Project number: QLK2-CT-2001-02018
EC contribution: 2,300,000 Euros
Duration: 36 months
Starting date: January, 2002
Contract type: Shared cost RTD

The general objective of the X-TB project is to use an integrated, multidisciplinary approach combining proteomics with structural and functional genomics to define the proteome of the tubercle bacillus with the explicit aims of identifying novel drug targets and developing new chemotherapeutic compounds to treat tuberculosis. Attempts will be made to identify lead compounds for new drugs that might help to reduce the duration of therapy through their greater potency and show activity against the current multidrug resistant strains of Mycobacterium tuberculosis.

Given the current global socio-political climate, the importance of curing and preventing tuberculosis cannot be overstated. While the development of a new vaccine to replace BCG is a laudable if long-term objective, the design of new drugs is a more tangible goal. Although directly observed short course chemotherapy (DOTS) exists to treat the disease, this treatment has not been improved for over 30 years. DOTS is singularly inefficient by the standards of today’s pharmaceutical industry in terms of drug activity and toxicity, and its efficacy is threatened by increasingly widespread drug resistance. Here, we wish to employ state-of-the-art post-genomics technology to initiate the development of enhanced tuberculosis chemotherapy for the new millenium. In particular, we would like to harness the powerful new tools of structural and functional genomics for drug and drug target discovery.

The principal objective is to further the development of new drugs to combat the growing menace of tuberculosis by using a post-genomics approach. The proteome of Mycobacterium tuberculosis will be intensively studied to expand our knowledge base and identify proteins or enzymes that could serve as targets for new antibiotics. Essentiality will be determined by creating appropriate mutants and specificity appraised by bioinformatics. Mycobacterial proteins that could serve as novel targets for chemotherapy will be identified, and inhibitors will either be isolated from libraries using novel screens of natural or (semi)synthetic products or, by using three dimensional structures as templates for drug design.

1. Biochemically and structurally characterised potential drug targets of known function
2. High-throughput production of unknown proteins for library screening and structural studies
3. Function predictionsand refined protein families from advanced bioinformatics
4. Definition of the proteome under different physiological conditions
5. Linkage maps for intraproteome and host-pathogen protein-protein interactions
6. Databases for proteomics and structural genomics
7. Confirmation of essentiality by gene replacement
8. Protein structures determined by NMR and X-ray crystallography
9. Novel automated screens using fluorescent two-hybrid systems
10. Screens of chemical and natural product libraries to identify potential lead compounds
11. Leads for drugs from rational design and molecular modelling

This project will lead to better definition of the proteins present in M. tuberculosis together with detailed understanding of their activities, regulation, structures and interactions. It is probable that many of these proteins play essential roles and therefore represent attractive drug targets. Screening of chemical and natural product libraries will identify potential ligands and inhibitors that could correspond to lead compounds for the development of new drugs to treat tuberculosis, in all its forms.

Unité de Génétique Moléculaire Bactérienne,
Institut Pasteur,
25-28, rue du Docteur Roux,
F-75724 Paris Cedex 15,
FRANCE
Tel : 33 - 1 45 68 84 46
Fax : 33 - 1 40 61 35 83
Email : stcole@pasteur.fr
http://www.pasteur.fr/recherche/unites/Lgmb/

Prof. Stewart T. Cole, Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15, France, Tel : 33 - 1 45 68 84 46, Fax : 33 - 1 40 61 35 83 Email : stcole@pasteur.fr

Dr. Pedro Alzari, Unité de Biochimie Structurale, Institut Pasteur, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15, France, Tel : 33 - 1 45 68 86 07 , Fax : 33 - 1 45 68 86 04 Email : alzari@pasteur.fr

Prof. Brigitte Gicquel, Unité de Génétique Mycobactérienne, Institut Pasteur, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15, France, Tel : 33 - 1 45 68 88 28 , Fax : 33 - 1 45 68 88 43 Email : bgicquel@pasteur.fr

Prof. Stefan H. E. Kaufmann Max-Planck-Institut for Infection Biology Monbijoustrasse 2 D-10117 Berlin Germany Tel : 49 - 30 28 460 502 Fax : 49 - 30 28 460 501 Email : kaufmann@mpiib-berlin.mpg.de

Dr. Ida Rosenkrands Statens Seruminstitut Bacterial Vaccine Department 5 Artillerivej DK-2300 Copenhagen S Denmark Tel : 45 - 32 68 37 21 Fax : 45 - 32 68 30 35 Email : idr@ssi.dk

