Fraxetin Inhibits UGT1A1 and UGT1A9 Activities In Vitro: Inhibition Kinetics, Molecular Dynamics Simulation, and Prediction of Herb-Drug Interaction Risk.

Background/Objectives: Fraxetin (7,8-dihydroxy-6-methoxycoumarin), a coumarin constituent of Cortex Fraxini (Qinpi) used in traditional Chinese medicine, is metabolised mainly by UGT1A9, but its potential to inhibit UGT enzymes and cause herb-drug interactions (HDIs) is largely unstudied. Methods: Fraxetin and four related coumarins were screened against 11 recombinant human UGTs; isoforms inhibited ≥80% underwent full kinetic analysis with 4-methylumbelliferone as probe. Binding was examined by molecular docking on AlphaFold structures with PLIP, triplicate 100 ns molecular dynamics, and MM/GBSA and MM/PBSA free-energy calculations, and interaction risk by FDA 2020 in vitro-in vivo extrapolation (IVIVE). Results: Fraxetin alone inhibited both UGT1A1 and UGT1A9 by >80% and was characterised in detail, acting as a mainly competitive mixed-type inhibitor (UGT1A1 IC50 15.99 μM, Ki 8.32 μM; UGT1A9 IC50 8.44 μM, Ki 5.90 μM). A structure-activity comparison identified a dual-element pharmacophore comprising the C-6 methoxy group and the 7,8-dihydroxycoumarin aglycone. MM/GBSA favoured UGT1A9 over UGT1A1 (ΔΔG = -4.06 kcal/mol, p = 0.005), concordant with the kinetic ranking. IVIVE predicted a borderline systemic signal (R1 > 1.02) but an intestinal R1,gut approximately five- to seven-fold above the high-risk threshold of 11 after capping the luminal concentration at fraxetin aqueous solubility. Conclusions: This is the first characterisation of fraxetin as a moderate-potency inhibitor of UGT1A1 and UGT1A9 and points to a previously under-recognised herb-drug interaction risk concentrated in the intestinal lumen rather than systemically; the finding constitutes an interaction signal requiring clinical confirmation rather than an established risk.