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Kinetic characterization of HCV NS3 inhibitors using an SPR biosensor
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. (Helena Danielson)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
(English)Manuscript (Other academic)
Abstract [en]

An SPR biosensor-based assay for studies of the interactions with full-length NS3 protease from hepatitis C virus (HCV) has been developed. It was used to characterize the interaction kinetics for a series of NS3 protease inhibitors. Moreover, the interaction between NS3 and the NS4A cofactor could be studied.

The KD of the NS3-NS4A interaction was 30 nM in the standard buffer. It was reduced 600-fold by increasing the ionic strength to 300 mM NaCl. By using surfaces with only NS3 or with NS3 and co-immobilised NS4A, the effect of this protein cofactor on the interaction with several protease inhibitors was investigated. NS4A increased the affinity for all compounds, between 2 to 40 times, indicating that the NS3-NS4A complex binds inhibitors better than only NS3. The obtained interaction data was also compared with inhibition data, revealing a very good correlation between koff or KD with Ki (r = 0.92 and r = 0.90 respectively) over a broad range of affinities and potencies, showing that this biosensor based assay is a good and powerful tool for detailed studies of NS3 protease inhibitors which can serve as a future cure for HCV infection.

Keyword [en]
Hepatitis C virus, NS3, NS4A, protease, biosensor, SPR, inhibition
Identifiers
URN: urn:nbn:se:uu:diva-98863OAI: oai:DiVA.org:uu-98863DiVA: diva2:201396
Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2009-11-24
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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