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Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery
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, 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, Organic Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
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2008 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 16, no 10, p. 5501-5513Article in journal (Refereed) Published
Abstract [en]

A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC(50) of 610+/-15microM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.

Place, publisher, year, edition, pages
2008. Vol. 16, no 10, p. 5501-5513
Keywords [en]
virtual screening, glutamine synthetase, gamma-glutamyl, ammonia ligase
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-102946DOI: 10.1016/j.bmc.2008.04.015ISI: 000256052400015PubMedID: 18462943OAI: oai:DiVA.org:uu-102946DiVA, id: diva2:217091
Available from: 2009-05-13 Created: 2009-05-13 Last updated: 2018-01-13Bibliographically approved
In thesis
1. Structural Studies of Glutamine Synthetases – Towards the Development of Novel Antitubercular Agents
Open this publication in new window or tab >>Structural Studies of Glutamine Synthetases – Towards the Development of Novel Antitubercular Agents
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutamine synthetase (GS) plays an important role in nitrogen metabolism, where it catalyzes the ATP-dependent condensation of glutamate and ammonia to yield glutamine. Recent studies showed the importance of M. tuberculosis GS (MtGS) for growth and survival of the bacterium, and demonstrated its potential as a drug target.

This thesis presents structural studies of MtGS and mammalian GSs, which are aimed at identifying and developing novel inhibitors against the mycobacterial target.

The structure of MtGS was solved in complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium and ADP. The complex structure provides a detailed picture of the active site, offering several insights into catalysis and inhibition, as well as forming a solid basis for structure-based drug design.

The apo canine GS and liganded human GS structures described in this thesis represent the first structures of the mammalian enzymes. Comparison of the structures revealed substrate-induced conformational changes. Inspection of the nucleotide-binding site showed that it differs from that of MtGS, thus offering good opportunities to design specific and selective inhibitors of the mycobacterial enzyme.

The amino acid-binding site of MtGS was evaluated as a target for inhibition, using a combination of a literature survey, structure-based virtual screening and the synthesis of a small library of compounds. As a result, several new inhibitors of MtGS could be identified.

Finally, the structural basis for inhibition of MtGS by a purine analogue (PA) is provided. PA, an analogue of a class of compounds found to inhibit MtGS in a high-throughput screening assay, targets the nucleotide-binding site. The architecture of the HsGS nucleotide-binding site indicates that PA would not be able to bind to the human enzyme, offering good prospects for selective inhibition of MtGS.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. p. 48
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 553
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-9286 (URN)978-91-554-7283-2 (ISBN)
Public defence
2008-10-17, B21, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2008-09-25 Created: 2008-09-25 Last updated: 2009-06-01Bibliographically approved
2. Hit Identification and Hit Expansion in Antituberculosis Drug Discovery: Design and Synthesis of Glutamine Synthetase and 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase Inhibitors
Open this publication in new window or tab >>Hit Identification and Hit Expansion in Antituberculosis Drug Discovery: Design and Synthesis of Glutamine Synthetase and 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase Inhibitors
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Since the discovery of Mycobacterium tuberculosis (Mtb) as the bacterial agent causing tuberculosis, the permanent eradication of this disease has proven challenging. Although a number of drugs exist for the treatment of tuberculosis, 1.7 million people still die every year from this infection. The current treatment regimen involves lengthy combination therapy with four different drugs in an effort to combat the development of resistance. However, multidrug-resistant and extensively drug-resistant strains are emerging in all parts of the world. Therefore, new drugs effective in the treatment of tuberculosis are much-needed.

The work presented in this thesis was focused on the early stages of drug discovery by applying different hit identification and hit expansion strategies in the exploration of two new potential drug targets, glutamine synthetase (GS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR).

A literature survey was first carried out to identify new Mtb GS inhibitors from compounds known to inhibit GS in other species. Three compounds, structurally unrelated to the typical amino acid derivatives of previously known GS inhibitors, were then discovered by virtual screening and found to be Mtb GS inhibitors, exhibiting activities in the millimolar range. Imidazo[1,2-a]pyridine analogues were also investigated as Mtb GS inhibitors. The chemical functionality, size requirements and position of the substituents in the imidazo[1,2-a]pyridine hit were investigated, and a chemical library was designed based on a focused hierarchical design of experiments approach. The X-ray structure of one of the inhibitors in complex with Mtb GS provided additional insight into the structure–activity relationships of this class of compounds.

Finally, new α-arylated fosmidomycin analogues were synthesized as inhibitors of Mtb DXR, exhibiting IC50 values down to 0.8 µM. This work shows that a wide variety of aryl groups are tolerated by the enzyme. Cinnamaldehydes are important synthetic intermediates in the synthesis of fosmidomycin analogues. These were prepared by an oxidative Heck reaction from acrolein and various arylboronic acids. Electron-rich, electron-poor, heterocyclic and sterically hindered boronic acids could be employed, furnishing cinnamaldehydes in 43–92% yield.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. p. 86
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 148
Keywords
Tuberculosis, Glutamine synthetase, Imidazo[1, 2-a]pyridine analogues, Focused hierarchical design of experiments, DXR, Fosmidomycin analogues, 1-Deoxy-D-xylulose-5-phosphate reductoisomerase, Cinnamaldehydes, Oxidative Heck reaction
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-155428 (URN)978-91-554-8151-3 (ISBN)
Public defence
2011-10-21, B22, Husargatan 3, SE-751 23, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2011-09-30 Created: 2011-06-22 Last updated: 2018-01-12Bibliographically approved

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Nilsson, Mikael T.Odell, Luke R.Larhed, MatsMowbray, Sherry L.

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