Methods for assessing binding of a small-molecule radioligand to soluble enzyme isocitrate dehydrogenase 1 variants

Isocitrate dehydrogenase 1 (IDH1) is a cytosolic NADP⁺-dependent enzyme involved in cellular redox homeostasis and 2-oxoglutarate (2-OG) metabolism. Glioma-associated mutations in IDH1 confer a neomorphic enzymatic activity, resulting in the production and accumulation of the oncometabolite D-2-hydroxyglutarate (2-HG), which contributes to metabolic and epigenetic alterations in IDH-mutant tumors. Small-molecule inhibitors of mutant IDH1 have emerged as promising therapeutic agents. In parallel, radiolabeled derivatives of these inhibitors are being investigated as potential positron emission tomography (PET) tracers for non-invasive imaging of mutant IDH1. Characterizing the binding of small-molecule radioligands to soluble intracellular proteins such as IDH1, however, presents specific methodological challenges. Appropriate experimental approaches are therefore needed to characterize radioligand binding to IDH1 enzymes.

Using several established biochemical and biophysical approaches, we found that the radiofluorinated inhibitor ivosidenib ([¹⁸F]AG-120) binds specifically to both IDH1 and IDH1^R132H at nanomolar radioligand concentrations. However, the investigated approaches yielded method-dependent apparent affinity estimates.

The findings support the use of radioligand-based approaches for characterizing soluble intracellular protein targets, while highlighting the need for assay-specific optimization and careful interpretation of quantitative binding parameters.