Browsing by Author "Lamprecht, Dirk Antonie"
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- ItemDevelopment of a drug discovery protocol through the expression of key mycothiol biosynthetic enzymes for Mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2008-04) Lamprecht, Dirk Antonie; Jardine, M. A.; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: This work focuses on mycothiol (MSH), the low molecular weight thiol of M tuberculosis, the causative agent of pulmonary TB. It has been proven through numerous studies that the enzymes involved in the biosynthesis and related reactions of MSH are good drug targets for the design of new antibiotics against M tuberculosis. Unfortunately, current screening methods are insufficient and do not allow for the high thought-put screening of potential inhibitors against these enzymes. In this work we laid the foundation for an improved method to expedite antitubercular drug discovery. During this study mycothiol disulfide reductase (Mtr) and the mycothiol biosynthetic enzyme MshB were recombinantly expres ed and purified from E. coli. The Mtr enzyme was hown to be active in the presence of des-myo-inositol mycothiol disulfide (DIM SM), a ubstrate analogue of Mtr. Taken together, these results should greatly facilitate the olution of the fir t crystal tructure of this essential M tuberculosis enzyme. Such a tructure would be an essential requirement of structure-based drug development effort directed at Mtr. Furthermore, we have developed a new ESVMS(TOF)-HPLC method for the quantitation of MSII and its pathway intermediates. This new analytical . method was employed to detect and quantitate three different pathway analytes from a ingle injection of M. megmati cell ly ate. It was al o u ed to determine the fluctuating MSH:M M level in M. megmatis cells growing under oxidative stress conditions. MshB enzyme reaction were al o analyzed u ing this method. A series of substrate analogues were also designed and tested again t both the expressed enzymes. The frrst of these, DI-MSSM, was used to test the activity of Mtr. Furthermore, two sets of substrate analogues of M hB - one of thioglycoside-di accharides and another containing a variety of fluorophores - were designed and synthesized with the goal of using the e analogues as scaffolds for the development of new inhibitor libraries. Among these analogues, one of the fluorogenic substrates showed better activity with MshB than its natural substrate. This molecule was also shown to undergo an intramolecular rearrangement, after reacting with MshB. This would be the fir t time this type of rearrangement is shown to be enzyme mediated. With further development this molecule could be u ed in a photometric-based as ay of MshB.