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Enzymes play central roles in cellular regulation, disease progression, and therapeutic response, making them ideal targets for diagnostic and therapeutic chemical probes. Among them, proteases and kinases are particularly valuable due to their dysregulation in cancer, immune disorders, and infectious diseases. In this Perspective, we highlight recent advances in chemical probe development aimed at these enzyme families, with a focus on imaging modalities and translational potential. Kinase-targeted probes are frequently derived from clinically approved inhibitors, facilitating their repurposing for diagnostic applications. In contrast, protease-responsive probes rely on detailed substrate specificity profiling, where the incorporation of unnatural amino acids serves as a key strategy for achieving selectivity and stability in biological systems. We explore the broad landscape of probe modalities, including activatable fluorescent substrates, PET tracers, and mass cytometry-compatible metal-tagged reagents, that collectively enable sensitive, real-time, and multiplexed analysis of enzyme function in cells and tissues. Notably, enzyme-activated theranostic systems are emerging that couple imaging with targeted drug release, expanding the functional scope of chemical probes beyond detection. We also discuss how artificial intelligence is beginning to support the design process, from structure prediction and binding affinity modeling to the generation of novel chemical scaffolds with favorable pharmacological properties. With several enzyme-targeted probes now in clinical trials or approved for human use, this field exemplifies the success of translational chemical biology and the potential of chemical tools to directly impact patient care.
Nguyen et al. (Wed,) studied this question.