Exploiting Electrode Nanoconfinement to Investigate the Catalytic Properties of Isocitrate Dehydrogenase (IDH1) and a Cancer-Associated Variant

Herold, Ryan A.; Reinbold, Raphael; Megarity, Clare F.; Abboud, Martine I.; Schofield, Christopher J.; Armstrong, Fräser A.

doi:10.1021/acs.jpclett.1c01517

“…To investigate the mechanism of IDH1 S280F mediated inhibitor resistance, we used established protocols 17 , 18 to produce highly purified forms of recombinant IDH1 R132C/S280F and R132H/S280F and, for comparison, IDH1 R132C, IDH1 R132H, IDH1 wildtype (wt), IDH1 S280F (without IDH1 R132C or R132H) and IDH1 R132H/Q277E (Supplementary Fig. 1a ).…”

Section: Resultsmentioning

confidence: 99%

“…To further investigate the mechanistic consequences of the S280F substitution, we studied the IDH1 variants using cyclic voltammetry (Supplementary Fig. 3g–l ), which can measure rates in each NADP(H) cycling direction and which can distinguish the conversion of isocitrate to 2-OG from the reduction of 2-OG to 2-HG 17 . The results imply that R132C and R132C/S280F catalyse the conversion of isocitrate to 2-OG with similar efficiency (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant protein production was conducted as described 17 , 18 . In brief, the recombinant proteins were produced in BL21(DE3)plysS E.coli cells with 1 mM Isopropyl β- d-1-thiogalactopyranoside (IPTG) with a post-induction temperature of 20 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Electrochemical experiments were performed in an anaerobic glovebox (Glove Box Technology) containing a nitrogen atmosphere (O 2 < 1 ppm). The electrochemical apparatus (a glass electrochemical cell and rotating pyrolytic graphite edge (PGE) electrodes) were as previously reported 17 . An Autolab PGSTAT 10 potentiostat using Nova software was used to conduct electrochemical experiments.…”

Section: Methodsmentioning

confidence: 99%

“…An Autolab PGSTAT 10 potentiostat using Nova software was used to conduct electrochemical experiments. Electrode potentials ( E ) were measured against a saturated calomel electrode (SCE) and converted to the standard hydrogen electrode (SHE) using a temperature-dependent potential conversion equation (at 25 °C the conversion is: E SHE = E SCE + 0.241 V) 17 . The counter electrode was a platinum wire.…”

Section: Methodsmentioning

confidence: 99%

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Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors

Reinbold

¹

,

Hvinden

²

,

Rabe

³

et al. 2022

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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.

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Electrochemical Nanoreactor Provides a Comprehensive View of Isocitrate Dehydrogenase Cancer‐drug Kinetics

Herold,

Schofield,

Armstrong

2023

Angewandte Chemie

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0

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The ability to control enzyme cascades entrapped in a nanoporous electrode material (the “Electrochemical Leaf”, e‐Leaf) has been exploited to gain detailed kinetic insight into the mechanism of an anti‐cancer drug. Ivosidenib, used to treat acute myeloid leukemia, acts on a common cancer‐linked variant of isocitrate dehydrogenase 1 (IDH1 R132H) inhibiting its “gain‐of‐function” activity—the undesired reduction of 2‐oxoglutarate (2OG) to the oncometabolite 2‐hydroxyglutarate (2HG). The e‐Leaf quantifies the kinetics of IDH1 R132H inhibition across a wide and continuous range of conditions, efficiently revealing factors underlying the inhibitor residence time. Selective inhibition of IDH1 R132H by Ivosidenib and another inhibitor, Novartis 224, is readily resolved as a two‐stage process whereby initial rapid non‐inhibitory binding is followed by a slower step to give the inhibitory complex. These kinetic features are likely present in other allosteric inhibitors of IDH1/2. Such details, essential for understanding inhibition mechanisms, are not readily resolved in conventional steady‐state kinetics or by techniques that rely only on measuring binding. Extending the new method and analytical framework presented here to other enzyme systems will be straightforward and should rapidly reveal insight that is difficult or often impossible to obtain using other methods.

show abstract

Electrochemical Nanoreactor Provides a Comprehensive View of Isocitrate Dehydrogenase Cancer‐drug Kinetics

Herold,

Schofield,

Armstrong

2023

Angew Chem Int Ed

Self Cite

3

0

View full text Add to dashboard Cite

The ability to control enzyme cascades entrapped in a nanoporous electrode material (the “Electrochemical Leaf”, e‐Leaf) has been exploited to gain detailed kinetic insight into the mechanism of an anti‐cancer drug. Ivosidenib, used to treat acute myeloid leukemia, acts on a common cancer‐linked variant of isocitrate dehydrogenase 1 (IDH1 R132H) inhibiting its “gain‐of‐function” activity—the undesired reduction of 2‐oxoglutarate (2OG) to the oncometabolite 2‐hydroxyglutarate (2HG). The e‐Leaf quantifies the kinetics of IDH1 R132H inhibition across a wide and continuous range of conditions, efficiently revealing factors underlying the inhibitor residence time. Selective inhibition of IDH1 R132H by Ivosidenib and another inhibitor, Novartis 224, is readily resolved as a two‐stage process whereby initial rapid non‐inhibitory binding is followed by a slower step to give the inhibitory complex. These kinetic features are likely present in other allosteric inhibitors of IDH1/2. Such details, essential for understanding inhibition mechanisms, are not readily resolved in conventional steady‐state kinetics or by techniques that rely only on measuring binding. Extending the new method and analytical framework presented here to other enzyme systems will be straightforward and should rapidly reveal insight that is difficult or often impossible to obtain using other methods.

show abstract

Exploiting Electrode Nanoconfinement to Investigate the Catalytic Properties of Isocitrate Dehydrogenase (IDH1) and a Cancer-Associated Variant

Cited by 12 publications

References 35 publications

Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors

Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors

Electrochemical Nanoreactor Provides a Comprehensive View of Isocitrate Dehydrogenase Cancer‐drug Kinetics

Electrochemical Nanoreactor Provides a Comprehensive View of Isocitrate Dehydrogenase Cancer‐drug Kinetics

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