Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 553 Structural Studies of Glutamine Synthetases – Towards the Development of Novel Antitubercular Agents WOJCIECH W. KRAJEWSKI ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 978-91-554-7283-2 2008 urn:nbn:se:uu:diva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ist of Papers This thesis is a comprehensive summary of the results presented in the fol- lowing publications and manuscript, which will be referred to by their Ro- man numerals: I Krajewski, W.W., Jones, T.A., and Mowbray, S.L. (2005) Struc- ture of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional in- sights. Proc. Natl. Acad. Sci. USA 102, 10499-10504. II Krajewski, W.W., Collins, R., Holmberg-Schiavone, L., Jones, T.A., Karlberg, T., and Mowbray, S.L. (2008) Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes, and provide opportunities for drug and herbicide design. J. Mol. Biol. 375, 217-228. III Nordqvist A., Nilsson, M.T., Röttger, S., Odell, L.R., Krajewski, W.W., Andersson, C.E., Larhed, M., Mowbray, S.L., and Karlen, A. (2008) Evaluation of the amino acid binding site of Mycobac- terium tuberculosis glutamine synthetase for drug discovery. Bioorg. Med. Chem. 16, 5501-5513. IV Nilsson, M.T.*, Krajewski, W.W.*, Srinivasa, B.R., Yahiaoui, S., Larhed, M., Karlen, A., Jones, T.A., and Mowbray, S.L. (2008) Structural basis for the inhibition of Mycobacterium tuberculosis glutamine synthetase by novel ATP-competitive inhibitors. Manuscript. * Joint first authorship Articles I, II, and III have been reproduced with permission from the respec- tive copyright holders. Contents Introduction.....................................................................................................9 Background...................................................................................................11 Glutamine synthetase and its role in nitrogen metabolism.......................11 Classification of GSs................................................................................11 Reaction mechanism of GS ......................................................................12 Other reactions catalyzed by GS ..............................................................12 Taut and relaxed forms of bacterial GS....................................................13 Regulation of bacterial GS activity ..........................................................13 GS inhibition by methionine sulfoximine and phosphinothricin .............14 Structures of bacterial and eukaryotic GSs ..............................................15 M. tuberculosis GS as a potential drug target...........................................18 General Methods...........................................................................................19 Protein expression and purification..........................................................19 Activity and inhibition assays ..................................................................19 X-ray crystallography...............................................................................20 Results and discussion ..................................................................................21 Structure of MtGS in complex with a transition-state mimic (Paper I)....21 Complex formation and crystallization................................................21 Overall structure ..................................................................................21 Active site............................................................................................22 Implications for catalysis and inhibition..............................................24 Structures of mammalian GSs (Paper II) .................................................25 Thermal shift assay..............................................................................25 Crystallization......................................................................................26 Overall structures.................................................................................27 Active site............................................................................................27 Substrate-induced conformational changes .........................................29 Comparison of HsGS and MtGS structures – implications for inhibitor design...................................................................................................29 Targeting the amino acid-binding site (Paper III) ....................................31 Literature survey..................................................................................31 Virtual screening..................................................................................32 Targeting the nucleotide-binding site (Paper IV).....................................35 Structure of a relaxed MtGS in complex with PA...............................35 MSO-P as a crystallization aid ............................................................35 Structure of a taut MtGS in complex with PA and MSO-P.................37 Prospects for selective inhibition of MtGS by PA...............................37 Future perspectives .......................................................................................39 Summary in Swedish ....................................................................................40 Acknowledgements.......................................................................................42 References.....................................................................................................44 Abbreviations ATase Adenylyl transferase CfGS GS from Canis familiaris GS Glutamine synthetase HsGS GS from Homo sapiens HTS High-throughput screening MSO L- Methionine-S-sulfoximine MSO-P Methionine sulfoximine phosphate Mtb Mycobacterium tuberculosis MtGS GS from Mycobacterium tuberculosis NCS Noncrystallographic symmetry PEG Polyethylene glycol PDB Protein Data Bank RAPID Rational Approaches to Pathogen Inhibitor Discovery rms Root mean square StGS GS from Salmonella typhimurium TB Tuberculosis UTase Uridylyl transferase -GCS -glutamyl:cysteine synthetase ZmGS GS from Zea mays Introduction Tuberculosis (TB) is a contagious disease caused by a pathogenic bacillus, Mycobacterium tuberculosis (Mtb). It has plagued humans for thousands of years, and despite the existence of treatment and control programs, continues to claim 1.6 million lives each year (The World Health Organization, http://www.who.org). In 2005, there were an estimated 8.8 million new cases of TB, 90% of which occurred in Asia and sub-Saharan Africa. While it is estimated that one-third of the world’s population is currently infected with M. tuberculosis, only 5-10% will develop the active form of TB. Particularly at risk are people with a weakened immune system. Although existing drugs can often cure TB, the treatment is lengthy, and requires taking a combination of drugs for at least 6 months. However, the therapy is not always successful and often results in patient relapsing. Fur- ther, poor patient compliance has led to the emergence of drug-resistant TB, which is even more difficult to treat. The situation is further exacerbated by the emergence of HIV/AIDS, which is a serious risk factor for TB (Corbett et al. 2003). The above facts indicate an urgent need for the development of novel, more effective drugs that would shorten the current treatment, and act on persistent and drug-resistant TB. Identification of novel antimicrobial compounds relies mainly on two ap- proaches, a so-called “empirical” process involving whole-cell screening, and a target-based approach, often referred to as a “mechanistic” path. Both approaches have advantages and disadvantages, but with the advances in genomics (Cole et al. 1998) and the availability of essentiality data, the latter seems to dominate the drug discovery landscape (Cole and Alzari 2007). During the past 10 years there has been an increase in research activities aimed at developing novel drugs against TB. The RAPID (Rational Ap- proaches to Pathogen Inhibitor Discovery) center that started at Uppsala University in 2003 is one such example. Research groups with expertise in structural biology, medicinal chemistry, and computational chemistry have combined to form the center. Within RAPID we target essential enzymes from Mtb, determine their three-dimensional structures and develop assays for monitoring their activities. With this in hand, the search for inhibitors can begin, either by