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Thermodynamic analysis of non-nucleoside inhibitor binding to HIV-1 reverse transcriptase
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
(English)Article in journal (Refereed) Submitted
Identifiers
URN: urn:nbn:se:uu:diva-93053OAI: oai:DiVA.org:uu-93053DiVA: diva2:166412
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2010-03-18Bibliographically approved
In thesis
1. Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes: Applications for Antiviral Drug Development
Open this publication in new window or tab >>Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes: Applications for Antiviral Drug Development
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs.

The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors.

Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors.

The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding.

Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.

Place, publisher, year, edition, pages
Uppsala: Institutionen för naturvetenskaplig biokemi, 2005. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 51
Keyword
Biochemistry, SPR biosensor, HIV-1 reverse transcriptase, HCMV protease, interaction kinetics, drug discovery, non-nucleoside inhibitor, resistance, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-5797 (URN)91-554-6250-2 (ISBN)
Public defence
2005-05-31, Room B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-05-04Bibliographically approved

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Danielson, U. Helena

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