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Peptide-Based Inhibitors of Hepatitis C Virus NS3 Serine Protease: Kinetic Aspects and Inhibitor Design
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
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. , p. 67
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 952
Keywords [en]
Biochemistry, serine protease, inhibitor, slow-binding, protein purification
Keywords [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-4127ISBN: 91-554-5905-6 (print)OAI: oai:DiVA.org:uu-4127DiVA, id: diva2:164244
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
List of papers
1. Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus
Open this publication in new window or tab >>Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus
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2002 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 25, no 3, p. 363-371Article in journal (Refereed) Published
Abstract [en]

Viral mRNA extracted from the serum of a patient infected with HCV strain I a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni2(+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K-M) for catalysis and the inhibitory potencies (IC50 and K-i) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-82149 (URN)10.1016/S1046-5928(02)00042-6 (DOI)000177967600001 ()12182815 (PubMedID)
Available from: 2008-06-15 Created: 2008-06-15 Last updated: 2017-12-14Bibliographically approved
2. Tetrapeptides as potent protease inhibitors of Hepatitis C Virus full-length NS3 (protease-helicase/NTPase)
Open this publication in new window or tab >>Tetrapeptides as potent protease inhibitors of Hepatitis C Virus full-length NS3 (protease-helicase/NTPase)
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2002 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 10, no 12, p. 3915-22Article in journal (Refereed) Published
National Category
Pharmaceutical Sciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-91487 (URN)10.1016/S0968-0896(02)00310-3 (DOI)
Available from: 2004-03-16 Created: 2004-03-16 Last updated: 2018-01-13Bibliographically approved
3. Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): a comparative study of different C-terminals
Open this publication in new window or tab >>Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): a comparative study of different C-terminals
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2003 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 11, no 12, p. 2551-2568Article in journal (Refereed) Published
National Category
Pharmaceutical Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-66026 (URN)10.1016/S0968-0896(03)00179-2 (DOI)
Available from: 2006-11-13 Created: 2006-11-13 Last updated: 2018-01-10Bibliographically approved
4. Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): model compounds towards small molecule inhibitors
Open this publication in new window or tab >>Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): model compounds towards small molecule inhibitors
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2003 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 11, no 13, p. 2955-2963Article in journal (Refereed) Published
National Category
Pharmaceutical Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-91489 (URN)10.1016/S0968-0896(03)00190-1 (DOI)
Available from: 2004-03-16 Created: 2004-03-16 Last updated: 2018-01-13Bibliographically approved
5. Structure-activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors
Open this publication in new window or tab >>Structure-activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors
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2004 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1672, no 1, p. 51-59Article in journal (Refereed) Published
Abstract [en]

The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure-activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.

Keywords
Hepacivirus/chemistry/*enzymology/metabolism, Protease Inhibitors/*pharmacology, Research Support; Non-U.S. Gov't, Structure-Activity Relationship, Viral Nonstructural Proteins/antagonists & inhibitors/*chemistry/*metabolism
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-91490 (URN)10.1016/j.bbagen.2004.02.008 (DOI)15056493 (PubMedID)
Available from: 2004-03-16 Created: 2004-03-16 Last updated: 2017-12-14Bibliographically approved

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