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Characterization of HCV Protease Inhibitors: Inhibition and Interaction Studies with Applications for Drug Discovery
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. (Helena Danielson)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, different approaches based on inhibition and interactions studies, have been used to characterize inhibitors of the non-structural protein 3 (NS3) from the hepatitis C virus (HCV). This involves identification of enzyme inhibitory effects and characterization of interaction mechanisms and kinetics, as well as effects on replication in a cell based system and serum protein binding. All this information contributes to HCV drug discovery.

By using an inhibition assay it was possible to evaluate the effects of NS3 protease inhibitors, tested or used in the clinic, on NS3 variants, representing different model systems often used for drug discovery. This study illustrates the importance of accounting for differences in catalytic properties in comparative analyses, for making relevant interpretations of inhibition data. An SPR biosensor-based assay expanded the first study, and provided kinetic and mechanistic information, by direct interaction analyses of the inhibitors. It revealed significant differences between the different genotypes and model systems, and provided data that can be used to better understand the efficacy of inhibitors.

Additionally, novel NS3 protease inhibitors were evaluated with respect to their potential to interfere with protease activity, their sensitivity to resistant mutants and effect on HCV replication. The most potent compounds were also characterized by their bioavailability, solubility and metabolic stability. This provides information for design of improved NS3 protease inhibitors, suggesting potential peptidomimetic structures for the backbone as well as for peptide substituents. These modification strategies allowed inhibitors to be truncated and less peptide-like, still with retained inhibitory effect.

A new strategy for analysis of serum protein binding, of importance for drug distribution was also developed. By defining and using the concept of binding efficiency, serum protein interactions of moderate affinity, as described by rapid kinetics, were characterized. This strategy is also applicable for analysis of low affinity interactions.

Taken together, all these studies provide knowledge and strategies for HCV drug discovery, and by using this information we might take a step closer to the final goal, which is to eradicate HCV.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1016
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-193256ISBN: 978-91-554-8591-7 (print)OAI: oai:DiVA.org:uu-193256DiVA: diva2:601620
Public defence
2013-03-15, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-02-22 Created: 2013-01-29 Last updated: 2013-02-28Bibliographically approved
List of papers
1. Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors
Open this publication in new window or tab >>Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors
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2014 (English)In: Journal of enzyme inhibition and medicinal chemistry (Print), ISSN 1475-6366, E-ISSN 1475-6374, Vol. 29, no 6, 868-876 p.Article in journal (Refereed) Published
Abstract [en]

Context: Natural strain variation and rapid resistance development makes development of broad spectrum hepatitis C virus (HCV) drugs very challenging and evaluation of inhibitor selectivity and resistance must account for differences in the catalytic properties of enzyme variants.

Objective: To understand how to study selectivity and relationships between efficacy and genotype or resistant mutants for NS3 protease inhibitors.

Materials and methods: The catalytic properties of NS3 protease from genotypes 1a, 1b and 3a, and their sensitivities to four structurally and mechanistically different NS3 protease inhibitors have been analysed under different experimental conditions.

Results: The optimisation of buffer conditions for each protease variant enabled the comparison of their catalytic properties and sensitivities to the inhibitors. All inhibitors were most effective against genotype 1a protease, with VX-950 having the broadest selectivity.

Discussion and conclusion: A new strategy for evaluation of inhibitors relevant for the discovery of broad spectrum HCV drugs was established.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-193252 (URN)10.3109/14756366.2013.864651 (DOI)000345518000013 ()24517372 (PubMedID)
Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2017-12-06Bibliographically approved
2. Identification of Weak Points of Hepatitis C Virus NS3 Protease Inhibitors Using Surface Plasmon Resonance Biosensor-Based Interaction Kinetic Analysis and Genetic Variants
Open this publication in new window or tab >>Identification of Weak Points of Hepatitis C Virus NS3 Protease Inhibitors Using Surface Plasmon Resonance Biosensor-Based Interaction Kinetic Analysis and Genetic Variants
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2014 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 57, no 5, 1802-1811 p.Article in journal (Other academic) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-193251 (URN)10.1021/jm401690f (DOI)000333005800012 ()
Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2017-12-06Bibliographically approved
3. Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors with alkenylic prime-side substituents
Open this publication in new window or tab >>Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors with alkenylic prime-side substituents
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2010 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 18, no 14, 5413-5424 p.Article in journal (Refereed) Published
Abstract [en]

Phenylglycine has proved to be a useful P2 residue in HCV NS3 protease inhibitors. A novel pi-pi-interaction between the phenylglycine and the catalytic H57 residue of the protease is postulated. We hypothesized that the introduction of a vinyl on the phenylglycine might strengthen this pi-pi-interaction. Thus, herein is presented the synthesis and inhibitory potency of a series of acyclic vinylated phenylglycine-based HCV NS3 protease inhibitors. Surprisingly, inhibitors based on both D- and L-phenylglycine were found to be effective inhibitors, with a slight preference for the d-epimers. Furthermore, prime-side alkenylic extension of the C-terminal acylsulfonamide group gave significantly improved inhibitors with potencies in the nanomolar range (approximately 35 nM), potencies which were retained on mutant variants of the protease.

Keyword
HCV, Protease inhibitors, Peptidomimetics, Phenylglycine, Resistance, Alkenylic acylsulfonamides
National Category
Natural Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-129431 (URN)10.1016/j.bmc.2010.05.027 (DOI)000279744700060 ()20541424 (PubMedID)
Available from: 2010-08-15 Created: 2010-08-15 Last updated: 2017-12-12Bibliographically approved
4. Novel peptidomimetic HCV NS3 protease inhibitors spanning the P2-P1´ region
Open this publication in new window or tab >>Novel peptidomimetic HCV NS3 protease inhibitors spanning the P2-P1´ region
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-193250 (URN)
Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2014-03-09
5. Quantification of interactions between drug leads and serum proteins by use of "binding efficiency"
Open this publication in new window or tab >>Quantification of interactions between drug leads and serum proteins by use of "binding efficiency"
2011 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 409, no 2, 163-175 p.Article in journal (Refereed) Published
Abstract [en]

To develop efficient and reliable methods for prediction of serum protein binding of drug leads, the kinetic characteristics for the interactions between selected compounds and human serum albumin and α(1)-acid glycoprotein have been explored using a surface plasmon resonance biosensor. Conventional methods for quantification of interactions (i.e., using rate constants or affinities determined on the basis of a reasonable mechanistic model) were applicable for only a few of the compounds. The affinity of a primary interaction and the contribution of lower affinity secondary interactions could be estimated for some compounds, but the affinity of many compounds could not be quantified by either of these methods. To have a quantification method that could be used for all compounds, independent of affinity and complexity of interaction mechanisms, the concept of "binding efficiency," analogous to "catalytic efficiency" used for enzymes, was developed. It allowed the quantification of the binding of compounds interacting with weak affinity and for which saturation is not reached within a concentration range where the compound is soluble or when the influence of interactions with secondary sites makes interpretations difficult. In addition, compounds with large fractional binding can be identified by this strategy and simply quantified relative to reference compounds. This approach will enable ranking and identification of structure-activity relationships of compounds with respect to their serum protein binding profile.

Keyword
α1-Acid glycoprotein (AGP), Binding efficiency, Biosensor, Human serum albumin (HSA), Serum protein, Surface plasmon resonance (SPR)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-140682 (URN)10.1016/j.ab.2010.10.028 (DOI)000287176600001 ()21036137 (PubMedID)
Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2017-12-11Bibliographically approved

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