Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.755291
Title: Evaluation and identification of novel inhibitors of Mycobacterium tuberculosis InhA
Author: Ahmed, Aneesa
ISNI:       0000 0004 7428 2899
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2016
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Abstract:
Tuberculosis (TB) caused approximately 1.5 million deaths worldwide in 2014 and is the second biggest killer due to a single infectious agent. Issues with current TB treatment such as interactions with antiretroviral therapy for HIV co-morbidities, poor compliance and the emergence of resistance to current antibiotics mean that there is an urgent need for new antitubercular drugs. InhA is an enoyl reductase that is the primary target for isoniazid, a first-line antitubercular drug. This enzyme plays a key role in the synthesis of mycolic acids which are key components of the TB cell wall. As such, InhA is an attractive target for future antitubercular drugs. Isoniazid is a pro-drug which is activated by the enzyme, katG, and resistance to isoniazid has been primarily attributed to mutations in katG. Thus, drug discovery for InhA to date has focused on direct inhibitors which do not require activation, mainly using virtual screening and structure-based drug design approaches. In recent years a structural mutation, S94A InhA, has been detected in clinical isolates conferring resistance to isoniazid in the absence of katG mutations, suggesting it is becoming clinically relevant in the context of isoniazid resistance. To date, no direct InhA inhibitors are available clinically, and only two S94A InhA inhibitors have been reported. In this thesis, two main approaches were used to evaluate and identify novel InhA inhibitors. Firstly, recently discovered wild-type (WT) InhA inhibitors at the University of Nottingham were further evaluated by determining their ability to inhibit the activity of the S94A InhA mutant, given the apparent increasing importance of this mutation in isoniazid resistance. In addition, their ability to inhibit the growth of Mtb, E. coil and S. aureus was determined in order to provide a more relevant inhibitory profile. Secondly, novel fragment-linking drug discovery tools were investigated as this may provide an alternative route for drug discovery and has not yet been reported for InhA. A series of WT InhA inhibitors previously discovered at the University of Nottingham were evaluated by determining their inhibitory activity for S94A InhA and determining their ability to inhibit the growth of Mtb, E. coli and S. aureus to provide a more relevant inhibitory profile as lead compounds. Two compounds were found to inhibit the activity of S94A InhA more strongly than WT InhA, which is of significant interest since only two S94A InhA inhibitors have been reported in the literature. Another compound was discovered that displayed an ability to inhibit the growth of Mtb and M. bovis at 25 ug.m1-1 whilst displaying no inhibitory activity for E. coli or S. aureus at the concentrations tested. To further explore the S94A InhA mutant, crystallization studies were then performed. The ultimate aim was to obtain structural information for the binding modes of the two inhibitors that displayed a more promising inhibitory activity for S94A InhA compared to WT InhA as this would aid future structure-based drug design. Successful crystallization conditions were determined for WT InhA-NADH and S94A InhA-NADH, which were similar to those reported in the literature. Novel fragment-linking drug discovery approaches were then explored for InhA. In order to explore the feasibility of this approach for InhA, molecular docking studies were conducted on a series of deconstructed InhA inhibitors to compare the binding poses of the fragments to the complete inhibitor. Findings suggested that the binding position of some of the fragments were preserved, when compared to the corresponding portion of the inhibitor. The structures of these fragments provide a starting point for further investigation and the possible de novo design of fragments for a fragment-linking approach. An experimental fragment-linking approach, in situ click chemistry, was also developed for InhA with the aim of identifying novel inhibitors. The method development consisted of using molecular docking studies to select azide and alkyne fragments, developing and optimizing an in situ method, and developing a suitable LC-MS/MS analytical method to detect the formation of 1,2,3-triazoles. A novel disubstituted 1,2,3-triazole was detected in a larger amount in the presence of InhA, by approximately twelve-fold, suggesting the enzyme was catalyzing the formation of this triazole. Attempts were made to validate the precise site of binding for the azide and alkyne fragments, but were unsuccessful and further work is required to determine the inhibitory activity of this compound. This in situ click chemistry tool will provide an exciting novel drug discovery approach in addition to the current reported methods for InhA.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.755291  DOI: Not available
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