title: Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease.
abstract: The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.
authors.affiliation: Departments of Biochemistry, Medicinal Chemistry, and Computational Chemistry, Istituto di Ricerche di Biologia Molecolare (IRBM) "P. Angeletti", Via Pontina Km 30 600, 00040 Pomezia, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Narjes
authors.forename: M
authors.initials: M
authors.lastname: Brunetti
authors.forename: S
authors.initials: S
authors.lastname: Colarusso
authors.forename: B
authors.initials: B
authors.lastname: Gerlach
authors.forename: U
authors.initials: U
authors.lastname: Koch
authors.forename: G
authors.initials: G
authors.lastname: Biasiol
authors.forename: D
authors.initials: D
authors.lastname: Fattori
authors.forename: R
authors.initials: R
authors.lastname: De Francesco
authors.forename: V G
authors.initials: VG
authors.lastname: Matassa
authors.forename: C
authors.initials: C
authors.lastname: Steinkühler
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000613:Aminobutyrates / Q000737:chemistry; D001665:Binding Sites; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D020128:Inhibitory Concentration 50; D007651:Keto Acids / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000409:Alanine; D000613:Aminobutyrates; D007651:Keto Acids; D009842:Oligopeptides; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C086477:2-amino-3,3'-difluoroisobutyric acid; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases