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Interaction kinetic characterization of HIV-1 reverse transcriptase non-nucleoside inhibitor resistance
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2006 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, Vol. 49, no 8, 2375-87 p.Article in journal (Refereed) Published
Abstract [en]

To decipher the mechanism for non-nucleoside inhibitor resistance of HIV-1 reverse transcriptase, the kinetics of the interaction between wild type and drug-resistant variants of the enzyme and structurally diverse inhibitors were determined. Substitution of amino acid residues in the inhibitor binding site resulted in altered rate constants for the pre-equilibrium between two unliganded forms of the enzyme, and for the association and dissociation of the inhibitor-enzyme interaction. The Y181C, V108I, and P225H substitutions affected primarily the association and dissociation rate constants, while the K103N and the L100I substitutions also influenced the equilibrium between the two forms of the free enzyme. The K103N and the L100I substitutions were found to facilitate both the entry of the inhibitor into the binding pocket as well as its exit, in contrast to what has been reported elsewhere. Interaction kinetic-based resistance profiles showed that phenethylthiazolylthiourea compounds were relatively insensitive to the studied substitutions.

Place, publisher, year, edition, pages
2006. Vol. 49, no 8, 2375-87 p.
Keyword [en]
Binding Sites, Biosensing Techniques/methods, Crystallography; X-Ray, Drug Resistance; Viral, Enzyme Activation/drug effects, HIV-1 Reverse Transcriptase/*antagonists & inhibitors/chemistry, Kinetics, Molecular Structure, Protein Conformation, Protein Structure; Tertiary, Reverse Transcriptase Inhibitors/chemistry/*pharmacology, Structure-Activity Relationship
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-93052DOI: 10.1021/jm0504050PubMedID: 16610781OAI: oai:DiVA.org:uu-93052DiVA: diva2:166411
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2009-11-24Bibliographically 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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