title: Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase.
abstract: A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K(d) values ranging from 30 to 60 microM. In addition, these peptides inhibited the NS5B activity in vitro with IC(50) ranging from 6 to 48 microM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase alpha, human polymerase beta, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC(50) of 50 microM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis.
authors.affiliation: Department of Infectious Disease, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Amin
authors.forename: Joe
authors.initials: J
authors.lastname: Zaccardi
authors.forename: Stanley
authors.initials: S
authors.lastname: Mullen
authors.forename: Stephane
authors.initials: S
authors.lastname: Olland
authors.forename: Mark
authors.initials: M
authors.lastname: Orlowski
authors.forename: Boris
authors.initials: B
authors.lastname: Feld
authors.forename: Patrick
authors.initials: P
authors.lastname: Labonte
authors.forename: Paul
authors.initials: P
authors.lastname: Mak
mesh_terms: D004220:Disulfides; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016174:Hepacivirus / Q000201:enzymology*; D019151:Peptide Library; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004220:Disulfides; D004791:Enzyme Inhibitors; D019151:Peptide Library; D010455:Peptides; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase