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Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
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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.

Place, publisher, year, edition, pages
2016. Vol. 24, no 12, 2603-2620 p.
Keyword [en]
Hepatitis C virus; Drug resistance; Pyrazinone; NS3 protease inhibitors; R155K
National Category
Organic Chemistry
Research subject
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-243315DOI: 10.1016/j.bmc.2016.03.066ISI: 000376727800002PubMedID: 27160057OAI: oai:DiVA.org:uu-243315DiVA: diva2:787013
Funder
Swedish Research Council, D0571301
Available from: 2015-02-08 Created: 2015-02-08 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors: Targeting Different Genotypes and Drug-Resistant Variants
Open this publication in new window or tab >>Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors: Targeting Different Genotypes and Drug-Resistant Variants
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several combination therapies, based on different DAAs, with or without the need of pegylated interferon-α injection, are available for chronic HCV infections. The chemical foundation of the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors. The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of the virus and were less potent against other genotypes.

The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical trials or approved, potentially with a unique resistance profile and with a broad genotypic coverage. Successive modifications were performed around the pyrazinone core structure to clarify the structure-activity relationship; a P3 urea capping group was found valuable for inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket. Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme. Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with respect to improved PK properties, were also performed on an advanced class of HCV NS3 protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic proline urea scaffold with nano-molar cell based activities.

Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic scaffolds and carboxylic acid bioisosteres.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 108 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 197
Keyword
hepatitis C virus, HCV, NS3 protease inhibitors, structure-activity relationship, 2(1H)-pyrazinone, quinazoline, resistance, Pd catalysis
National Category
Organic Chemistry Other Chemistry Topics
Research subject
Medicinal Chemistry; Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-243317 (URN)978-91-554-9166-6 (ISBN)
Public defence
2015-03-27, B41 BMC, Husargatan 3, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2015-03-05 Created: 2015-02-08 Last updated: 2015-03-12Bibliographically 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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Belfrage, Anna KarinAbdurakhmanov, EldarBrandt, PeterGising, JohanSkogh, AnnaDanielson, U. HelenaSandström, Anja

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