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

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, UPro2B7 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, UPro2B7 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. UPro2B7 serves as a practical and reliable tool for in situ imaging and inhibitor assessment of UGT2B7 activity.