Spermidine-Based Activity Probe for ALDH1A1 in Cells

We report a spermidine analog that engages aldehyde dehydrogenase 1A1 (ALDH1A1) in cellulo. Chemoproteomic profiling demonstrated high selectivity, identifying ALDH1A1 as the main detectable cellular target. The analog undergoes intracellular bioactivation and then irreversibly modifies the enzyme within its catalytic/substrate-binding region, enabling activity-dependent labeling. We used this activity-dependent chemical probe to determine the minimal effective concentration of the ALDH1A1-selective inhibitor NCT-501 in two cancer cell lines, A549 and MDA-MB-468, in which this or structurally related inhibitors had been tested previously. At the optimized concentration, NCT-501 attenuated ALDH1A1 activity without changing protein abundance. Under these selective, activity-targeted conditions, phenotypes commonly attributed to ALDH1A1 inhibition, previously observed with pan-ALDH inhibitors or high-dose regimens, were not detected. These findings indicate that loss of ALDH1A1 catalytic activity alone is insufficient to produce the widely ascribed cellular responses, pointing instead to functional redundancy among ALDH isoforms and/or noncatalytic contributions of ALDH1A1. Our spermidine-based probe provides an activity-aware readout of target engagement using widely adopted methods (fluorescence microscopy, in-gel fluorescence, western blotting) and offers a practical framework for dissecting ALDH1A1 biology in living cells.