Development of antibacterials directed against the thymidylate synthase ThyX in Mycobacterium tuberculosis : mechanism kinetic and in silico inhibitor optimisation

Tuberculosis is a global health issue as the recent data from World Health Organization indicates that one third of the human population is infected with a dormant form of tuberculosis. Consequently, 9 million new cases and 1.5 million deaths caused by Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, were reported in 2014. The treatment for tuberculosis lasts at least 6 months and excessive or misuse of currently available anti-tuberculosis agents has led to the rise of multi and extensively resistant forms of this important human pathogen. The dissemination of Mtb resistant strains is alarming and the need for new therapeutic agents and targets to fight these Mtb strains is urgent. During my thesis, I have investigated reaction and inhibition mechanism of Mtb ThyX. ThyX catalyses the synthesis of deoxythymidine monophosphate (dTMP) by the methylation of carbone 5 of deoxyuridine monophosphate (dUMP), using N5,N10-methylenetetrahydrofolate and NADPH as carbon and hydride donors respectively. As ThyX is absent in humans and they are structurally and mechanistically distinct from analogous human enzyme, these proteins are a promising therapeutic target in Mtb and many other pathogenic bacteria. Moreover, recent genome wide studies of clinical isolates have revealed overexpression of Mtb thyX in some multi-resistant isolates of Mtb suggesting an involvement in a compensatory pathway in the mechanism of acquisition of drug resistance. I have studied the kinetics of Mtb ThyX mechanism under steady-state conditions and by using time resolved spectroscopy. I showed the selectivity of the enzyme for NADPH, and studied the dynamics of dUMP and NADPH binding in the Mtb ThyX active site as well as the reactivity of the flavin cofactor with molecular oxygen. These studies have explained how Mtb, a strict aerobe, can synthetize thymidylate even in the presence of molecular oxygen, despite the reduced flavin-factor (obligatory intermediate of the reaction) is highly oxygen reactive. A second part of my work focused on identifying Mtb ThyX inhibitors with antimycobacterial activity. Two approaches were used: i) selecting naphthoquinone derivatives, by similarity searching, or by picking previously reported compounds with anti-Mtb activity but of unknown target(s), and testing them for Mtb ThyX inhibition ; ii) Developing a pharmacophore model that can be used for screening chemical libraries in silico and/or prioritizing further compounds for additional experimental testing. These approaches have led to the identification of several new Mtb ThyX inhibitors with low micromolar inhibition constants while no significant inhibition was observed on human thymidylate synthase. The inhibition modes of this new set of compounds suggest a novel binding site on Mtb ThyX than previously observed on other ThyX proteins. The most potent compounds like idebenone or 2-[(4-methoxyphenyl)methyl]-1,4-dihydronaphthalene-1,4-dione also exhibit moderate activity against clinical strains of Mtb. The precise binding site of these compounds remains unknown and will require further investigation to guide SAR studies to optimize these inhibitors. Updating the pharmacophore model will allow to select new compounds to be tested in vitro. Idebenone has already passed phase 1 and 2 clinical trials for duchenne muscle dystrophy, exhibiting good ADME-tox properties. Repurposing idebenone derivatives to target ThyX in Mtb will be the focus of future work.