Dr. Ute Möllmann Hans-Knöll-Institut für Naturstoff-Forschung Beutenbergstr. 11 D-07745 Jena Germany Tel : 49 - 3641 65 6656 Fax : 49 - 3641 65 6652 Email : moellman@pmail.hki-jena.de

Dr. Andrew Munro University of Leicester, University Road LE1 7RH Leicester UK Tel : 44 116 252 3464 Fax : 44 116252 3369 E mail : awm9@le.ac.uk

Dr. Michael Arand Institute of Toxicology University of Mainz Obere Zahlbacher Str. 67 D-55131 Mainz Germany Tel : 49-6131 3934376 Fax : 49-6131 230506 Email : arand@mail.uni-mainz.de

Prof. Kai Johnsson Institute of Organic Chemistry University of Lausanne CH-1015 Lausanne Switzerland Tel : 41 - 21 692 3956 Fax : 41 - 21 692 3965 Email : kai.johnsson@epfl.ch

Dr. Nicholas Keep Department of Crystallography, Birkbeck College, University of London Malet Street London WC1E 7HX UK Tel : 44 - 20 7631 6852 Fax : 44 - 20 7631 6803 Email : n.keep@bbk.ac.uk

Dr. Keith S Wilson Structural Biology Laboratory Department of Chemistry University of York Heslington, York YO1 5DD UK Tel : 44 1904 432519 Fax : 44 1904 410519 Email : keith@yorvic.york.ac.uk

Dr. Yves Bourne AFMB, CNRS UPR9039 31, Chemin Joseph Aiguier F-13402 Marseille Cedex 20, France Tel : 33 - 4 91 82 86 24 Fax : 33 - 4 91 82 86 21 Email : yves@afmb.cnrs-mrs.fr

Dr. Alwyn Jones Department of Cell and Molecular Biology Biomedical Center Uppsala University Box 596 SE-751 Uppsala Sweden Tel : 46 - 18 4714982 Fax : 46 - 18 536971 Email : alwyn@xray.bmc.uu.se

The principal objective is to further the development of new chemotherapeutic agents to combat the growing menace of tuberculosis by using a post-genomic strategy that combines the expertise of mycobacteriologists with the technical competence and innovative skills of (bio)chemists, structural biologists and bioinformatics experts. The proteome of Mycobacterium tuberculosis will be intensively studied to expand our knowledge base and identify proteins that could serve as targets for new inhibitors. Essentiality will be determined by creating appropriate mutants and specificity appraised by bioinformatics. Lead compounds for new drugs will be identified by screening libraries of natural or synthetic compounds then refined by molecular modelling. Inhibitors will also be designed rationally using the protein structure as template. Activity of new antitubercular agents will be tested in vitro and in animal models.

We are studying a series of known proteins from M. tb, with demonstrable biochemical activities, that are being subjected to structural analysis and rational drug design. In parallel, many genes of unknown function have been cloned and expressed in E. coli to facilitate further biochemical and structural studies. These include both proteins, selected for their potential as drug targets, or unknown proteins belonging to large gene families. All proteins that overexpress in soluble form will be purified and used in chemical library screenings and structural studies. In parallel, novel targets and pathways for the development of antibacterial agents are being highlighted by establishing linkage maps between the proteomes of M. tb and the human host. These protein-protein interactions will in turn serve as the basis of target-oriented drug screening assays.

Several potential drug targets of known function have been characterized biochemically and structural information obtained, including high resolution 3D-structures of a cytochrome P450, serine-threonine protein kinase, inositol-1-P synthase and an epoxide hydrolase. High throughput expression, purification and crystallization platforms have been established to further the study of proteins of known or unknown function. Many of the latter have been localized in the cell and on the proteome map, and are now being characterized structurally. This work is complemented by the construction of mutants by gene knock-out and signature-tagged mutagenesis, and by establishing protein interaction maps both within the pathogen and with host proteins. A relational database has been constructed to present the results of 2D-protein analysis and the proteome studied under different physiological conditions. Novel protein fusion technology has been developed that enables proteins to be specifically labelled and used to screen chemical and natural product libraries.

Structural Genomics of Mycobacteria
http://www.pasteur.fr/SGM
The SGM web site is dedicated to share data concerning the progress status of the structural genomics project on mycobacterial proteins.

Information for group members
Further information for registered members of the Structural and functional genomics of Mycobacterium tuberculosis group can be found here. To register, please contact Fabrice Guillemot, fguillem@pasteur.fr.

