Advances in Activity‐Based Sensing Probes for Isoform‐Selective Imaging of Enzymatic Activity

Until recently, there were no generalizable methods for assessing the effects of post‐translational regulation on enzymatic activity. Activity‐based sensing (ABS) has emerged as a powerful approach for monitoring small‐molecule and enzyme activities within living systems. Initial examples of ABS were applied for measuring general enzymatic activity; however, a recent focus has been placed on increasing the selectivity to monitor a single enzyme or isoform. The highest degree of selectivity is required for differentiating between isoforms, where the targets display significant structural similarities as a result of a gene duplication or alternative splicing. This Minireview highlights key examples of small‐molecule isoform‐selective probes with a focus on the relevance of isoform differentiation, design strategies to achieve selectivity, and applications in basic biology or in the clinic. The development of isoform‐selective activity‐based probes enables the identification of enzyme activity within living systems. In this Minireview, key examples are highlighted and the relevance of isoform differentiation, design strategies, and applications are discussed.