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Identification of Weak Points of Hepatitis C Virus NS3 Protease Inhibitors Using Surface Plasmon Resonance Biosensor-Based Interaction Kinetic Analysis and Genetic Variants
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
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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
Place, publisher, year, edition, pages
2014. Vol. 57, no 5, 1802-1811 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-193251DOI: 10.1021/jm401690fISI: 000333005800012OAI: oai:DiVA.org:uu-193251DiVA: diva2:601600
Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Characterization of HCV Protease Inhibitors: Inhibition and Interaction Studies with Applications for Drug Discovery
Open this publication in new window or tab >>Characterization of HCV Protease Inhibitors: Inhibition and Interaction Studies with Applications for Drug Discovery
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:nbn:se:uu:diva-193256 (URN)978-91-554-8591-7 (ISBN)
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
2. Novel Procedures for Identification and Characterization of Viral Proteases Inhibitors
Open this publication in new window or tab >>Novel Procedures for Identification and Characterization of Viral Proteases Inhibitors
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay.

The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, the compounds were analyzed under conditions optimized for each variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs.

A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target.  

The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1115
Keyword
drug discovery, viral proteases, inhibitors, Hepatitis C, NS3, cytomegalovirus
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-215700 (URN)978-91-554-8855-0 (ISBN)
Public defence
2014-02-28, B42, BMC, Uppsala Universitet, Husargatan 3, 751 23, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-02-07 Created: 2014-01-15 Last updated: 2014-02-10

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