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Tetrapeptides as potent protease inhibitors of Hepatitis C Virus full-length NS3 (protease-helicase/NTPase)
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 Biochemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
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2002 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 10, no 12, 3915-22 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2002. Vol. 10, no 12, 3915-22 p.
National Category
Pharmaceutical Sciences Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-91487DOI: 10.1016/S0968-0896(02)00310-3OAI: oai:DiVA.org:uu-91487DiVA: diva2:164240
Available from: 2004-03-16 Created: 2004-03-16 Last updated: 2015-02-05Bibliographically approved
In thesis
1. Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors
Open this publication in new window or tab >>Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hepatitis C Virus (HCV) is the leading cause of chronic liver disease worldwide as well as the primary indication for liver transplantation. More than 3% of the world’s population is chronically infected with HCV and there is an urgent need for effective therapy. NS3 protease, a viral enzyme required for propagation of HCV in humans, is a promising target for drug development in this area. This thesis addresses the design, synthesis and biochemical evaluation of new HCV NS3 protease inhibitors.

The main objective of the thesis was the synthesis of peptide-based protease inhibitors of the bifunctional full-length NS3 enzyme (protease-helicase/NTPase). Three types of inhibitors were synthesized: i) classical serine protease inhibitors with electrophilic C-terminals, ii) product-based inhibitors with a C-terminal carboxylate group, and iii) product-based inhibitors with C-terminal carboxylic acid bioisosteres.

The developmental work included the establishment of an improved procedure for solid-phase peptide synthesis (SPPS) in the N-to-C direction, in contrast to the C-to-N direction of classical SPPS methods. This inverse method facilitated synthesis of the peptides modified at the C-terminal.

The potency of more than seventy newly synthesized inhibitors was assessed in an in vitro assay using the native form of the protease, i.e. the full-length NS3. The structure-activity relationship (SAR) data achieved was different from SAR data obtained from the more widely used truncated NS3 (protease domain) assay, indicating that the helicase domain of NS3 participates in the binding of the inhibitors.

The most potent inhibitors identified in this study contained a C-terminal phenyl acyl sulfonamide moiety, i.e. a carboxylic acid bioisostere. It is concluded that the acyl sulfonamide moiety is a promising P1-P1´ spanning entity, which may have potential for use in the development of more drug-like HCV protease inhibitors.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 79 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 282
Keyword
Pharmaceutical chemistry, Farmaceutisk kemi
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-3311 (URN)91-554-5538-7 (ISBN)
Public defence
2003-03-21, Lecturehall B41, BMC, Uppsala, 10:15 (English)
Opponent
Available from: 2003-02-26 Created: 2003-02-26 Last updated: 2015-02-05Bibliographically approved
2. Peptide-Based Inhibitors of Hepatitis C Virus NS3 Serine Protease: Kinetic Aspects and Inhibitor Design
Open this publication in new window or tab >>Peptide-Based Inhibitors of Hepatitis C Virus NS3 Serine Protease: Kinetic Aspects and Inhibitor Design
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs.

In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies.

A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values.

The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases.

Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond.

The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.

Place, publisher, year, edition, pages
Uppsala: Institutionen för naturvetenskaplig biokemi, 2004. 67 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 952
Keyword
Biochemistry, serine protease, inhibitor, slow-binding, protein purification, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-4127 (URN)91-554-5905-6 (ISBN)
Public defence
2004-04-16, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2004-03-16 Created: 2004-03-16 Last updated: 2017-05-04Bibliographically approved

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Johansson, AnjaÅkerblom, EvaLindeberg, GunnarDanielson, HelenaHallberg, Anders

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