title: Inhibition and binding kinetics of the hepatitis C virus NS3 protease inhibitor ITMN-191 reveals tight binding and slow dissociative behavior.
abstract: The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.
authors.affiliation: InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, USA. rrajagopalan@intermune.com
authors.forename: Ravi
authors.initials: R
authors.lastname: Rajagopalan
authors.forename: Shawn
authors.initials: S
authors.lastname: Misialek
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Stevens
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Brandhuber
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Ballard
authors.forename: Steven W
authors.initials: SW
authors.lastname: Andrews
authors.forename: Scott D
authors.initials: SD
authors.lastname: Seiwert
authors.forename: Karl
authors.initials: K
authors.lastname: Kossen
mesh_terms: D019943:Amino Acid Substitution; D016174:Hepacivirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases