Herpesvirus protease inhibition by dimer disruption.
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 approximately 200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 microM. 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.
Shimba
Nobuhisa
N
Department of Pharmaceutical Chemistry, University of California-San Francisco, 600 16th St., Box 2280, San Francisco, CA 94143-2280, USA.
Nomura
Anson M
AM
Marnett
Alan B
AB
Craik
Charles S
CS
D000595:Amino Acid Sequence; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D019288:Herpesvirus 8, Human / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity
D010455:Peptides; D011480:Protease Inhibitors; D010450:Endopeptidases