A Fluorogenic Probe for Screening and Kinetic Evaluation of Aldehyde Dehydrogenase 1A1 (ALDH1A1) Inhibitors

A fluorogenic competitive enzyme inhibitor emitting in the visible range enables enzyme assays to screen new potential inhibitors of the target enzyme (ALDH1A1) by displacement method, providing kinetic parameters and more. We intro-duce the first fluorogenic ALDH1A1 inhibitor synthesized by combining a fluorescent napthalimide derivative with an isatin motif. Isatin derivatives are known to bind strongly to the ALDH1A1 substrate binding site, allowing a broad win-dow for future modifications. The presented compound competes for the enzyme-substrate binding site, allowing the evaluation of new competitive inhibitors by monitoring its displacement. Its strong binding affinity (KD = 127 ± 16 nM) and inhibitory concentration (IC50 = 370 ± 8 nM), along with its visible-range fluorescence, make it compatible with standard fluorimeters, supporting assay development and precise measurements. Furthermore, ALDH1A1's ubiquitous presence in cells and the compound's uniform distribution upon cellular entry makes it a valuable tool for evaluating the impact of ALDH1A1 inhibition in cellular systems. Our findings with pancreatic ductal adenocarcinoma (MIA PaCa-2) cells having a dominant ALDH1A1 expression indicate that ALDH1A1 inhibition may not necessarily diminish cancer stemness, contrary to common findings in various cancer types.