Computational Design of CYP102A1 Variants for Biosynthesis of a Next-Generation Antiplatelet Drug DT-678

Clopidogrel is a widely used antiplatelet prodrug to treat acute coronary syndromes. However, its clinical efficacy is hampered by ineffective bioactivation to produce the pharmacologically active metabolite (AM), leading to variability in the antiplatelet response among different ethnic groups. To overcome the shortcomings of clopidogrel, DT-678 was developed by conjugating AM to 3-nitropyridine-2-thiol via a mixed disulfide bond. It has been challenging to produce the conjugate in a high yield by chemical synthesis. Here, we report the first de novo biosynthesis of DT-678 using engineered CYP102A1 variants. We applied structure-based computational design using UniDesign to generate three variants (UD4, UD5, and UD6) that enhanced the catalytic activity and selectivity toward DT-678 synthesis. Among them, UD6 demonstrated the highest total turnover number and DT-678-specific productivity under the optimized conditions. Mechanistic analysis revealed that rapid enzyme inactivation, driven by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, limited the overall yield. Remarkably, we found that ascorbic acid significantly protected CYP102A1 variants from inactivation and, hence, increased production yield. This work establishes a scalable enzymatic strategy for DT-678 biosynthesis and highlights the importance of combining protein engineering with redox control to overcome limitations in CYP-catalyzed reactions.