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Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of  Protease Inhibitors
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Helena Danielson)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 48, 11592-11602 p.Article in journal (Refereed) Published
Abstract [en]

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a   bifunctional enzyme with a protease and a helicase functionality   located in each of the two domains of the single peptide chain. There   is little experimental evidence for a functional role of this   unexpected arrangement since artificial single domain forms of both   enzymes are catalytically competent. We have observed that low   concentrations of certain protease inhibitors activate the protease of   full-length NS3 from HCV genotype 1a with up to 100%, depending on the   preincubation time and the inhibitor used. The activation was reduced,   but not eliminated, by increased ionic strength, lowered glycerol   concentration, or lowered pH. In all cases, it was at the expense of a   significant loss of activity. Activation was not seen with the   artificial protease domain of genotype 1b NS3 fused with a fragment of   the NS4A cofactor. This truncated and covalently modified enzyme form   was much less active and exhibited fundamentally different catalytic   properties to the full-length NS3 protease without (he fused cofactor.   The most plausible explanation for the activation was found to involve   a slow transition between two enzyme conformations, which differed in   their catalytic ability and affinity for inhibitors. Equations derived   based on this assumption resulted in better fits to the experimental   data than the equation for simple competitive inhibition. The mechanism   may involve an inhibitor-induced stabilization of the helicase domain   in a conformation that enhances the protease activity, or all improved   alignment of the catalytic triad in the protease. The proposed mnemonic   mechanism and derived equations are viable for both these explanations   and can serve as a basic framework for future studies of enzymes   activated by inhibitors or other ligands.

Place, publisher, year, edition, pages
2009. Vol. 48, no 48, 11592-11602 p.
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-98859DOI: 10.1021/bi9016928ISI: 000272083900028OAI: oai:DiVA.org:uu-98859DiVA: diva2:201383
Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2010-06-28Bibliographically approved
In thesis
1. Kinetic studies of NS3 and NS5B from Hepatitis C virus: Implications and applications for drug discovery
Open this publication in new window or tab >>Kinetic studies of NS3 and NS5B from Hepatitis C virus: Implications and applications for drug discovery
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV.

By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided.

Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease.

This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays.

Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor.

Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen.

The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.


Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 65 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 615
Hepatitis C virus, NS3, NS5B, enzyme kinetics, inhibition, resistance, drug
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
urn:nbn:se:uu:diva-98868 (URN)978-91-554-7445-4 (ISBN)
Public defence
2009-04-17, BMC, Sal B7:101a, Husargatan 3, UPPSALA, 09:00 (English)
Available from: 2009-03-26 Created: 2009-03-04 Last updated: 2012-04-13

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