Lead Compounds Discovery And Their Anti-tumor Mechanism Against Apoptosis-related Targets

Escape of apoptosis is one of the six hallmarks of tumor.Inducing apoptosis of tumor cell has been one of the main strategies of cancer therapy in the past decades.Tumor cell apoptosis is a sophisticated network regulated by numerous signaling pathway,proteins and intrinsic molecules.Researches started from apoptosis pathway related proteins,such as the inhibitor of apoptosis protein(IAP)family.Currently,several small molecules targeting IAP family proteins have entered phase I or phase II clinical trials.Orphan nuclear receptor is a key member in the regulation of apoptosis.Orphan nuclear receptor can be activated by specific endogenous ligand and then modulates the transcription of apoptosis genes.Specially,apoptosis signals promote the translocation of Nur77 from nucleus to mitochondria.Nur77 interacts and changes BCL-2 protein from anti-apoptosis to pro-apoptosis.Recently,reactive oxygen species(ROS)has been considered as a member of second messengers,which regulates massive proteins and cell signal pathways.Targeting ROS directly regulating proteins is a new strategy for tumor apoptosis regulation.Small molecules originated from natural products have various scaffolds and extensive biological activities including pro-apoptosis acvitity on tumor.This work integrated the above factors in tumor apoptosis regulation and then carried out three aspects of studies.For the first aspect of study,considering previously reported key mutations of cIAP1 dimerization,we revealed that the hydrogen bond network and electrostatic interaction between E447 and peripheral amino acid play a vital role in cIAP1 dimerization and degradation.Molecular dynamics suggested that D303 and autoubiquitination related T612 form hydrogen bond.We designed D303 A mutation of cIAP1 and validated the suggestion via D303 A mutant dimerization.To explore the ability of natural products in targeting cIAP1,we screened natural product compound library by cIAP1 specific protein thermal shift assay,which discovered Helenine(targeting cIAP1 kinetic dissociation is 163.5 nM)and 6-Shogaol(targeting cIAP1 kinetic dissociation is 359.8 nM).cIAP1,which is rarely modulated by natural products,is the most potent target of the two compounds.Cell based assays further proved that Helenine and 6-Shogaol are cIAP1 targeting natural products.Overall,in vitro and ex vivo assays indicated that Helenine and 6-Shogaol is cIAP1 targeting natural products,and they have potential for further structural modification.For the second aspect of study,comparative analysis of Nur77 inflammatory regulatory site and apoptotic regulatory site by molecular dynamics simulation revealed different effects on protein conformation.Meanwhile,we discovered novel natural product compounds of Nur77,demethoxycurcumin and baohuoside I.The affinity of demethoxycurcumin with Nur77 is 1.18 ?M,while the affinity of baohuoside I with Nur77 is 3.07 ?M.So far,Nur77 is the most potent target of demethoxycurcumin and baohuoside I reported.Cell based assays proved that these two natural products showed pro-apoptosis effect.For the third aspect of study,using covalent docking combined with enzymatic inhibition assay,we discovered a potent and selective peroxiredoxin 1(PRDX1)inhibitor,which was named P1-L02.PRDX1 is a significant enzyme in regulating reactive oxygen species(ROS).The half maximal inhibitory concentration of P1-L02 targeting PRDX1 enzymatic activity is 89.3 nM and P1-L02 showed modest selectivity of enzymatic activity inhibition among PRDX family.Through molecular dynamics simulation and crystallization conditions screening of mutant proteins,we obtained high-resolution crystal structure of PRDX1 and then successfully solved the complex crystal structure of P1-L02 with PRDX1.Cell based assays verified that the phenotype of P1-L02 is consistent with the biological function of PRDX1.Further experiments showed that P1-L02 induced apoptosis through disrupting the protein-protein interaction of PRDX1-ASK1.Currently,P1-L02 is the most potent PRDX1 inhibitor,which may serve as tool compound for further PRDX1 related mechanistic study.It also laid a foundation for the anti-tumor lead compound development targeting PRDX1.