Hypericin and hyperforin from St. John's Wort are potent intestinal UGT inhibitors with distinct binding modes: mechanistic and quantitative prediction of clinical drug-drug interaction risk.

St. John's Wort (SJW; Hypericum perforatum) is widely used for mild-to-moderate depression and is known to induce certain cytochrome P450 enzymes (notably CYP3A4, with reported effects on CYP2C9 and CYP2C19) and intestinal P-glycoprotein, but its effects on UDP-glucuronosyltransferases (UGTs), the principal phase-II drug-metabolizing enzymes, remain largely uncharacterized. Using recombinant human UGTs with 4-methylumbelliferone as the probe substrate, we characterized inhibition by the two major SJW constituents, hypericin and hyperforin, across eleven isoforms, and combined AlphaFold2-based molecular docking, molecular dynamics, and MM/PBSA analyses with FDA 2020 static-model in vitro-in vivo extrapolation (IVIVE) to predict drug-drug interaction (DDI) risk. Both compounds were broad-spectrum UGT inhibitors, with hyperforin the most potent (UGT1A1 K i = 1.46 μM; UGT2B7 K i = 6.70 μM). On the clinically critical UGT1A1, hyperforin and hypericin occupied the aglycone pocket through two structurally distinct binding modes that both produced noncompetitive kinetics. Under a complete-dissolution assumption, IVIVE flagged hyperforin as a high-risk intestinal UGT inhibitor (R 1,gut = 69.49 for UGT1A1 and 15.93 for UGT2B7, far above the FDA threshold of 11); however, incorporating hyperforin's poor (∼2%) dissolution and acid instability lowered the corrected R 1,gut to ∼2.0-2.9 (UGT1A1) and ∼1.2-1.4 (UGT2B7), indicating a potential rather than definitive intestinal interaction whose magnitude depends on the SJW formulation. These findings provide mechanistic and quantitative support for reported clinical SJW-drug interactions and highlight intestinal glucuronidation as a previously underappreciated SJW-mediated DDI pathway.