The studies on the mechanism and inhibition of Isocitrate Dehydrogenases

Thesis

Isocitrate dehydrogenases (IDHs) are human metabolic enzymes producing NADPH (IDH1/2) or NADH (IDH3). IDHs are involved in cancer metabolism and progress; upregulation and mutations to human IDH1/2 that catalyse reduction of 2-oxoglutarate (2OG) to 2-hydroxyglutarate have been identified in multiple cancers. The work described in this thesis attempted to gain a better understanding of IDH1 and IDH2 biochemistry by studying the inhibition and kinetics of wild type (wt) IDH1/2 and mutant IDH1/2 (mIDH1/2), with the aim of enabling improved IDH1/2 targeting treatments.

By comparison with IDH1, there are only limited inhibition studies reported on IDH2. The work described in this thesis began with profiling of reported mIDH1/2 inhibitors with IDH2 using biochemical and biophysical assays. Allosterically binding mIDH2 targeting inhibitors were found to be Mg²⁺ or Mg²⁺-2OG complex competitive, as is the case for IDH1. NaCl was observed to affect the kinetics of both Mg²⁺ and 2OG (lower their affinities) with mIDH1/2 in a manner that increases the inhibition potencies of mIDH1/2 inhibitors, particularly with R172K IDH2.

In the assay optimisation of the inhibition assays for wt IDH1/2, the results support the proposal that the substrate for wt IDH2 is the isocitrate-Mg²⁺ complex as recently shown for IDH1. Screening for wt IDH1/2 inhibitors was performed. As a result, several wt IDH1 and wt IDH2 selective inhibitors and a pan-wt IDH1/2 inhibitor were identified. Mechanism of action studies suggested the pan-wt IDH1/2 inhibitors are likely to be Mg²⁺ or Mg²⁺-isocitrate competitive.

To date, all potent mIDH1/2 inhibitors and the wt IDH1/2 inhibitors identified here are likely to be allosteric binders; it is of interest to find an active site interacting inhibitor competing with isocitrate/2OG binding. TCA cycle intermediates and their analogues, and synthetic 2OG derivatives were studied with wt and mutant IDH1/2 to identify inhibitors and competitive substrates. A 2OG derivative and N-oxalylglycine were identified as IDH1/2 variant inhibitors. Some synthetic 2OG derivatives bearing C3-substitution manifested substantial activity as substrates with IDH1/2 variants. Mechanistic studies with synthetic 2OG derivatives and R132H IDH1 are consistent with half-site activity and positive cooperativity processes involving the IDH dimers.

Overall, the work described in this thesis reveals that the allosteric inhibition of both wt and variant IDH1/2s leads to Mg²⁺ or Mg²⁺-substrate competition. In addition, 2OG derivatives can act as both inhibitors and alternative substrates for IDH1/2 variants; they will be useful as biochemical probes to study IDH catalysis.

Authors: Liu, X

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English