Rational Design of an Isoform-Specific Fluorogenic Probe for Human UGT2B7: Enabling Functional Analysis and Inhibitor...

UDP-glucuronosyltransferase 2B7 (UGT2B7), a pivotal drug-metabolizing enzyme, plays a critical role in conjugating and detoxifying diverse nonpolar xenobiotics. However, few methods exist for conveniently monitoring UGT2B7 function in biological systems. Herein, we developed an integrated strategy, combining structure-based virtual screening with biochemical validation, to create the first UGT2B7-activatable fluorogenic probe that enables in situ detection of UGT2B7 activity. A series of 1,8-naphthalimide derivatives were designed and synthesized based on the PET mechanism. Following the preliminary screening, UPro^2B7 demonstrates outstanding isoform specificity and sensitivity. It enables highly effective functional imaging of endogenous UGT2B7, demonstrating specific endoplasmic reticulum (ER) colocalization and high spatial resolution. Critically, UPro^2B7 serves as a superior alternative to conventional drug substrates for screening of UGT2B7 inhibitors. Utilizing this probe, we identify Licoagrochalcone A (Lic A) and Glycycoumarin (Gly) as potent UGT2B7 inhibitors, validated in live cells, rat tissues, and zebrafish. Collectively, this work demonstrates an integrated strategy for rationally engineering isoform-specific, UGT2B7-activatable fluorogenic substrate. UPro^2B7 serves as a practical and reliable tool for in situ imaging and inhibitor assessment of UGT2B7 activity.