title: Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors.
abstract: The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.
authors.affiliation: Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, 751 23 Uppsala, Sweden.
authors.forename: Matthis
authors.initials: M
authors.lastname: Geitmann
authors.forename: Göran
authors.initials: G
authors.lastname: Dahl
authors.forename: U Helena
authors.initials: UH
authors.lastname: Danielson
mesh_terms: D002219:Carbamates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003521:Cyclopropanes; D004789:Enzyme Activation / Q000187:drug effects*; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D054833:Isoindoles; D007700:Kinetics; D007769:Lactams / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D047028:Macrocyclic Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002219:Carbamates; D003521:Cyclopropanes; D054833:Isoindoles; D007769:Lactams; D047029:Lactams, Macrocyclic; D047028:Macrocyclic Compounds; D011392:Proline; D011480:Protease Inhibitors; D011804:Quinolines; D013449:Sulfonamides; D013844:Thiazoles; D017361:Viral Nonstructural Proteins; C479555:BILN 2061; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C553752:danoprevir