Abstract
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.
Highlights
Human isocitrate dehydrogenase (IDH1) and its cancer-associated variant (IDH1 R132H) are rendered electroactive through coconfinement with a rapid NADP(H) recycling enzyme in nanopores formed within an indium tin oxide electrode
Genes encoding isocitrate dehydrogenases (IDH) are the most frequently mutated metabolic genes associated with cancer.[1,2]
IDH1 R132H is associated with >80% of grade II/III gliomas and is a common mutant allele associated with acute myeloid leukemia (AML).[3,9−11] To treat IDH1-associated cancers, small molecule drugs inhibiting IDH1 variants have been developed: Ivosidenib (AG-120) is FDA-approved to treat AML,[12] and other inhibitors are in clinical trials.[2,7,13]
Summary
Enzyme expression and purification, electrode fabrication, enzyme loading method, electrochemical quantification of FNR, electrochemical measurement details, live buffer exchange protocol, enzyme solution assays and IC50 measurement, data smoothing. Herold − Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom; orcid.org/ 0000-0001-7935-6269. Raphael Reinbold − Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom. Megarity − Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom. Abboud − Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom; Present Address: (M.I.A.) Department of Natural Sciences, Lebanese American University, Byblos/Beirut, Lebanon.; orcid.org/0000-0003-2141-5988.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call