Structural and biochemical studies on potential drug targets from Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis, the cause of tuberculosis (TB), presents a major threat to human health worldwide. The major goals for future anti-TB therapies are to develop new drugs that will shorten and simplify TB treatment, to discover compounds that are effective against multi-drug resistant TB (MDRTB) and to improve treatment of latent TB infection. The aim of this project was to determine the structures and assess the functionality of M. tuberculosis proteins deemed to be potential drug targets. The application of structural and biochemical knowledge was hoped to aid in the discovery of potential inhibitors of validated targets. The protein of unknown function, Rv0343 (IniC), found to be up-regulated in response to current antibiotic therapies, was studied with a view to carrying out structural analyses and elucidation of its molecular function. Constructs of this protein were successfully expressed, and partially purified, but due to its instability and tendency to aggregate, crystallisation trials could not be performed. However, comparative studies have led to an improved understanding of this protein and its possible defensive role in M. tuberculosis. Enzymes from the essential tryptophan biosynthetic pathway may also serve as potential M. tuberculosis drug targets. The product of the trpD gene, the enzyme that catalyses the second step in tryptophan biosynthesis, was targeted for structural and biochemical analysis. The structure of TrpD from M. tuberculosis in complex with its substrate phosphoribosylpyrophosphate (PRPP) and Mg2+ was solved by X-ray crystallography at 2.3 A resolution (7?=0.194, 7?free=0.255). Knowledge of the active site and of the interactions made by PRPP with the protein enabled in silico modelling to correctly identify the binding mode of anthranilate prior to catalysis. A biochemical activity assay was developed so that target-specific inhibitor screening of the TrpD enzyme could be carried out. NMR studies have tentatively suggested that the M. tuberculosis enzyme HisA (annotated to be involved in histidine biosynthesis), is also involved in the third step of tryptophan biosynthesis, an enzyme that may likewise serve as a potential drug target.