Inhibition Studies on MenD, an essential enzyme from the human pathogen Mycobacterium Tuberculosis

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dc.contributor.advisor Johnston, J en
dc.contributor.advisor Lott, S en Chuang, Cheng-Han en 2017-09-04T00:05:53Z en 2017 en
dc.identifier.uri en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract Tuberculosis (TB), caused primarily by Mycobacterium tuberculosis (Mtb), is one of the world’s deadliest infectious diseases affecting approximately one-third of the world’s population. TB can persist in a host for decades and relapse particularly if the immune system becomes compromised. Current treatments for TB can be problematic, and cases of multi-drug resistant (MDR) and extreme drug resistant (XDR) TB are on the rise. This calls for an improvement on the current treatment or a development of new anti-TB therapeutics. Menaquinone (MQ) is an electron carrier important for electron transport across the membrane and energy production during aerobic and anaerobic respiration in bacteria. MenD (2-succinyl-5- enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase) is the MQ biosynthesis enzyme that catalyses the first committed step in the classical MQ biosynthesis pathway. Previous work has expressed soluble Mtb MenD (Rv0555) using the Mycobacterium smegmatis expression system and solved the crystal structure in various states of the reaction cycle. The aim of this thesis was to explore various techniques and identify those most suitable for screening of Mtb MenD inhibitor candidates that had been previously identified by in silico screening and structure-based drug design approaches. Both ligand binding and inhibition assays were explored. Binding screen approaches included differential scanning fluorimetry and intrinsic protein fluorescence binding. A current existing inhibition assay following the loss of the second substrate, isochorismate, was converted into a higher-throughput plate-reader format, optimised and tested against a series of inhibitors. Also, ferricyanide reduction assay was tested for the first time. Finally, X-ray crystallography was used to understand the mode of binding that potential candidates exhibited with the enzyme. Of the two binding assays explored, intrinsic protein fluorescence binding was identified as the most useful. The translation and optimisation of the inhibition assay into a higher throughput format were relatively successful however the inhibition assay tests suggested low signal to noise to be a problem requiring further optimisation. X-ray crystallography proved to be a useful technique to identify protein binding compounds and define their binding mode. Three compounds were successfully identified in this way and found to bind to the allosteric site of the protein. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof Masters Thesis - University of Auckland en
dc.relation.isreferencedby UoA99264957413702091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
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dc.title Inhibition Studies on MenD, an essential enzyme from the human pathogen Mycobacterium Tuberculosis en
dc.type Thesis en Biological Science en The University of Auckland en Masters en
dc.rights.holder Copyright: The author en
pubs.elements-id 657348 en
pubs.record-created-at-source-date 2017-09-04 en

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