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Resistance profiles of cyclic and linear inhibitors of HIV-1 protease
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
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2002 (English)In: Antiviral Chemistry & Chemotherapy, ISSN 0956-3202, E-ISSN 2040-2066, Vol. 13, no 1, 27-37 p.Article in journal (Refereed) Published
Abstract [en]

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

Place, publisher, year, edition, pages
2002. Vol. 13, no 1, 27-37 p.
National Category
Pharmaceutical Sciences Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-64332PubMedID: 12180647OAI: oai:DiVA.org:uu-64332DiVA: diva2:92243
Available from: 2006-03-04 Created: 2006-03-04 Last updated: 2017-11-30Bibliographically approved
In thesis
1. Interaction Characteristics of Viral Protease Targets and Inhibitors: Perspectives for drug discovery and development of model systems
Open this publication in new window or tab >>Interaction Characteristics of Viral Protease Targets and Inhibitors: Perspectives for drug discovery and development of model systems
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Viral proteases are important targets for anti-viral drugs. Discovery of protease inhibitors as anti-viral drugs is aided by an understanding of the interactions between viral protease and inhibitors. This thesis addresses the characterization of protease-inhibitor interactions for application to drug discovery and model system development.

The choice of a relevant target is essential to molecular interaction studies. Therefore, full-length NS3 protein of hepatitis C virus (HCV) was obtained, providing a more relevant target and a better model for the development of HCV protease inhibitors. In addition, resistance to anti-viral drugs, a serious problem in the treatment of AIDS, prompted the investigation of resistant variants of human immunodeficiency virus (HIV) protease.

Drug resistance was initially explored by characterization of the interactions between a series of closely related inhibitors and resistant variants of HIV protease, using an inhibition assay to determine the inhibition dissociation constants (Ki). The relationship between structure, activity and resistance profiles was not clarified, indicating that the effect of structural changes in the inhibitors and the protease are not predictable and must be analyzed case wise. It was proposed that additional kinetic characterization of the interactions was required and a biosensor-based method allowing for determination of affinity, KD, and interaction rate constants, kon and koff, was adopted. The increased physiological relevance of this method was confirmed, and the affinity data have better correlation with cell culture data. In addition, interactions between clinical inhibitors of HIV protease and enzyme variants indicate that increased dissociation rates (koff) are associated with the development of resistance.

Thermodynamic characterization of the interactions between HIV-1 protease and clinically relevant inhibitors revealed distinct energetic characteristics for inhibitors. The resolution of the energetics of association and dissociation identified an inhibitor with unique interaction characteristics and confirmed the validity of using this method for further characterization of molecular interactions.

This work resulted in the development of model systems for the analysis of kinetics, resistance and thermodynamic characteristics of protein-inhibitor interactions. The results give increased understanding of the biomolecular interactions and can be applied to drug discovery.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 49 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 822
Keyword
Biochemistry, viral proteases, biomolecular interactions, kinetics, thermodynamics, biosensors, drug discovery, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-3342 (URN)91-554-5568-9 (ISBN)
Public defence
2003-04-25, lecture hall B42, Uppsala, 10:15 (English)
Opponent
Available from: 2003-04-01 Created: 2003-04-01 Last updated: 2017-05-04Bibliographically approved
2. Exploring Inhibitors of HIV-1 Protease: Interaction Studies with Applications for Drug Discovery
Open this publication in new window or tab >>Exploring Inhibitors of HIV-1 Protease: Interaction Studies with Applications for Drug Discovery
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A variety of HIV-1 protease inhibitors and their interactions with the enzyme have been characterized in order to identify novel and improved drugs against AIDS. The investigated inhibitors were represented by clinical and non-clinical inhibitors, active site and allosteric inhibitors, transition-state analogues and metal-ions. In addition, different enzyme variants were used to investigate the contribution of different amino acid residues to the interaction with different ligands. The problem of resistance has been addressed by exploring novel types of inhibitors, and resistant mutants of HIV-1 protease. A study resolving the inhibition of HIV-1 protease by Cu2+ showed that the enzyme can be allosterically inhibited and that copper inhibition is a result of an interaction with His-69 and a subsequent conformational change. Several types of transition-state analogues were analyzed with respect to their inhibition of wild-type and resistant mutants of HIV-1 protease. Unfortunately cyclic compounds were not found to be better than linear compounds. Moreover, it was not possible to identify structure-activity relationships that clearly correlated with efficacy towards mutants and a biosensor based method for more detailed kinetic studies was therefore adopted. By cross-linking the immobilized enzyme on the biosensor matrix, a stable surface was obtained and kinetic rate constants could be determined for the interaction between the enzyme and inhibitors. Additional improvements in the methodology involved identification of a more representative interaction model, allowing more detailed studies of interactions with resistant mutants and varying conditions. Finally, absorption to lipid membranes and interaction with human serum albumin and α1-glycoprotein by clinical drugs were studied in a simplified ADME model system for improvement of the earlier stages of drug development. These studies have revealed important characteristics of these drugs that can potentially be modeled into new compounds that have improved efficacy of both wild-type and resistant mutants of HIV-1 protease.

Place, publisher, year, edition, pages
Uppsala: Institutionen för naturvetenskaplig biokemi, 2004. 47 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 1038
Keyword
Biochemistry, kinetics, biosensors, ADME, drug discovery, SPR, resistance, inhibition, copper, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-4655 (URN)91-554-6087-9 (ISBN)
Public defence
2004-12-03, B41, BMC, Husargatan 3, Uppsala, 13:30 (English)
Opponent
Supervisors
Available from: 2004-11-12 Created: 2004-11-12 Last updated: 2017-05-04Bibliographically approved

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