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Peptidomimetic Enzyme Inhibitors: Targeting M. tuberculosis Ribonucleotide Reductase and Hepatitis C Virus NS3 Protease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
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. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 119
Keywords [en]
enzyme inhibitor, peptidomimetics, structure-activity relationship, tuberculosis, ribonucleotide reductase, hepatitis C virus, NS3 protease
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-112345ISBN: 978-91-554-7716-5 (print)OAI: oai:DiVA.org:uu-112345DiVA, id: diva2:290634
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: 2018-01-12Bibliographically approved
List of papers
1. Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
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2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 13, no 12, p. 822-832Article in journal (Refereed) Published
Abstract [en]

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that TrP5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain., as compared to Trp5.

Keywords
mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, alanine scan, statistical molecular design, structure activity relationships, FHDoE
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-14261 (URN)10.1002/psc.906 (DOI)000252000600007 ()17918768 (PubMedID)
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2018-01-12Bibliographically approved
2. Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
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2010 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 16, no 3, p. 159-164Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors.

Keywords
Fluorescence polarization, Mycobacterium tuberculosis, Peptide inhibitors, Ribonucleotide reductase
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-112344 (URN)10.1002/psc.1214 (DOI)000275448300007 ()20127854 (PubMedID)
Available from: 2010-01-26 Created: 2010-01-13 Last updated: 2018-01-12Bibliographically approved
3. Novel pseudopeptides incorporating a benzodiazepine-based turn mimetic – targeting Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Novel pseudopeptides incorporating a benzodiazepine-based turn mimetic – targeting Mycobacterium tuberculosis ribonucleotide reductase
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2013 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 21, no 7, p. 1992-2000Article in journal (Refereed) Published
Abstract [en]

Peptides mimicking the C-terminus of the small subunit (R2) of Mycobacterium tuberculosis ribonucleotide reductase (RNR) can compete for binding to the large subunit (R1) and thus inhibit RNR activity. Moreover, it has been suggested that the binding of the R2 C-terminus is very similar in M. tuberculosis and Salmonella typhimurium. Based on modeling studies of a crystal structure of the holocomplex of the S. typhimurium enzyme, a benzodiazepine-based turn mimetic was identified and a set of novel compounds incorporating the benzodiazepine scaffold was synthesized. The compounds were evaluated in a competitive fluorescence polarization assay and in an RNR activity assay. These studies revealed that the compounds incorporating the benzodiazepine scaffold have the ability to compete for the M. tuberculosis R2 binding site with low-micromolar affinity.

National Category
Biological Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-112341 (URN)10.1016/j.bmc.2013.01.020 (DOI)000316770300041 ()
Available from: 2010-01-26 Created: 2010-01-13 Last updated: 2017-12-12Bibliographically approved
4. β-Amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors
Open this publication in new window or tab >>β-Amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors
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2008 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 16, no 10, p. 5590-5605Article 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.

Keywords
hepatitis C, HCV, NS3, protease inhibitor, beta-amino acid, 3D-QSAR, CoMFA, docking
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-96053 (URN)10.1016/j.bmc.2008.04.005 (DOI)000256052400023 ()18434166 (PubMedID)
Available from: 2007-09-06 Created: 2007-09-06 Last updated: 2018-01-13Bibliographically approved

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