Structure based design of type II dehydroquinase inhibitors against Mycobacterium tuberculosis

Robinson, David Alexander (2003) Structure based design of type II dehydroquinase inhibitors against Mycobacterium tuberculosis. PhD thesis, University of Glasgow.

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Abstract

There is currently an alarming increase in the world-wide incidence of tuberculosis (TB), the disease caused due to infection by Mycobacterium tuberculosis. There is no easy treatment for TB which currently requires a regime of four drugs administered over a six month time course. The rise in strains of M. tuberculosis resistant to one or more of these frontline anti-tubercular drugs, is a strong incentive to develop novel anti-tubercular agents. The shikimate pathway has been identified as a potential target for the development of new antimicrobial drugs and knockout strains of M. tuberculosis lacking shikimate kinase have been shown to be non-viable.In this study the third enzyme in the pathway, type II dehydroquinase is considered an attractive target as many pathogenic bacteria possess the type II enzyme whereas many bacteria that inhabit the human gut posses the type I form. Structure based ligand design was used to identify potential inhibitors of the enzyme type II 3-dehydroquinate deydratase (DHQase) from M. tuberculosis (MTDHQase). This methodology allows the identification of non-substrate like inhibitors that have solubility profiles more amenable to development as potential drugs. The crystal structures of MTDHQase available in house, were analysed to generate Pharmacophore models, which were subsequently used to screen a virtual compound library, to generate a list of potential inhibitors. This process was repeated for the type II DHQases from Streptomyces coelicolor (SCDHQase) and Helicobacter pylori (HPDHQase) and the hit lists for each enzyme compared. The hits unique to MTDHQase were screened in vitro, identifying six compounds that were active inhibitors. In tandem the cloning and structural analysis of the type II 3-dehydroquinase from Helocobacter pylori was undertaken as a series of inhibitors of this enzyme obtained from a traditional high throughput screen (HTS) against a compound library was available from GlaxoSmithKline. The structure of HPDHQase in complex with the transition state analogue 2,3-anhydroquinic acid was determined to 3.1A resolution. While the structure of HPDHQase in complex with inhibitor AH9095, an HPDHQase specific non-substrate-like inhibitor, was determined to 1.5A resolution. Both structures were solved using the molecular replacement method. Comparison of the structures of HPDHQase with those of SCDHQase and MTDHQase provided significant insight into the factors that affect ligand specificity. The complex of HPDHQase and AH9095 provided the first crystal structure of a type II 3-dehydroquinase with a non-substrate like ligand. This structure allowed the identification of new, unexploited areas of the type II 3-dehydroquinase active site which may be utilised to develop specificity and potency of inhibitors. The present assay for 3-dehydroquinase activity is based upon UV absorbance at 234nm from the product 3-dehydroshikimate. Detection of UV absorbance at this wavelength is not possible when the assay is carried out in plastic 96 well plates, therefore not amenable for high throughput methods. Development of a simple colorimetric assay for 3-dehydroquinase activity was investigated to permit high throughput testing of compounds. By coupling the dehydroquinase step to dehydroshikimate dehydratase, the third enzyme in the quinate degradation pathway, the product protoctaechuate can be detected by a strong colour change in the presence of ferric chloride or sodium molybdate. The amount of protocatechuate detectable by this method, in 96 well plate format, was investigated and expression studies were carried out upon the coupling enzyme dehydroshikimate dehydratase from Acinetobacter coalceticus.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Advisers: John Coggins; Adrian Lapthorn
Keywords: Pharmacology
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-72499
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72499

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