Understanding and targeting isocitrate-metabolising enzymes involved in tuberculosis and cancer

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In 2016, 1.3 million people died from tuberculosis (TB),(1) which is caused by Mycobacterium tuberculosis (Mtb), a pathogen that is rarely treated with common antibiotics. Multi-drug treatments are available but the long-term (at least six months) strict treatment regimens pose challenges for patients. The emergence of Mtb resistant strains is making treatment increasingly ineffective. In 2016, 490,000 cases of multidrug-resistant TB were reported.(1) Key characteristics for the survival of Mtb involve its metabolic flexibility and its ability to persist in a highly antibiotic-resistant dormant state.(2) An important metabolic feature of mycobacteria is the glyoxylate shunt, a pathway that exists in most prokaryotes and enables Mtb to grow on C2 carbon sources such as fatty acids. It partly bypasses the TCA cycle, in which isocitrate dehydrogenase (IDH) catalyses the oxidative decarboxylation of 2-oxoglutarate (2OG) giving isocitrate, which is then decarboxylated to succinyl-CoA by 2OG dehydrogenase. Succinate is then generated via an oxidative phosphorylation as catalysed by succinyl-CoA synthetase. While the decarboxylation steps in the TCA cycle lead to the loss of two carboxyl groups as CO2, the glyoxylate shunt bypasses these steps, hence enabling simple carbon source compounds to be used for macromolecule synthesis (e.g. glucose).Isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, converts isocitrate (instead of 2OG) into glyoxylate and succinate directly. ICL is upregulated in Mtb in certain non-replicating states. An icl knockout attenuated the persistence and virulence of Mtb.(2) Thus, the glyoxylate shunt is important for Mtb to survive inside the host and finding inhibitors of IDH and/or ICL may enable new treatment strategies. Detailed biochemical and biophysical studies of the bifurcating point between the TCA cycle and the glyoxylate shunt are required for understanding the metabolic pathways in Mtb and developing inhibitors for them are important for validating isocitrate metabolising enzymes as drug targets. Therefore, my work is aimed at biochemical and biophysical studies on IDH and ICL and their regulation, including kinetic, mass spectrometry (MS), crystallography and nuclear magnetic resonance spectroscopy (NMR) studies, as well as early stage inhibitor development. Accordingly, I will be screening existing in-house libraries against these enzymes as well as inhibitor libraries provided by GSK. The potency, selectivity and binding-mode of hit molecules will be determined by biochemical, biophysical and crystallographic studies; and will further inform on chemical inhibitor optimisation. Potent inhibitors identified in my screens will be tested against mycobacteria cells at the Francis-Crick Institute. The results will yield a more detailed understanding of the role of the glyoxylate shunt and its regulation as well as yield inhibition information of the metabolism in mycobacteria. Alongside the development of inhibitors for Mtb IDH/ICL, analogous biochemical and biophysical studies will be carried out on human IDH2 and its variants which are involved in cancer (R140Q, R172K). These variants are reported to gain a neomorphic function to produce 2-hydroxyglutarate (2HG), an oncometabolite which is also produced by Mtb IDH(3). These investigations will support our understanding of the isocitrate metabolism in both tuberculosis and humans; and thus, could additionally pave the way to new cancer therapeutics.This project falls within the BBSRC research area "Combatting antimicrobial resistance" and aligns with the priorities to "Understand the fundamental microbiology of organisms with known resistance prevalence in order to understand how resistance develops and is maintained, and to develop mitigation strategies" and "Underpin the development of novel antimicrobials and alternatives to antimicrobials". It involves the University of Oxford, the Francis Crick Institute and GSK.