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Development of a drug discovery protocol through the expression of key mycothiol biosynthetic enzymes for Mycobacterium tuberculosis

Lamprecht, Dirk Antonie (2008-04)

Thesis (MSc) -- University of Stellenbosch, 2008.


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.

AFRIKAANSE OPSOMMING: Die werkstuk fokus op mikotiol (MSH), wat 'n Jae molekulere gew1g tiol van M tuberculosis is. Verskeie studies het alreeds bewys dat die ensieme wat betrokke is by die biosintese en sellulere funksies van MSH, goeie teikens is vir die ontwikkeling van nuwe M tuberculosis antibiotikums. Huidige metodes vir die evalueering van inhibeerders teen hierdie ensieme is onvoldoende en laat nie die toets van groot hoeveelhede inhibeerders gelyktydig toe nie. In hierdie betrokke werkstuk word die grondslag gele vir die onwikkeling van 'n verbeterde metode vir die ontdekking van antituberkurale middels. Gedurende die studie is rnikotiol disulfied reduktase (Mtr) en die rnikotiol biosintetiese ensiem, MshB, in E. coli uitgedruk en gesuiwer. Dit is die eerste suksesvolle uitdrukking van Mtr in E. coli en maak dit moontlik om die eerste kristalstruktuur van hierdie es ensiele M tuberculosis ensiem op te los. Die Mtr kristalstruktuur sal 'n belangrike hulpmiddel wees vir truktuurgebaseerde ontwikkeling van middele teen Mtr. Yerder, i daar ook 'n nuwe ESI/MS(TOF)-HPLC metode ontwikkel vrr die kwantifisering van MSll en sy tus engangers in die biosintese pad. Die nuwe analitiese . mctodc is gebruik om drie ver killende tus engangers op te spoor en te kwantifi eer. Al drie tu sengangers is in 'n enkele in puiting van M smegmatis sel vervloeiing opgespoor. Die metode i ook gebruik om die verandering in M H:MSSM verhoudings te ondersoek in M. smegmatis elle wat onder oksidatiewe toestande opgegroei is. Ensiemreaksies met MshB i ook geanaliscer met die mctode. 'n Reeks ub traatanaloc i ook ontwikkel en getoet teen Mtr en M hB. Des-myo-ino itol mikotiol disulfied (DI-M SM) is gebruik om die aktiwiteit van Mtr te toets. Verder is twee groepe ub traatanaloe teen M hB ontwikkel tiogliko idie e-disakkariede en f1uoroscrende substrate, met die doe! om as raamwerk te dien vir die ontwikkeling van inhibeerders teen MshB. Daar is gcvind <lat een van die fluoroserende substrate beter aktiwiteit toon met MshB as die natuurlike substraat. Dit is ook gewys <lat die molekule intramolekulere herrang kiking ondergaan na die inwerking van MshB. Hierdie ondekking dien ecrste bewys <lat die tipe intramolekulere herrangskiking deur 'n ensiem gemedieer kan word. Met verdere onwikkeling kan hierdie molekule gebruik word in die fotometriese analiese van MshB ensiemreaksies.

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