Human isocitrate dehydrogenase (IDH1) and its cancer-associated variant (IDH1 R132H) are rendered electroactive through coconfinement with a rapid NADP(H) recycling enzyme (ferredoxin-NADP + reductase) in nanopores formed within an indium tin oxide electrode. Efficient coupling to localized NADP(H) enables IDH activity to be energized, controlled, and monitored in real time, leading directly to a thermodynamic redox landscape for accumulation of the oncometabolite, 2-hydroxyglutarate, that would occur in biological environments when the R132H variant is present. The technique enables time-resolved, in situ measurements of the kinetics of binding and dissociation of inhibitory drugs.

Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.

Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP + /NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP + reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo—the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 10 2 to 10 3 -fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the “gain of function” reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.

Isocitrate dehydrogenase (IDH) 1/2 gain-of-function variants catalyze the production of the oncometabolite 2hydroxyglutarate and are validated targets for leukemia treatment. We report binding and inhibition studies on 13 IDH1/2 variant inhibitors, including clinical candidates and drugs, with wild-type (wt) IDH1 and its cancer-associated variant, IDH1 R132H. Interestingly, all the variant inhibitors bind wt IDH1 despite not, or only weakly, inhibiting it. Selective inhibition of the IDH1 R132H variant over wt IDH1 does not principally relate to the affinities of the inhibitors for the resting forms of the enzymes. Rather, the independent binding of Mg 2+ and 2-oxoglutarate to the IDH1 variant makes the variant more susceptible to allosteric inhibition, compared to the tighter binding of the isocitrate−Mg 2+ complex substrate to wt IDH1. The results highlight that binding affinity need not correlate with inhibition selectivity and have implications for interpretation of inhibitor screening results with IDH and related enzymes using turnover versus binding assays.

Formaldehyde (HCHO) is a simple and highly reactive human metabolite but its biochemistry is poorly defined. A limiting factor in HCHO research is lack of validated quantification methods for HCHO relevant to biological samples. We describe spectroscopic studies on a reported fluorescence-based HCHO detection method involving its reaction with ampicillin. The results validate the structure and fluorescence properties of the HCHO-ampicillin reaction product. However, the same adduct is observed after reaction of ampicillin with glyoxylate. Related fluorophores were formed with other biologically relevant carbonyl compounds. Overall, our studies suggest the ampicillin method is not reliable for selective detection and quantification of HCHO in biological samples.