1. Banu, S., Honoré, N., Saint-Joanis, B., Philpott, D., Prèvost, M.C. and Cole, S.T. (2002)
   Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens?
   Mol. Microbiol. 44: 9-19.
2. Cole, S.T. (2002)
   Comparative and functional genomics of the Mycobacterium tuberculosis complex.
   Microbiology 48: 2919-2928.
3. McLean, K.J., Cheesman, M.R., Rivers, S.L., Richmond, A., Leys, D., Chapman, S.K., Reid, G.A., Price, N.C., Kelly, S.M., Clarkson, J., Smith, W.E. and Munro, A.W. (2002)
   Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis.
   J. Inorg. Biochem. 91: 527-541.
4. McLean, K.J., Marshall, K.R., Richmond, A., Hunter, I.S., Fowler, K., Kieser, T., Gurcha, S.S., Besra, G.S. and Munro, A.W. (2002)
   Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes.
   Microbiology 148: 2937-2949.
5. Sinha, S., Arora, S., Kosalai, K., Namane, A., Pym, A.S. and Cole, S.T. (2002)
   Proteome analysis of the plasma membrane of Mycobacterium tuberculosis.
   Comp. Funct. Genom. 3: 470-483.

6. Arand, M., Hallberg, M., Zou, J-Y., Bergfors, T., Oesch, F, van der Werf, M.J., de Bont, J.A.M., Jones, T.A. and Mowbray, S.L. (2003)
   The 1.2 Å structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel mechanism.
   EMBO J. 22: 2583-2592.
7. Boitel, B., Ortiz-Lombardia, M., Duran, R., Pompeo, F., Cole, S.T., Cervenansky, C., and Alzari, P.M. (2003)
   PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis.
   Mol. Microbiol. 49: 1493-1508.
8. Arand, M., Cronin, A., Oesch, F., Mowbray, S.L., and Jones, T.A., (2003)
   The tell tale structures of epoxide hydrolases.
   Drug Metab. Rev. 35: 365-383, 2003.
   
   
9. Jungblut, P.R., (2003)
   Proteome Analysis: Elucidating the Blueprint of Life.
   Biomedical Progress 16: 1-7.
10. Jungblut. P.R. (2003)
    Bacterial Proteomes.
    In Encyclopedia of the Human Genome, Macmillan Publishers Ltd, Nature Publishing Group, 1-5.
11. Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H. and Johnsson, K. (2003)
    A general method for the specific labeling of fusion proteins with small molecules in vivo.
    Nat. Biotechnol. 21: 86-89.
12. Leys, D., Mowat, C.G., McLean, K.J., Richmond, A., Chapman, S.K., Walkinshaw, M.D. and Munro, A.W. (2003)
    Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 Å reveals novel features of cytochrome P450.
    J. Biol. Chem. 278: 5141-5147.
13. Mattow, J., Schaible, U.E., Schmidt, F., Hagens, K., Siejak, F., Brestrich, G., Haeselbarth, G., Müller, E.C., Jungblut, P.R., and Kaufmann, S.H.E. (2003)
    Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG Copenhagen.
    Electrophoresis 24 3405-3420.
14. McLean, KJ, Scrutton, NS and Munro, AW. (2003)
    Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA.
    Biochem. J. 372: 317-327.
15. Ortiz-Lombardia, M., Pompeo, F., Boitel, B. and Alzari, P.M. (2003)
    Crystal structure of the catalytic domain of the PknB serine/threonine kinase from Mycobacterium tuberculosis.
    J. Biol. Chem. 278: 13094-13100.
16. Vincentelli et al. (2003)
    Medium-scale structural genomics: strategies for protein expression and crystallization.
    Acc. Chem. Res. 36:165-172.
17. Recchi, C., Sclavi, B., Rauzier, J., Gicquel, B. and Reyrat, J.-M. (2003).
    Mycobacterium tuberculosis Rv1395 is a class III transcriptional regulator of the AraC family involved in cytochrome P450 regulation.
    J. Biol. Chem. 278: 33763-33773.
18. Okkels LM, Brock I, Follmann F, Agger EM, Arend SM, Ottenhoff TH, Oftung F, Rosenkrands I, and Andersen P. (2003)
    PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family.
    Infect. Immun. 71: 6116-6123.
    
