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.
Keywords: activity-based sensing; fluorescence; molecular imaging; probe development; rational design.
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