Cavity-Guided Chemiluminescent Probes for Drug-Metabolizing Enzymes: Rational Design , Activity Imaging, and Translational Applications

Drug-metabolizing enzymes (DMEs), which are crucial for xenobiotic biotransformation, profoundly influence therapeutic efficacy, drug safety, and physiological homeostasis. However, conventional analytical methods such as LC-MS/MS and fluorescence-based assays face limitations in background interference, spatiotemporal resolution, and in vivo imaging, while the structurally diverse catalytic cavities and overlapping substrate preferences of DMEs complicate isoform-specific activity assessment. In this context, chemiluminescence (CL) probes, particularly programmable Schaap-type 1,2-dioxetanes that operate without external light excitation, offer near-zero background and high sensitivity for monitoring DME activity. Here, cavity-guided CL probes are defined as probes whose recognition triggers, steric dimensions, electronic properties, and linker architectures are selected or optimized according to the substrate-binding and catalytic features of a target enzyme’s catalytic cavity. This review summarizes reported DME-targeted CL probes and discusses how active-pocket architecture and substrate-recognition rules can guide probe design for oxidases, hydrolases, and reductases. Representative applications in drug-metabolism analysis, drug–drug interaction assessment, disease-related activity imaging, and translational studies are highlighted. On this basis, we propose a design framework that matches active-pocket properties, substrate archetypes, and CL triggering mechanisms. A cross-case comparison now summarizes detection limits, linear ranges, emission wavelengths, signal enhancement, response characteristics, selectivity, precision, or reproducibility where reported, and biological applications; unreported parameters are explicitly identified as evidence gaps.