Unraveling the Biosynthesis and Anti-Inflammatory Bioactivity of Tripdiolide from Tripterygium wilfordii

Abstract Triptolide (TP), a promising anti-inflammatory drug candidate, is limited by its severe multiorgan toxicity. To reduce toxicity while maintaining pharmacological efficacy, various modified derivatives have been employed. Tripdiolide (TPD), a natural product discovered alongside TP, holds the potential to be more potent and safer than TP. Here, we discover tandemly duplicated CYP71BEs in the chromosomes of Tripterygium wilfordii and reveal that CYP71BE272 catalyzes the C-2 hydroxylation of TP to produce TPD, thereby elucidating the final biosynthetic step of TPD. Pharmacokinetic characterization shows that TPD is rapidly cleared from plasma and preferentially distributes to bone and joint tissues. This specific C-2 modification enables TPD to maintain TP’s anti-inflammatory activity both in a collagen-induced arthritis mouse model and in vitro while exhibiting a significantly improved safety profile and reduced systemic and reproductive toxicity in both sexes, suggesting that TPD has the potential to replace TP for further drug development. By elucidating the catalytic mechanism of CYP71BE272, we identify a mutant CYP71BE272A370G/I443M, which exhibits an improved conversion rate and catalytic efficiency, and ultimately achieve the microbial production of TPD. Our study not only addresses the supply constraints of TPD and comprehensively evaluates its pharmacological efficacy in RA but also provides a viable research model for developing other highly effective new drugs.