Precision Design of Fluorogenic Probes via Orthogonal Tuning of Binding and Photophysics for Isoform-Selective ALDH2 Imaging.
TL;DR
A5 is developed, a fluorogenic substrate for aldehyde dehydrogenase 2 (ALDH2) that exhibits high isoform selectivity and a >240-fold signal enhancement over the standard NADH assay, and enables quantitative imaging of ALDH2 activity across multiple biological scales.
Abstract
Fluorogenic probes that report enzyme activity are essential for studying biological functions. However, designing them for targets with low catalytic turnover and narrow substrate specificity remains a significant challenge. Here, we present a precision design framework that separates the requirements for sensitivity and selectivity by integrating molecular docking, quantum chemical modeling of fluorogenic mechanisms, and targeted fine-tuning of the probe structures. As a proof of concept, we developed A5, a fluorogenic substrate for aldehyde dehydrogenase 2 (ALDH2) that exhibits high isoform selectivity and a >240-fold signal enhancement over the standard NADH assay. A5 enables quantitative imaging of ALDH2 activity across multiple biological scales─in blood samples, live cells, and intact mouse brains─and supports the identification of small-molecule activators with therapeutic potential in an Alzheimer's disease model. This work establishes a modular strategy for creating activity-based probes tailored to challenging enzymatic targets, with broad applications in precision imaging, drug discovery, and mechanistic biochemistry.
