Inhibition and regulation of Mycobacterium tuberculosis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The shikimate pathway is responsible for the biosynthesis of the aromatic amino acids and other aromatic metabolites in plants, micro-organisms and apicomplexan parasites. The shikimate pathway is essential in bacteria and plants, but absent from mammals, which has led to interest in the enzymes of the pathway as targets for the design of antimicrobial and herbicidal agents. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first commit¬ted step of the shikimate pathway, the condensation of phosphoenolpyruvate and erythrose 4-phosphate to yield 3-deoxy-D-arabino-heptulosonate 7-phosphate. The subject of this thesis is the investigation of inhibition and allosteric regulation of the DAH7PS enzyme from Myco¬bacterium tuberculosis (MtuDAH7PS), the pathogen that causes tuberculosis. Tuberculosis remains a major health threat to the global community, and the emergence of multi-drug resistant strains highlights the need for new tuberculosis treatments. Inhibitors of MtuDAH7PS have the potential to be developed into new anti-tuberculosis drugs. Chapter 2 describes the design, synthesis and evaluation of active site inhibitors based on intermediate mimic scaffolds. The intermediate mimics synthesised represent the first reported example of inhibitors targeting the active site of MtuDAH7PS. The most active compounds tested displayed inhibition constants in the sub-micromolar range, making them the most potent inhibitors of any DAH7PS enzyme reported to date. MtuDAH7PS displays a complex and subtle mechanism of synergistic regulation: The enzyme is inhibited by binary combinations of the aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr). Three allosteric binding sites were identified using X-ray crystallo¬graphic analysis of MtuDAH7PS in complex with Trp and Phe. While these crystal structures led to the identification of an allosteric binding site which preferentially binds Trp, the role and selectivity of the other two sites with respect to Phe and Tyr remained unclear. The results described in Chapter 3 provide structural and biochemical evidence for the hypothesis that each of the three allosteric binding sites has a preference for binding one of the aromatic amino acids Trp, Phe and Tyr, respectively. The results furthermore show that the ternary combination of Trp, Phe and Tyr synergistically regulates MtuDAH7PS, leading to almost complete loss of enzymatic activity in the presence of all three allosteric ligands. In Chapter 4, the interaction of MtuDAH7PS with the naturally less common D-enantiomers of the aromatic amino acids is described. It was found that the D-enantiomers of the aromatic amino acids have no effect on enzymatic activity of MtuDAH7PS, suggesting an efficient mechanism by which the enzyme can discriminate between allosteric ligands of opposite configuration. Studies of the binding affinity of the D-amino acids to MtuDAH7PS as well as structural characterisation of MtuDAH7PS-D-amino acid complexes by X-ray crystallographic analysis suggest that D-Trp and D-Phe can still bind to their respective sites. The lack of inhibition is attributed to subtle differences in the binding mode of the D-enantiomers of the ligands compared to the L-enantiomers. Chapter 5 details the discovery of alternative ligands and inhibitors targeting the allosteric sites of MtuDAH7PS using virtual screening. Libraries of potential alternative ligands were docked into the allosteric sites of MtuDAH7PS and the predicted docking poses were used to guide the selection of compounds for physical screening. Using this approach, a number of ligands and inhibitors of MtuDAH7PS were discovered and their interaction with the enzyme structurally characterised. Comparison of the crystallographically observed binding modes of new ligands with the docking poses predicted by computational docking highlighted potential improvements to the virtual screening method. The analysis of the correlation between ligand binding modes and inhibition of enzymatic activity provided further insight into which interactions between the allosteric ligand and the binding site are crucial in order to achieve inhibition. The crystal structures of MtuDAH7PS in complex with the new alternative ligands can serve as a starting point for the design of ligands with increased affinity and potency.