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Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
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2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 13, no 12, 822-832 p.Article 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.

Place, publisher, year, edition, pages
2007. Vol. 13, no 12, 822-832 p.
Keyword [en]
mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, alanine scan, statistical molecular design, structure activity relationships, FHDoE
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-14261DOI: 10.1002/psc.906ISI: 000252000600007PubMedID: 17918768OAI: oai:DiVA.org:uu-14261DiVA: diva2:42031
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Development and Application of Computational Methods in Antitubercular Drug Design: Identification of Novel Inhibitors of Ribonucleotide Reductase
Open this publication in new window or tab >>Development and Application of Computational Methods in Antitubercular Drug Design: Identification of Novel Inhibitors of Ribonucleotide Reductase
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tuberculosis kills approximately 1.7 million people each year around the world making it one of the most lethal infectious diseases. This thesis concerns the development of two computational tools that can support the early stages of drug discovery, and their use in an anti-tubercular drug discovery program.

One of the tools developed is a statistical molecular design (SMD) approach that generates information-rich libraries biased towards a lead structure. The other metod is a post-filtering technique to increase the success of virtual screening, has also been developed. Both methods have been validated using literature data.

Ribonucleotide reductase (RNR) has been identified as a potential anti-tubercular target, and our focus has been to develop small-molecule inhibitors of this target. The enzyme consists of two subunits (a large R1 and a small R2 subunit) that have to associate in order to generate a bioactive complex. It had previously been shown that a heptapeptide corresponding to the small R2 subunits C-terminal inhibited the enzyme. In order to investigate the requirements for inhibitory effect of the peptide a library was designed using the developed SMD approach. The designed library was synthesized and evaluated for biological activity and an OPLS-DA model was derived to understand which positions were most important for activity.

In order to identify small-molecule inhibitors of RNR a combined shape- and structure-based virtual screen was performed, employing ROCS, GlideXP and the developed post-filtering technique. Starting from a library of 1.5 million compounds 24 was acquired and evaluated for enzymatic activity. The best compounds were almost as potent as the starting peptide, but considerably more drug-like.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 68 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 91
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-99173 (URN)978-91-554-7460-7 (ISBN)
Public defence
2009-04-24, B22, BMC, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-04-02 Created: 2009-03-09 Last updated: 2009-09-04Bibliographically approved
2. Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes
Open this publication in new window or tab >>Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes structural and binding studies in enzymes from two different  organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA).

RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show 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. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors

Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 684
Keyword
Mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, fluorescence polarization, lipase, interfacial activation, hydrolase, X-ray structure, substrate specificity
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-109697 (URN)978-91-554-7638-0 (ISBN)
Public defence
2009-12-05, B42, Uppsala Biomedical Center (BMC), Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2009-11-12 Created: 2009-10-22 Last updated: 2012-09-18Bibliographically approved
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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Nurbo, JohannaMuthas, DanielLundstedt, TorbjörnUnge, TorstenKarlén, Anders

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