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Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Helena Danielson)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Helena Danielson)
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2013 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 97, no 3, 356-368 p.Article in journal (Other academic) Published
Abstract [en]

Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

Place, publisher, year, edition, pages
2013. Vol. 97, no 3, 356-368 p.
Keyword [en]
HCV, NS5B, filibuvir, VX-222, tegobuvir, allosteric inhibitor, induced fit, kinetics, chemodynamics, thermodynamics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-171996DOI: 10.1016/j.antiviral.2012.12.027ISI: 000317709400018OAI: oai:DiVA.org:uu-171996DiVA: diva2:513817
Available from: 2012-04-03 Created: 2012-03-31 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Reducing Attrition via Improved Strategies for Pre-clinical Drug Discovery: SPR-biosensor Aided Interaction Studies
Open this publication in new window or tab >>Reducing Attrition via Improved Strategies for Pre-clinical Drug Discovery: SPR-biosensor Aided Interaction Studies
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The efficacy of a drug is tightly intertwined with its interaction mechanism with the drug target. The mechanism is dependent on the physicochemical and structural characteristics of both target and drug molecule.

Drug discovery is plagued by a high attrition rate, whereas in the clinic, a major issue is drug resistance. To improve the quality of the lead compounds in the pre-clinical phase of drug discovery, and thereby reducing the attrition, a deeper understanding of interaction mechanisms is needed. We have adopted new strategies and techniques for this purpose.

A compound library was compiled for the purpose of fragment-based drug discovery. Its compatibility with the SPR platform, along with its interaction profile, was validated. The library was subsequently used in a screening campaign for novel scaffolds of human immunodeficiency virus-1 protease, not sensitive to common resistance mutations. This was achieved by the use of a target panel containing signature resistance mutations towards already approved HIV-1 protease inhibitors. 10 scaffolds were identified and deemed novel. These constitute interesting starting points for development of a new generation of HIV-1 protease inhibitors with different resistance mechanisms, which is very valuable in combination therapies.

The cause of difference in anti-viral potency in cell cultures was investigated for two iso-affinity compounds acting on the hepatitis C viral polymerase, NS5B. By SPR-aided interaction analysis with chemo- and thermodynamic characterization, filibuvir and VX-222, both same-site allosteric inhibitors in phase II clinical trials, were identified to have two different interaction mechanisms. This was ultimately suggested to cause the differences in potency.

A structure-kinetic relationship study, with a thermodynamic characterization, was performed for an approved thrombin inhibitor and five close P3-analogues. This study had the aim to better understand the basic mechanisms of the interactions. Stopped-flow spectroscopy, SPR, and calorimetry were used in parallel and their results compared before evaluation with x-ray crystallography data.

Thus, this thesis has demonstrated successful use of fragment-based drug discovery and high resolution techniques to advance projects in most stages of pre-clinical drug discovery with the aim to reduce the future drug attrition and to understand molecular interactions on a fundamental level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 923
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-171997 (URN)978-91-554-8340-1 (ISBN)
Public defence
2012-05-30, B41, BMC, Uppsala University, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2012-05-04 Created: 2012-03-31 Last updated: 2012-08-01Bibliographically approved
2. Discovery and evaluation of direct acting antivirals against hepatitis C virus
Open this publication in new window or tab >>Discovery and evaluation of direct acting antivirals against hepatitis C virus
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued.

In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants.

By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3.

In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV.

An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate.

Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1312
Keyword
Direct acting antivirals, Hepatitis C, NS3-4A protease, NS5B polymerase, structure-based drug discovery, fragment-based drug discovery, surface plasmon resonance
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-265299 (URN)978-91-554-9398-1 (ISBN)
Public defence
2015-12-18, B21, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-11-27 Created: 2015-10-26 Last updated: 2016-01-13

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Winquist, JohanAbdurakhmanov, EldarDanielson, Helena

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