| [Background and significance]Cell death plays a crucial role in the development and homeostasis maintenance in multicellular organism.Apoptosis is recognized as the major programmed cell death under physiological condition,while necrosis was traditionally thought to be an accidental cell death.Recently,necroptosis(programmed necrosis)was identified as a regulated form of necrosis that is regulated by receptor-interacting kinase(RIP),RIP3 and mixed lineage kinase domain-like protein(MLKL).Increasing evidences demonstrate that necroptosis was associated to many fatal diseases such as acute pancreatitis,ischemic cardio-cerebrovascular disease,neurodegeneration disease,atherosclerosis and tumor metastasis.Therefore,the discovery and administration of potent necroptosis inhibitors may provide a new therapeutic for necroptosis associated diseases.[Objective]Our project is aimed to discover novel small molecule inhibitors for necroptosis to illustrate their mechanisms underlying the inhibitory action and to explore their therapeutic potency in necroptosis associated diseases.[Methods]We utilized a HT-29 cell based screening assay to identify small molecules of ability to inhibit necroptosis from a small molecule compounds library.To improve the efficiency of the compounds derived from the screening assay,much effort on chemical modifications has been made.Consequently,a series of variants including a compound named PK68 which has a potent efficiency against necroptosis were generated.To test at which step of the necroptosis pathway the small molecules exerts its effect,we monitored the activation of RIP3 and MLKL by western blot analysis,checked the formation of amyloid-like structures which appear as punctate substances by immunofluorescence technique.Comprehensive analysis of its effect on multiple necroptosis pathways including TNF-α induced,HSV-1 triggered necroptosis,lead to a hypothesis that kinase activity of RIP1 or RIP3 may be the target of compound PK68.In-vitro non-radioactive luminescent kinase assay was performed to verify the hypothesis.As might have been expected,the kinase activity of RIP1 but not RIP3 was inhibited by PK68 dramatically in a dose dependent manmer.To exclude off-target effect of PK68,we checked its effect on RIP3 or MLKL oligomerization induced necroptosis system.We evaluated the effect of PK68 on multiple organs by histological analysis of tissue sections from mice administrated different doses of PK68 daily for one week.To investigate whether PK68 possesses therapeutic potency in necroptosis associated diseases,we tested its effect on TNF-α induced systemic inflammatory response and renal ischemia reperfusion injury in vivo.[Results](1)a small molecule PK6 is an inhibitor of TNF-α induced necroptosis identified from cell based screening assay;(2)chemical modifications and SAR(structure activity relation)result in a series of variants capable of inhibiting necroptosis,particularly PK68 with potent efficiency;(3)PK68 blocks the activation of RIP3 and MLKL;(4)PK68 prevents the formation of RIP1/RIP3 necrosome;(5)PK68 inhibits Toll like receptor mediated necroptosis;(6)PK68 represses HSV-1 induced necroptosis;(7)PK68 specifically inhibits he kinase activity of RIP1;(8)PK68 has no effect on RIP3 dimerization or MLKL oligomerization induced necroptosis;(9)PK68 has no effect on TNF-α induced NF-κB activation;(10)There is no damage on mice organs caused by PK68 administration at the indicated doses;(11)PK68 protects mice from TNF-α induced systemic inflammatory response;(12)PK68 attenuates ischemia reperfusion induced renal injury.[Conclusion]Taken all together,we identified the necroptosis inhibitor PK68 which possesses potent inhibition on RIP1 kinase activity.Therapeutic potency in necroptosis associated diseases is also observed in two different animal models respectively associated with acute inflammation and ischemia reperfusion injury.Our study suggests that PK68 could be a promising agent for the development of new anti-necroptosis therapy. |
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