Transition from classical methods to new strategies: Mechanistic evaluation of inhibitors against Mycobacterium tuberculosis shikimate kinase
Type of DegreePhD Dissertation
Chemistry and Biochemistry
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Tuberculosis (TB) still stands as one of the leading causes of mortality resulting from an infectious agent. In part, this stems from the emergence of multidrug and extensive drug-resistant strains of tubercle bacilli, demonstrating the pressing need for the development of novel anti tubercular agents. Interestingly, the shikimate pathway, found in plants, bacteria, fungi and algae, is essential for the survival of Mycobacterium tuberculosis. This pathway produces precursors to aromatic amino acids and other aromatic cellular metabolites. Fortunately, it has no mammalian analogue. This makes the enzymes of this pathway suitable targets for the development of novel anti-microbial agents. The research presented in this dissertation focuses on Mycobacterium tuberculosis shikimate kinase (MtSK). Our objectives were to develop LC-MS-based screening and characterization of potential anti tubercular agents – natural, synthetic or semi synthetic, develop fluorescence methods for rapid mechanistic evaluation of MtSK inhibitors and strategies for screening out promiscuous inhibitors. Using mass spectrometry, we characterized a group of marine derived compounds, manzamine alkaloids. These inhibitors showed mixed-noncompetitive mode of inhibition, with time-dependent and slow-binding components. Of this group, one, 6-cyclohexamidomanzamine A, a derivative of the parent manzamine A scaffold, showed slow, tight-binding kinetics with an inhibition constant in the nanomolar range. Another marine-derived inhibitor we characterized was ilimaquinone, which showed irreversible inhibition evident from formation of a covalent adducts with primarily serines and threonines, and to a lesser extent lysines and tyrosines. It is worth noting that due to the nonspecific nature of this inhibitor, as it has shown to modify other proteins (not only MtSK), further exploitation of this molecule as a potential antibacterial, antiviral, or anticancer agent should be approached with great care. With respect to developing rapid screening tools, we have generated a panel of MtSK variants with intrinsic fluorescent properties. Substitutions were made on the lid domain, V116W, the adenine binding domain, N151W, and the shikimate binding domain, E54W. These variants show differential responses to substrates and inhibitors. Interestingly, we noted that the binding of one of the substrates, shikimate produced changes in fluorescence in only one of the variants, supporting previously reported data that substrate binding induces conformational changes. Using these, we evaluated the inhibition kinetics of previously characterized inhibitor, so-called Compound 1. Our data supported a mixed-type mode of inhibition, with a slow, reversible component. Competition experiments indicated Compound 1 binds at or near the shikimate binding site, evident from spectral shifts in E54W in the presence of the inhibitor. Together, we have shown that the 6-cyclohexamido derivative of manzamine A is a potent inhibitor of MtSK, and this may be an indication of potential success in later drug discovery stages. The use of ilimaquinone for further drug screening and discovery purposes is at the discretion of the investigator. Our combinatorial approach to weeding out nonspecific inhibitors together with our panel of variants would increase turnover in the early drug screening stages and prevent wastage of long hours on fruitless drug investigations. Lastly, our panel of variants shows great potential for further development into high throughput screen platforms and possibly as sensors.