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β-Amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors
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.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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2008 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 16, no 10, 5590-5605 p.Article in journal (Refereed) Published
Abstract [en]

In an effort to develop a new type of HCV NS3 peptidomimetic inhibitor, a series of tripeptide inhibitors incorporating a mix of alpha- and beta-amino acids has been synthesized. To understand the structural implications of beta-amino acid substitution, the P(1), P(2), and P(3) positions of a potent tripeptide scaffold were scanned and combined with carboxylic acid and acyl sulfonamide C-terminal groups. Inhibition was evaluated and revealed that the structural changes resulted in a loss in potency compared with the alpha-peptide analogues. However, several compounds exhibited muM potency. Inhibition data were compared with modeled ligand-protein binding poses to understand how changes in ligand structure affected inhibition potency. The P(3) position seemed to be the least sensitive position for beta-amino acid substitution. Moreover, the importance of a proper oxyanion hole interaction for good potency was suggested by both inhibition data and molecular modeling. To gain further insight into the structural requirements for potent inhibitors, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model has been constructed using comparative molecular field analysis (CoMFA). The most predictive CoMFA model has q(2)=0.48 and r(pred)(2)=0.68.

Place, publisher, year, edition, pages
2008. Vol. 16, no 10, 5590-5605 p.
Keyword [en]
hepatitis C, HCV, NS3, protease inhibitor, beta-amino acid, 3D-QSAR, CoMFA, docking
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96053DOI: 10.1016/j.bmc.2008.04.005ISI: 000256052400023PubMedID: 18434166OAI: oai:DiVA.org:uu-96053DiVA: diva2:170498
Available from: 2007-09-06 Created: 2007-09-06 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Improved CoMFA Modeling by Optimization of Settings: Toward the Design of Inhibitors of the HCV NS3 Protease
Open this publication in new window or tab >>Improved CoMFA Modeling by Optimization of Settings: Toward the Design of Inhibitors of the HCV NS3 Protease
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hepatitis C virus (HCV), with a global prevalence of roughly 2%, is among the most serious diseases today. Among the more promising HCV targets is the NS3 protease, for which several drug candidates have entered clinical trials. In this work, computational methods have been developed and applied to the design of inhibitors of the HCV NS3 protease.

Comparative molecular field analysis (CoMFA) modeling and molecular docking are the two main computational tools used in this work. CoMFA is currently the most widely used 3D-QSAR method. Methodology for improving its predictive performance by evaluating 6120 combinations of non-default parameters has been developed. This methodology was tested on 9 data sets for various targets and found to consistently provide models of enhanced predictive accuracy. Validation was performed using q2, r2pred and response variable randomization.

Molecular docking was used to develop SARs in two series of inhibitors of the HCV NS3 protease. In the first series, preliminary investigations indicated that replacement of P2 proline with phenylglycine would improve potency. Docking suggested that phenylglycine-based inhibitors may participate in two additional interactions but that the larger, more flexible phenylglycine group may result in worse ligand fit, explaining the loss in potency. In the second series, β-amino acids were explored as α-amino acid substitutes. Although β-amino acid substitution may reduce the negative attributes of peptide-like compounds, this study showed that β-amino acid substitution resulted in reduced potency. The P3 position was least sensitive to substitution and the study highlighted the importance of interactions in the oxyanion hole.

Finally, docking was used to provide the conformations and alignment necessary for a CoMFA model. This CoMFA model, derived using default settings, had q2 = 0.31 and r2pred = 0.56. Application of the optimization methodology provided a more predictive model with q2 = 0.48 and r2pred = 0.68.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 80 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 61
Keyword
Pharmaceutical chemistry, CoMFA, 3D-QSAR, model validation, Docking, SAR, hepatitis C virus, HCV, NS3 protease inhibitor, Farmaceutisk kemi
Identifiers
urn:nbn:se:uu:diva-8140 (URN)978-91-554-6932-0 (ISBN)
Public defence
2007-09-28, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2007-09-06 Created: 2007-09-06 Last updated: 2009-11-30Bibliographically approved
2. Kinetic studies of NS3 and NS5B from Hepatitis C virus: Implications and applications for drug discovery
Open this publication in new window or tab >>Kinetic studies of NS3 and NS5B from Hepatitis C virus: Implications and applications for drug discovery
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV.

By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided.

Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease.

This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays.

Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor.

Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen.

The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.

 

Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 615
Keyword
Hepatitis C virus, NS3, NS5B, enzyme kinetics, inhibition, resistance, drug
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-98868 (URN)978-91-554-7445-4 (ISBN)
Public defence
2009-04-17, BMC, Sal B7:101a, Husargatan 3, UPPSALA, 09:00 (English)
Opponent
Supervisors
Available from: 2009-03-26 Created: 2009-03-04 Last updated: 2017-05-04
3. Peptidomimetic Enzyme Inhibitors: Targeting M. tuberculosis Ribonucleotide Reductase and Hepatitis C Virus NS3 Protease
Open this publication in new window or tab >>Peptidomimetic Enzyme Inhibitors: Targeting M. tuberculosis Ribonucleotide Reductase and Hepatitis C Virus NS3 Protease
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on the design and synthesis of inhibitors targeting Mycobacterium tuberculosis ribonucleotide reductase (RNR) and hepatitis C virus (HCV) NS3 protease; enzymes that have been identified as potential drug targets for the treatment of tuberculosis and hepatitis C, respectively. Small peptides have been recognized as inhibitors of these enzymes. However, the use of peptides as drugs is limited due to their unfavorable properties. These can be circumvented by the development of less peptidic molecules, often referred to as peptidomimetics. When this work was initiated, only a few inhibitors targeting M. tuberculosis RNR had been identified, whereas the HCV NS3 protease was an established drug target. Therefore, early peptidomimetic design strategies were applied to inhibitors of RNR while the NS3 protease inhibitors were subjected to modifications in a later stage of development.

It has previously been shown that peptides derived from the C-terminus of the small subunit of M. tuberculosis RNR can compete for binding to the large subunit, and thus inhibit enzyme activity. To investigate the structural requirements of these inhibitors, different series of peptides were evaluated. First, peptides from an N-terminal truncation, an alanine scan and a designed library were synthesized and evaluated to examine the importance of the individual amino acid residues. Then, a set of N-terminally Fmoc-protected peptides was evaluated, and it was found that the N-terminal group improved the affinity of the peptides even when the length of the compounds was reduced. Furthermore, potential inhibitors of less peptidic character were generated by the introduction of a benzodiazepine-based scaffold.

To further reduce the peptidic character and investigate the binding properties of HCV NS3 protease inhibitors, a series of tripeptides incorporating a β-amino acid was synthesized. Inhibition was evaluated and docking studies were performed to understand how the structural changes affected inhibitory potency. The results illustrated the importance of preserving the hydrogen bonding network and retaining electrostatic interactions in the oxyanion hole between inhibitor and protein.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 119
Keyword
enzyme inhibitor, peptidomimetics, structure-activity relationship, tuberculosis, ribonucleotide reductase, hepatitis C virus, NS3 protease
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-112345 (URN)978-91-554-7716-5 (ISBN)
Public defence
2010-03-12, B21, BMC, Husargatan 3, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2010-02-18 Created: 2010-01-13 Last updated: 2010-02-18Bibliographically approved

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Danielson, U. HelenaSandström, Anja

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