The long term objective of the work is to develop novel inhibitors against drug resistant Mycobacteria Tuberculosis. Drug resistant Mycobacteria Tuberculosis poses a significant threat to the health and well being of the world's population. The drug resistant form is particularly hard to treat requiring expensive and prolonged treatment. Death and serious prolonged illness can result from infection even in healthy individuals. Immuno compromised individuals, such as the eldery, those undergoing cancer or transplant therapy and those with diseases which supress immune function, are particularly at risk of becoming infected. Drug resistant M Tuberculosis has been identified as a BAND C priority pathogen by the National Institute of Allergy and Infectious Disease biodefence program. This means there is serious concern that the organism could be weaponised and used against the United States of America or its Allies. To achieve this goal, this project will use protein X-ray crystallography to provide structural data to drive forward rational inhibitor design and synthesis studies.
The specific aims of this project are: 1. To determine the structure of RmlD from M. tuberculosis. 2. Prepare and analyze co-crystals of RmlC, RmlD, and GIf with active compounds prepared by Richard Lee. 3. Determine the structure of farnesyl diphosphate synthase from M. tuberculosis. Determine the structure of this enzyme co-crystallized with inhibitors. 4. Determine the structure of decaprenyl diphosphate synthase from M. tuberculosis. Determine the structure of this enzyme co-crystallized with inhibitors. To realize these aims we will overexpress these proteins in E. coll. The proteins will then be purified to heterogeneity by chromatographic methods. Once pure the protein will be crystallized by incubation with precipitants. Crystals will be examined by X-ray diffraction using in-house sources or synchrotron. Novel structures will be solved with multiple wavelength methods other by molecular replacement methods. Complexes of the proteins with the compounds made by Lee will be formed by either soaking or co-crystallisation. Visual and computer based analysis of these structures will be used to guide computation and synthetic efforts.
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