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.