19. Agger EM, Brock I, Okkels LM, Arend SM, Aagaard CS, Weldingh KN, and Andersen P. (2003)
    Human T-cell responses to the RD1-encoded protein TB27.4 (Rv3878) from Mycobacterium tuberculosis.
    Immunology 110:507-512.
    
20. Gendreizig, S., Kindermann, M. and Johnsson, K. (2003)
    Induced protein dimerization in vivo through covalent labelling.
    J. Am. Chem. Soc. 125: 14970-14971.
    
21. Kindermann, M, George N, Johnsson N, and Johnsson K, (2003)
    Selective and covalent immobilization of fusion proteins for the creation of functional protein microarrays.
    J. Am. Chem. Soc. 125: 7810-7811.
    
22. Juillerat A, Gronemeyer T, Pick H, Vogel H, and Johnsson K. (2003)
    Directed evolution of human O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo.
    Chemistry & Biology 10:313-317.
23. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K. Johnsson,
    Nat Biotechnol 2003, 21, 86.
24. Kindermann, N. George, N. Johnsson, K. Johnsson (2003)
    J Am Chem Soc 125, 7810.

25. Roos AK, Andersson CE, Bergfors T, Jacobsson M, Karlen A, Unge T, Jones TA, and Mowbray SL. (2004)
    Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site.
    J. Molec. Biol. 335: 799-809.
    
26. Bentley, S.D., Brosch, R., Gordon, S.V., Hopwood, D.A., Cole, S.T. (2004)
    Genomics of Actinobacteria, The High G+C Gram-Positive Bacteria.
    In Microbial Genomes (Eds. Fraser, C.M. Read, T. Nelson, K.E.) Humana Press Inc., Totowa, NJ, pp. 333-360.
    
27. Cohen-Gonsaud, M., Keep, N.H., Davies, A.P, Ward, J., Henderson, B. and Labesse, G. (2004)
    Resuscitation promoting factors possess a lysozyme-like domain.
    Trends Biochem. Sci. 29:7-10.
    
28. Rousseau, C., Winter, N., Pivert, E., Bordat, Y., Neyrolles, O., Avé, P., Huerre, M., Gicquel, B. and Jackson, M. (2004).
    Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection.
    Cell. Microbiol. 6: 277-287.
    
29. Schmidt, F., Donahoe, S.,Hagens, K., Mattow, J., Schaible,U.E., Kaufmann, S.H.E., Aebersold, R., and Jungblut, P.R. (2004)
    Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope coded affinity tag technology.
    Mol. Cell. Prot. 3: 24-42.
    
30. Demangel, C., P. Brodin, Cockle, P.J., Brosch, R., Majlessi, L., Leclerc, C. and Cole, S.T. (2004).
    Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of CFP-10 and ESAT-6.
    Infect. Immun. 72: 2170-2176.
    
31. Okkels, L.M., and Andersen, P. (2004)
    Protein-Protein Interactions of Proteins from the ESAT-6 Family of Mycobacterium tuberculosis.
    J. Bacteriol. 186: 2487-2491.
32. Brodin, P., Rosenkrands, I., Andersen, P., Cole, S.T. and Brosch, R. (2004)
    ESAT-6 proteins: protective antigens and virulence factors?
    Trends Microbiol.12:500-508.
33. Alzari PM. (2004)
    First structural glimpse at a bacterial Ser/Thr protein phosphatase.
    Structure, 12:1923-1924.
34. Pleissner, K.-P, Eifert, T., Buettner, S., Schmidt, F., Boehme, M., Meyer T.F., Kaufmann, S.H.E., Jungblut P.R. (2004)
    Web-accessible proteome databases for microbial research.
    Proteomics 4, 1305-1313.
35. Krah, A., Wessel, R., Pleissner, K.-P.(2004)
    Assessment of Protein Spot Components Applying Correspondence Analysis for Peptide Mass Fingerprint Data.
    Proteomics 4, 2982-2986.
36. Pleissner, K.-P., Schmelzer, P., Wehrl, W., Jungblut, P.R. (2004)
    Presentation of differentially regulated proteins within a web-accessible proteome database system of microorganisms.
    Proteomics 4, 2987-2990.
37. Okkels L. M., Müller, E.C., Schmid, M., Rosenkrands, I., Kaufmann S.H.E., Andersen P., Jungblut P. R. (2004)
    CFP10 discriminates between non-acetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction.
    Proteomics 4, 2954-2960
38. A. Keppler, H. Pick, C. Arrivoli, H. Vogel, K. Johnsson (2004)
    Proc Natl Acad Sci USA 101, 9955.
39. Kindermann, I. Sielaff, K. Johnsson (2004)
    Bioorg Med Chem Lett 14, 2725.
40. P. Tafelmeyer, N. Johnsson, K. Johnsson, (2004)
    Chem Biol 11, 681.
41. Arand, M., Cronin, A., Adamska, M., and Oesch, F
    Epoxide hydrolases.
    Methods Enzymol., in press
42. Stéphane Canaan, Damien Maurin, Henri Chahinian, Bénédicte Pouilly, Cécile Durousseau, Frédéric Frassinetti, Loréna Scappuccini-Calvo, Christian Cambillauand Yves Bourne (2004)
    Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis: a novel carboxyl esterase structurally related to the HSL family.
    Eur. J. Biochem. 271 3953-3961.
43. Renaud Vincentelli, Stéphane Canaan, Valérie Campanacci, Christel Valencia, Damien Maurin, Frédéric Frassinetti, Loréna Scappucini-Calvo, Christian Cambillau and Christophe Bignon (2004)
    High-Throughput Automated Refolding Screening of Inclusion Bodies.
    Protein Sci. 13 2782-2792.
44. Cohen-Gonsaud, M., Barthe, P., Pommier; F., Harris, R., Driscoll, P.C., Keep, N.H. and Roumestand, C. (2004).
    Letter to the Editor: 1H, 15N, and 13C chemical shift assignments of the resuscitation promoting factor domain of Rv1009 from Mycobacterium tuberculosis.
    J. Biomol. NMR 30, 373-374.

