Leveraging protein dynamics to drug filovirus protein-nucleic acid interactions using simulations and experiments

Project Summary Many proteins are classified as ‘undruggable,’ especially those that engage in protein-protein and protein- nucleic acid interactions as they interact with their binding partners through flat interfaces. This is particularly true in the case of many viral proteins that interact with host factors and viral nucleic acids and lack enzymatic activity. In the highly lethal filovirus family one protein, viral protein 35 (VP35) is primarily responsible for the viruses’ immune evasion. In this family the ebolavirus is the most fatal with a case fatality rate at 66% in recent outbreaks. Thus, there is a great need for discovering new therapeutic targets in this family of viruses. Although proteins are known to be dynamic, only recently has considering protein dynamics for drug design become tractable through advances in molecular dynamics methods. As such, discovering ‘cryptic’ pockets that are absent in available structures but open due to protein dynamics could provide new druggable sites. Here, I propose integrating atomically-detailed simulations, biophysical, and cellular experiments to understand the cryptic pocket in viral protein 35 (VP35) from the highly lethal filoviruses. VP35 plays multiple essential roles in these viruses’ replication cycles, including binding the viral RNA genome to block hosts’ innate immunity and acting as a polymerase co-factor. However, available crystal structures of VP35 lack appealing pockets for drug discovery and the protein has so far remained undruggable. We recently have applied adaptive sampling simulations to preferentially sample conformations with large pocket volumes. This revealed a potentially druggable cryptic pocket. While the pocket does not directly coincide with the crucial interface for binding RNA, we have shown that the pocket can allosterically modulate RNA binding and is a good drug target. To further test this, I will use a thiol labeling experiment to directly test for this cryptic pocket in VP35 homologs. Then, to determine if this cryptic pocket is allosterically coupled to function, I will test if stabilizing the open form allosterically disrupts RNA binding using a fluorescence anisotropy assay. I will then screen for small-molecule inhibitors of RNA binding that bind to the cryptic pocket and confirm this by X-ray crystallography and mutational tests. Finally, I will assess the effect of stabilizing the open pocket on VP35’s interferon antagonism, and viral replication activities using an in-vitro ATPase assay and cellular minigenome assay. Successful completion of these experiments will further the National Institute of General Medical Sciences’ mission to increase our understanding of biological processes for laying the foundation of advancing disease treatments. These results will demonstrate the power of fusing simulations and experiments to characterize hidden conformations and dynamics, uncovering cryptic pockets and allostery that present new therapeutic opportunities.