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

BACKGROUND: 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. RESULTS: Using several established biochemical and biophysical approaches, we found that the radiofluorinated inhibitor ivosidenib ([¹⁸F]AG-120) binds specifically to both IDH1 and IDH1R132H at nanomolar radioligand concentrations. However, the investigated approaches yielded method-dependent apparent affinity estimates. CONCLUSIONS: 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.