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Discovery and evaluation of direct acting antivirals against hepatitis C virus
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
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 [en]
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: urn:nbn:se:uu:diva-265299ISBN: 978-91-554-9398-1 (print)OAI: oai:DiVA.org:uu-265299DiVA: diva2:865085
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
List of papers
1. Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
Open this publication in new window or tab >>Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
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2016 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, no 12, 2603-2620 p.Article in journal (Refereed) Published
Abstract [en]

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

Keyword
Hepatitis C virus; Drug resistance; Pyrazinone; NS3 protease inhibitors; R155K
National Category
Organic Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-243315 (URN)10.1016/j.bmc.2016.03.066 (DOI)000376727800002 ()27160057 (PubMedID)
Funder
Swedish Research Council, D0571301
Available from: 2015-02-08 Created: 2015-02-08 Last updated: 2017-12-04Bibliographically approved
2. Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
Open this publication in new window or tab >>Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-265295 (URN)
Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
3. 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
Open this publication in new window or tab >>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
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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.

Keyword
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:nbn:se:uu:diva-171996 (URN)10.1016/j.antiviral.2012.12.027 (DOI)000317709400018 ()
Available from: 2012-04-03 Created: 2012-03-31 Last updated: 2017-12-07Bibliographically approved
4. Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
Open this publication in new window or tab >>Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-265287 (URN)
Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
5. A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
Open this publication in new window or tab >>A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-265290 (URN)
Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
6. Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
Open this publication in new window or tab >>Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-265292 (URN)
Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13

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