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Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria.

The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined.

The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods.

The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 949
Keyword [en]
Tuberculosis, Mycobacterium tuberculosis, MEP pathway, CoA pathway, drug development, crystal structure, DXR, IspD, PanK
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry; Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-179057ISBN: 978-91-554-8416-3 (print)OAI: oai:DiVA.org:uu-179057DiVA: diva2:543281
Public defence
2012-09-21, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2012-08-30 Created: 2012-08-06 Last updated: 2013-01-22Bibliographically approved
List of papers
1. The 1.9 Å resolution structure of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a potential drug target
Open this publication in new window or tab >>The 1.9 Å resolution structure of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a potential drug target
2006 (English)In: Acta Crystallogr. Section D Biol. Crystallogr., ISSN 0907-4449, Vol. 62, 807-813 p.Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-96292 (URN)
Available from: 2007-10-19 Created: 2007-10-19 Last updated: 2015-04-02
2. Structural and functional studies of mycobacterial IspD enzymes
Open this publication in new window or tab >>Structural and functional studies of mycobacterial IspD enzymes
Show others...
2011 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 67, 403-414 p.Article in journal (Refereed) Published
Abstract [en]

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-154127 (URN)10.1107/S0907444911006160 (DOI)000290235200002 ()21543842 (PubMedID)
Available from: 2011-05-26 Created: 2011-05-26 Last updated: 2017-12-11Bibliographically approved
3. Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase
Open this publication in new window or tab >>Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase
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2011 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 14, 4964-4976 p.Article in journal (Refereed) Published
Abstract [en]

The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

National Category
Biochemistry and Molecular Biology Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-156614 (URN)10.1021/jm2000085 (DOI)000292892300003 ()21678907 (PubMedID)
Available from: 2011-08-07 Created: 2011-08-04 Last updated: 2017-12-08Bibliographically approved
4. Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
Open this publication in new window or tab >>Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
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2012 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 68, 134-143 p.Article in journal (Refereed) Published
Abstract [en]

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.

Keyword
tuberculosis, DXR, IspC, MEP pathway
National Category
Medical and Health Sciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-169337 (URN)10.1107/S0907444911052231 (DOI)000299469100006 ()
Available from: 2012-03-05 Created: 2012-02-28 Last updated: 2017-12-07Bibliographically approved
5. Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase
Open this publication in new window or tab >>Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 25, 18260-18270 p.Article in journal (Refereed) Published
Abstract [en]

Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

Keyword
Tuberculosis, Mycobacterium tuberculosis, drug development, pantothenate kinase, PanK
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Molecular Biology; Biochemistry
Identifiers
urn:nbn:se:uu:diva-179056 (URN)10.1074/jbc.M113.476473 (DOI)000320721900030 ()
Funder
Swedish Research Council
Available from: 2012-08-06 Created: 2012-08-06 Last updated: 2017-12-07Bibliographically approved

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