Peptide-to-Small Molecule: Discovery of Non-Covalent, Active-Site Inhibitors of β-Herpesvirus Proteases

Viral proteases, the key enzymes that regulate viral replication and assembly, are promising targets for antiviral drug discovery. Herpesvirus proteases are enzymes with no crystallographically confirmed noncovalent active-site binders, owing to their shallow and polar substrate-binding pockets. Here, we applied our previously reported "Peptide-to-Small Molecule" strategy to generate novel inhibitors of β-herpesvirus proteases. Rapid selection with a display technology was used to identify macrocyclic peptide 1 bound to the active site of human cytomegalovirus protease (HCMV^Pro) with high affinity, and pharmacophore queries were defined based on the results of subsequent intermolecular interaction analyses. Membrane-permeable small molecule 19, designed de novo according to this hypothesis, exhibited enzyme inhibitory activity (IC₅₀ = 10^-6 to 10^-7 M) against β-herpesvirus proteases, and the design concept was proved by X-ray cocrystal analysis.