Target Identification Of Lathyrane Diterpenoids Based On PTOTAC Technology And Study On Its An-ti-inflammatory Mechanism Through Targeting MAFF

Active natural products,including active components of Traditional Chinese Medicine(TCM),are an important source of drug discovery due to their unique biocompatibility,novelstructural 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.However,the biological activity of natural products comes not only from the regulation of a single protein or pathway,but also through the interaction with a large number of cellular components.The complexity of this mechanism brings great difficulties to the accurate target identification of natural products.PROtein TArgeting Chimera(PROTAC)technology can degrade target proteins through the the proteasome.Relying on chemical synthesis,the target protein(protein of interest,POI)ligand and the ubiquitin ligase E3 ligand are connected through a linker to form a bifunctional molecular compound that can spontaneously mediate the degradation of POI,which has broad research and application.Since PROTAC does not need strong binding affinity to degrade target proteins,we speculate that this technology can be suitable for identifying the exact targets of active natural products.Lathyrane diterpenoids are the main active components of the seeds of Euphorbia lathyris.They have a wide range of biological activities,including anti-inflammatory,anti-tumor,antitumor multidrug resistance and so on.In recent years,there are numbers of researches on the anti-inflammatory activities of lathyrane diterpenoids.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.Among them,(2S,3S,4S,5R,9S,11R,15R)-15-acetoxy-3-cinnamoyloxy-5-hydroxyl-14-oxolathyra-6(17),12Ediene(ZCY-001)demonstrated the strongest activity.However,their precise target and mechanism of action remain unclear.In this study,PROTAC technology combined with comparative quantitative proteomic was applied to determine the targets of lathyrane diterpenoids.Firstly,we synthesized a PROTAC bifunctional molecule with anti-inflammatory activity,ZCY-PROTAC,based on the core scaffold structure of the most active component ZCY-001,Lathyrol,which was used as the target protein(POI)ligand and thalidomide as the ligand of E3 ubiquitin ligase CRBN.Comparative quantitative proteomic was carried out on the cell lysate treated by ZCY-PROTAC,and the degradation mechanism of the candidate target proteins was verified.It was found that the target protein MAFF in mouse and human cells could be successfully degraded by ZCY-PROTAC.The in vitro target verification methods MST,SPR,CETSA and DARTS were confirmed that MAFF could bind directly both with the lathyrane diterpenoids lathyrol and ZCY020.In RAW264.7 cells and HEK293T cells,further mechanism studies showed that ZCY020 could inhibit the formation of MAFF homodimer and promote MAFF-Nrf2 heterodimerization.In LPS induced RAW264.7 cells,through targeting MAFF protein,ZCY020 activated MAFFNrf2/ARE pathway,regulated the transcription and expression of downstream protein HO-1,inhibited the activation of NF-κB pathway and promoted mitophagy to maintain mitochondrial quality control,thus exrting anti-inflammatory and antioxidant activities.Meanwhile,ZCY020 revealed significant therapeutic effects on LPS induced acute lung injury(ALI),IMQ induced psoriasis and collagen induced arthritis(CIA)mice,and the mechanism was also involved with the activation of MAFF-Nrf2/ARE pathway,the inhibition of NF-κB and the promotion of mitophagy.In summary,we creatively employed PROTAC technology to explore the targets of active components,by using PROTAC as the probe,combined with comparative quantitative proteomics to observe the changes of proteome before and after,and then confirmed that MAFF is a key target protein of lathyrane diterpenoids.Based on this evidence,we verified that in LPSinduced RAW264.7 macrophages,lathyrane diterpenoid ZCY020 activates the MAFF-Nrf2 pathway via targeting MAFF,and then decreases ROS production,inhibits NF-κB activation,and maintains mitochondrial quality to exert anti-inflammatory effects and achieve therapeutic effects on ALI,psoriasis and RA mice.These above results prove that PROTAC technology combined with comparative quantitative proteomics,named as(TCM)Targeted Degradomics,can be a novel and valuable supplementary approach for the target identification of natural products and other drugs.