Development of Allosteric Inhibitors Targeting Human Herpesviral Proteases

Human herpesviruses (HHVs) make up one of the most prevalent viral families and are the etiological agents of a variety of devastating human illnesses that lack safe and effective treatments. All HHVs express a dimeric serine protease that is essential to the viral life cycle. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), a gamma subfamily member, was chosen as the model system for developing broad-spectrum herpesviral inhibitors. Of particular interest are KSHV Pr inhibitors that act allosterically, either by dimer dissociation or other mechanisms. A small-molecule KSHV Pr inhibitor has been identified that traps a transient allosteric pocket at the protease dimer interface. The binding of this molecule (DD2) has been shown to prevent the dimerization of KSHV Pr, which is required for its activation. This novel binding pocket is bioinformatically predicted to be conserved across all HHV proteases and DD2 has been shown to have activity against the serine protease from cytolomegalovirus (CMV) a member of the beta herpesvirus subfamily. Additionally, methodologies have been developed to identify new inhibitors via a high throughput screen, elucidate the mechanism and binding modes of screening hits, and test for efficacy in a cell culture viral replication assay. This work presents methodologies and demonstrates the feasibility for identifying and using small-molecule allosteric inhibitors to target a conserved enzyme family that is conserved across a viral family.