45. DEMANGEL, C., GARNIER, T., ROSENKRANDS, I. and COLE, S.T. (2005)
    Differential effect of prior exposure to environmental mycobacteria on vaccination with Mycobacterium bovis BCG or a recombinant BCG expressing RD1 antigens.
    Infect. Immun. in press.
46. Majlessi, L., Brodin, P., Brosch, R., Rojas, M.J., Khun, H., Huerre, M., Cole, S.T. and Leclerc, C. (2005)
    Influence of ESAT-6 Secretion System 1 (RD1) of Mycobacterium tuberculosis on the Interaction between Mycobacteria and the Host Immune System.
    J Immunol. 174: 3570-3579.
47. Sinha, S. Kosalai, K., Arora, S., Namane, A., Sharma, P., Gaikwad, A.N., Brodin, P. and Cole, S.T. (2005)
    Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.
    Microbiology in press.
48. Cole ST, Alzari PM. (2005)
    TB – a new target, new drug.
    Science, 307:214-215.
49. McLean, K.J., Warman, A.J., Marshall, K.R. and Munro, A.W. (2005).
    Characterization of the M. tuberculosis ferredoxin encoded by gene Rv0763c, and analysis of its interactions with its cognate P450 CYP51.
    Biochem. J. (in preparation).
50. McLean, K.J., Sabri, M. and Munro, A.W. (2005).
    Analysis of the NADPH-binding site of the Mycobacterium tuberculosis adrenodoxin reductase FprA.
    Biochemistry (in preparation).
51. McLean, K.J., Leys, D. and Munro, A.W. (2005).
    Crystallization of CYP144 – a Mycobacterium tuberculosis P450 induced by oxidative stress.
    Acta Crystallographica (submitted).
52. Seward, H.E., McLean, K.J., Lewis, D.G., Munro, A.W. and Leys, D. (2005, in preparation).
    Atomic structure of the Mycobacterium tuberculosis CYP121:fluconazole complex.
53. Stéphane Canaan, Gerlind Sulzenbacher, Véronique Roig-Zamboni, Laurena Scappuccini-Calvo, Frédéric Frassinetti, Damien Maurin, Christian Cambillau, Yves Bourne (2005)
    Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis.
    FEBS Lett. 579 215-221.

54. Villarino A, Duran R, Wehenkel A, Fernandez P, England P, Brodin P, Cole ST, Simny-Arnat U, Jungblut PR, Cerveñansky C, Alzari PM.
    Activation loop-mediated substrate recruitment of Ser/Thr protein kinase PknB from Mycobacterium tuberculosis.
    Submitted.
55. Duran R, Villarino A, Wehenkel A, Fernandez P, BoitelB, Cole ST, Cerveñansky C, Alzari PM.
    Multiple autophosphorylation sites in the activation loop and juxtamembrane region of serine-threonine protein kinases from Mycobacterium tuberculosis.
    Submitted.