Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr alpha-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a similar to200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 muM. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity.

Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. An NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics.