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Mutations in human IDH genes occur in cancer and result in active site IDH variants with a gain-of-function ability to reduce the normal 2-oxoglutarate (2-OG) product of IDH catalysis to 2-hydroxyglutarate (2-HG). As reviewed in Chapter 1, elevated 2-HG levels are proposed to promote tumorigenesis via chromatin remodelling. Efficient IDH1 variant inhibitors bind in an allosteric manner at the dimer-interface and hinder binding of 2-OG and Mg2+. Ivosidenib is an IDH1 variant inhibitor that is approved for acute myeloid leukaemia (AML) treatment; however, acquired second-site S280F mutations to IDH1 render cancer cells resistant to ivosidenib treatment. The research described in this thesis investigated the mechanism of action of the second site IDH1 mutation and how to overcome resistance due to it. Kinetic analyses show that the IDH1 S280F substitution not only leads to resistance against ivosidenib but results in a higher affinity for 2-OG and Mg2+, and consequently, more efficient turnover of 2-OG to 2-HG. 1H Nuclear magnetic resonance (NMR) studies reveal that IDH1 cancer variants can turn over D-isocitrate to 2-HG. The rate of conversion of D-isocitrate to 2-HG by S280F substituted variants is more efficient than for IDH1 wildtype or active site variants without the S280F substitution. Mechanistic studies on IDH1 variants provide insights into the influence of various R132 substitutions and the role of the dimer-interface in IDH1 catalysis. In addition to resistance enabled by more efficient 2-HG production, ivosidenib binding is hindered by the loss of a hydrogen bond to S280, steric hindrance due to the S280F substitution, formation of a new hydrophobic pocket at the dimer-interface, and higher enzymatic affinity for 2-OG and Mg2+. Certain IDH1 variant inhibitors were shown to retain activity against isolated IDH1 R132C S280F and R132H S280F, some with high potency. Non-denaturing mass spectrometry (MS) reveals that inhibitors retaining activity bind with a stoichiometry of two inhibitors per IDH1 variant dimer, in contrast to ivosidenib, which binds with a stoichiometry of one inhibitor per dimer. Several inhibitors reduce 2-HG levels in cell lines overexpressing IDH1 R132C S280F or R132H S280F. Some of these inhibitors are in phase 2 clinical studies (FT-2102, DS-1001B) indicating that S280F-mediated ivosidenib resistance can be overcome by using alternative inhibitors. Targeting metabolism has also been of long-standing interest in the antibacterial field, including for Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm). After establishing production strategies and an activity assay for Mtb IDH1/IDH2 and Msm IDH, kinetic studies support the proposal that Mtb IDH2 is likely the essential IDH isoform for oxidative decarboxylation of isocitrate in Mtb metabolism. Mtb IDH2 activity is enhanced by several reactive carbonyl group containing metabolites. Most Hs IDH1 cancer variant inhibitors are not active against Mtb IDH1/IDH2 and Msm IDH but some exhibit weak activity. The overall results provide mechanistic insights into resistance to Hs IDH1 variant inhibitors and show how this can be overcome. The studies suggest that targeting IDH may be a viable strategy for mycobacterial treatment. |
