuclease

Abstract

Influenza is a serious hazard to human health that causes hundreds of thousands of deaths annually. Though vaccines and current therapeutics can blunt some of the perilous impact of this viral infection, new treatments are needed due to the constantly evolving nature of this virus. Recently, our growing understanding of an essential influenza viral protein, PA, has led to the development of focused libraries of new small molecules that specifically target the active site of the PA influenza endonuclease, which we report here. Our overarching approach has been to proactively develop lead inhibitors that are less likely to rapidly develop clinical resistance by optimizing inhibitors that retain activity against induced resistant mutants. Here, we report details behind the discovery of new potent inhibitors of wild type and resistant mutant endonucleases along with their high-resolution co-crystal structure-activity relationships. These results add to our understanding of nuclease protein targets and potentially serve as starting points for a new therapeutic approach to the treatment of influenza.

Crystallographically correct but confusing presentation of structural models deposited in the Protein Data Bank

“…An interesting case is a collection of 25 structures of different complexes of influenza virus endonuclease with various inhibitors that were published in a single paper (Beylkin et al, 2017). These structures were crystallized in three space groups, with a single protein molecule found in the asymmetric unit, but were presented in various locations around the unit cell (Table 1b).…”

Structural insights into the substrate specificity of the endonuclease activity of the influenza virus cap-snatching mechanism

“…The PA N endonuclease construct comprised residues 1–209 from the PA subunit of the pandemic H1N1 influenza virus A/California/04/2009 in which a flexible loop comprising residues 51–72 was replaced with a three-residue GGS linker. This choice of construct was based on previous work ( 24 ) and routinely produces high quality crystals suitable for analysis of protein-ligand complexes ( 29 ). The construct was cloned with an N-terminal His-tag into the pET28a+ expression vector.…”

New anti-viral drugs for the treatment of COVID-19 instead of favipiravir

“…As for the target protein, RNA polymerase is investigated in the Protein Data Bank (PDB) web tool. PDB IDs of selected target proteins, which belong to different virus types, are 5FDD, 5I13, 5W44, 6CFP, 6E6V, 6FS8, 6QWL, 6QX3 and 6QX8 (Beylkin et al, 2017;Credille et al, 2019;Fan et al, 2019;Kirchdoerfer & Ward, 2019;Omoto et al, 2018). Some of these proteins are multi-chain while others are single chain.…”