Rational design of a novel specific fluorescent substrate for monitoring hUGT1A4 activity and its application in identification of selective inhibitors

Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are a family of enzymes with highly similar amino acid sequences, making it challenging to distinguish between their roles. Developing selective probes and inhibitors is essential for understanding the unique functions of each isoform. In this study, we synthesized four novel naphthalimide-based fluorescent probes bearing nitrogen-containing substituents at the 4-position and identified N -(n-butyl)-4-(4-methylpiperazin-1-yl)-1,8-naphthalimide (BAD3) as a highly selective and sensitive substrate for UDP glucuronosyltransferase 1A4 (UGT1A4). Using BAD3, we established an inhibitor screening platform and identified ursolic acid (T7) as a promising lead compound from a natural product library. Structure-activity relationship (SAR) studies revealed that esterification at the 3-hydroxyl group significantly enhanced inhibitory activity, yielding two potent inhibitors, T25 and T26, while modifications at the 28-carboxyl group reduced activity. Further characterization confirmed T25 (inhibition constant ( K i ) = 0.64 μM) and T26 ( K i = 0.61 μM) as selective and competitive UGT1A4 inhibitors. Molecular docking revealed that the 28-carboxyl group plays a crucial role by forming a salt bridge with Arg258 in the UGT1A4 active site. In vivo studies demonstrated that T25 significantly altered the pharmacokinetic profile of BAD3, confirming its inhibitory effect on UGT1A4 in animals. Together, BAD3 and the selective inhibitors T25/T26 serve as valuable molecular tools for studying the physiological and pharmacological roles of UGT1A4. • Discovery of the first specific fluorescent substrate for UGT1A4 with 400-fold selectivity over other UGT isoforms. • Development of a new UGT1A4 inhibition screening system using the self-developed fluorescent substrate. • Identification of pentacyclic triterpenoids as the most promising natural inhibitors of UGT1A4. • Demonstration of the critical role of the 28-carboxylic acid group in ursolic acid derivatives for UGT1A4 inhibition. • Confirmation that 3-Acetylursolic acid and 3-Butyroylursolic acid act as competitive inhibitors through inhibition kinetics.