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Resistance profiling of hepatitis C virus protease inhibitors using full-length NS3
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. (ORGFARM)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. (ORGFARM)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2007 (English)In: Antiviral Therapy, ISSN 1359-6535, E-ISSN 2040-2058, Vol. 12, no 5, 733-740 p.Article in journal (Refereed) Published
Abstract [en]

Background: The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3.

Experimental: The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain.

Results: The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme.

Conclusions: These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.

Place, publisher, year, edition, pages
2007. Vol. 12, no 5, 733-740 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-17089ISI: 000248546900004PubMedID: 17713156OAI: oai:DiVA.org:uu-17089DiVA: diva2:44860
Available from: 2008-06-16 Created: 2008-06-16 Last updated: 2017-12-08Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 615
Keyword
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
Biochemistry
Identifiers
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)
Opponent
Supervisors
Available from: 2009-03-26 Created: 2009-03-04 Last updated: 2017-05-04

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Dahl, GöranSandström, AnjaÅkerblom, EvaDanielson, U. Helena

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