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Title: Identification of novel inhibitors for Mycobacterium tuberculosis InhA : towards structure-based drug design for tuberculosis
Author: Olotu-Umoren, Loyin
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2012
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Abstract:
Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of mortality among infectious diseases. Efforts to combat the disease have been hammered by the emergence of drug resistance as well as drug mismanagement and poverty. In view of the continuous worldwide spread of tuberculosis and declining effectiveness of drugs to fight the disease, there is a need to discover more efficacious anti-tuberculosis agents. InhA, the enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis, is one of the key enzymes involved in the synthesis of mycolic acids. Mycolic acids are known to be important for mycobacterial growth, survival and pathogenicity; thus making the pathway an attractive target for the development of novel anti-tubercular drugs. In this thesis, a combination of several techniques have been utilised to identify potential inhibitors of InhA. The first approach was to carry out a rapid in silico docking study of a library of chemical compounds (approximately 137,000) in order to identify compounds that are most likely to bind InhA. A structure-based virtual screening, using the docking program GOLD, led to the identification of fifteen top compounds which were selected for further experiments on the basis of top fitness scores, good binding mode, interactions and contacts with the protein. To explore further the binding of the selected ligands to InhA, the Molecular Mechanics / Poisson-Boltzmann and Surface Area method was used to estimate the relative binding free energy of the ligands to InhA following molecular dynamics simulations. The binding of all fifteen compounds were found favourable as reflected by negative binding free energy values. To perform the experimental validation of the 'hits’ by steady-state kinetics and inhibition studies, InhA was cloned, expressed, purified and characterised. The compounds were tested "experimentally for inhibitory effect on InhA activity. These studies revealed that seven of the compounds showed more than 20% inhibitory effect against InhA activity, with the highest inhibitory effect observed being 65.7 ± 0.9% inhibition. The compounds that exhibited more than 20% inhibitory activity were further characterised by determining the ICso values and the type of inhibition with respect to InhA substrate 2-£ra/7s-octenoyl-Co-enzyme-A. The most potent compound exhibited an ICso value of 28.06 ± 0.02pM. Submission of the compounds for evaluation as inhibitors of mycobacterial growth in culture revealed that all compounds showed modest antibacterial activity. In addition, crystallographic studies were carried out on InhA mutant and wild-type enzymes. At the time of this thesis work, no crystal structure is available on any InhA mutant ternary complex as well as for one of the reported InhA mutants, Isoleucinel6 to Threonine InhA. Studies were therefore carried out towards obtaining the mutant crystal structures, and on the wild-type enzyme in order to enhance the future structure determination of InhA protein-ligand complex with the identified hits. The mutant proteins were successfully crystallised, as well as the wild-type enzyme which produced a crystal that diffracted to 2.0A
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.754205  DOI: Not available
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