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UDP-glucuronosyltransferase 1A10 (UGT1A10) is an important phase II human metabolic enzyme. However, few methods are available to facilely monitor the function of UGT1A10 in biological systems. Here we report the first sensitive and selective fluorescent “off–on” UGT1A10 probe substrate UPr6. Using a collaborative modification strategy, we introduced a rigid propyl morpholine group to the amide and a methoxy group next to 4′-phenoxy of the 4-phenyl-1,8-naphthalimide skeleton to obtain UPr6. Visualization methods were established to evaluate the function of UGT1A10 in living cells, rat tissues, and zebrafish. From a Chinese herbal medicine compound library, two natural products EUC and DAP from licorice were discovered as potent UGT1A10 inhibitors, which remarkably improved the sensitivity of CPT-11 against a resistant human cancer cell line. This work established a feasible strategy to rationally design isoform-specific UGT probe substrates.