Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor.
The nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of herpes simplex virus (HSV) ribonucleotide reductases, specifically inhibits this enzyme activity. We report here that under standard reductase assay conditions, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity. Determination of the proteolytic susceptibility of HSV R2-(329-337) analogs showed that natural modifications which are present in the homologous varicella zoster virus (VZV) nonapeptide decreased its susceptibility to protease action 1.5-fold. Nx-acetylation, a modification known to protect peptides against aminopeptidase attacks, greatly improved the proteolytic resistance of HSV and VZV nonapeptides. Moreover, Ac-VZV R2-(298-306) exhibited a 15-fold higher potency on reductase inhibition than HSV R2-(329-337). The degradation process of HSV R2-(329-337) was partially inhibited by amastatin, bestatin, and leupeptin whereas it was completely abolished by bacitracin, suggesting a combined action of more than one aminopeptidase activity. Moreover, bacitracin protected most of these nonapeptide analogs from proteolysis, although it was less effective in preventing HSV R2-(332-337) degradation. Our results indicate that it is possible to determine, in the presence of bacitracin, the relative inhibitory potencies of HSV R2-(329-337) analogs with minimal error due to proteolytic susceptibility. Moreover, HSV R2-(329-337) modifications that were found to protect the peptide against degradation might be useful to increase its efficacy in vivo.
Paradis
H
H
Institut du Cancer de Montréal, Cańada.
Langelier
Y
Y
Michaud
J
J
Brazeau
P
P
Gaudreau
P
P
D000595:Amino Acid Sequence; D001414:Bacitracin / Q000494:pharmacology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D018139:Simplexvirus / Q000201:enzymology*
D010446:Peptide Fragments; D011480:Protease Inhibitors; D001414:Bacitracin; D012264:Ribonucleotide Reductases