PROTAC Technology As a Novel Tool to Identify the Target of Lathyrane Diterpenoids

Active natural products are an important source of drug discovery due to their unique biocompatibility, novel-structural framework and extensive pharmacological activities. The target identification of natural products is the key to thoroughly understanding their mechanism of action and guiding their subsequent structure optimization. Lathyrane diterpenoids isolated from the seeds of Euphorbia lathyris in our laboratory have been proved to possess good anti-inflammatory activities in lipopolysaccharide (LPS)-induced macrophages, but their precise target and mechanism of action remain unclear. Herein, we employed PROTAC technology combined with quantitative proteomic analysis to identify the potential targets of lathyrane diterpenoids in macrophages. ZCY-PROTAC, synthesized based on Lathyrol, the core scaffold structure of the most active natural compound (2S,3S,4S,5R,9S,11R,15R)-15-acetoxy-3- cinnamoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene, ZCY-001), intensively degraded a target protein MAFF in mouse and human cells. MST, CETSA and DARTS assays confirmed the direct binding of MAFF with Lathyrol or ZCY020, a natural product sharing the same core scaffold structure. Further mechanism studies showed that ZCY020 was capable of inhibiting the formation of a MAFF homodimer, promoting MAFF-Nrf2 heterodimerization, and thus regulating the transcription and expression of downstream protein HO-1, thereby exerting antioxidant, anti-inflammatory activity and promoting protective mitophagy in LPS-induced inflammation in macrophages and acute lung injury in mice. Together, our research indicates that MAFF is a potential target of ZCY020 and other lathyrane diterpenoids, and that PROTAC technology can be an innovative approach and a useful supplement for target identification of natural products.