Study Of The Pan-Bcl-2 Family Protein Drug Target By Small Molecules

Bcl-2 family proteins are the "switch" of intrinsic apoptosis pathways.The canonical Bcl-2 family proteins include anti-apoptotic Bcl-2-like proteins,which mainly contain Bcl-2,Mcl-1,etc;pro-apoptotic members Bax,Bak,and BH3-only proteins such as Bim,etc.The members of this family form a multi-level,redundancy,connectivity and compensatory molecular networks to regulate the balance of cell life and death by protein-protein interactions(PPIs)formed through BH3 domain of anti-apoptotic members and that of proapoptotic members.More and more non-canonical Bcl-2 family proteins containing BH3 domain have been discovered,which together with canonical Bcl-2 family proteins constitute pan-Bcl-2 family.This makes the composition and function of this molecular network more complex than before,which brings challenges for the discovery and confirmation of targeted anticancer drugs,as well as the development and rational application of targeted inhibitor anti cancer drugs.In this thesis,the different contributions in apoptosis network of Bcl-2 and Mcl-1 of the canonical Bcl-2 family through mutual compensatory were studied using small chemical molecules as tools by various experimental methods(cell biology,molecular biology,chemical biology,etc.)in conjunction with systematic analytical approach such as mathematical modeling.Furthermore,new non-canonical Bcl-2-like proteins were discovered by chemical proteomics approach and their molecular mechanisms as tumor targets were revealed through IPA(ingenuity pathway analysis)and other methods.We have finished the following work in the research fields of the mechanism study of combination drug target,discovery and confirmation of new drug target,and the development of new inhibitors of targeted anticancer drug candidates:Firstly,the contributions of anti-apoptotic Bcl-2 and Mcl-1 in the apoptotic network were quantitatively studied by mathematical modeling method,and were validated by experiments.The results showed that the combination ratio of specific Bcl-2 inhibitors and specific Mcl-1 inhibitors mainly depended on three factors,which were Bcl-2 protein expression level,Mcl-1 protein expression level,and the kinetic change of Mcl-1 protein.Two sets of equation guiding drug combination of Bcl-2 inhibitors and Mcl-1 inhibitors with different kinetic changes of Mcl-1 protein were obtained by fitting parameters to induce tumor cells apoptosis economically and efficiently.Secondly,A new pan-Bcl-2 family protein containing BH3 domain,Hsp70,was discovered by activity-based protein profiling(ABPP),as well as Bim was discovered as cochaperone to regulate Hsp70.The activity-based probes(ABPs)which were synthesized based on Bcl-2 family inhibitors coupled with chemical proteomics were used to ’fish’ new drug targets at the whole-proteome level and identify as Hsp70 by mass spectrometry.In vitro assay of affinity detection showed that Hsp70 formed PPIs with several canonical Bcl-2 family proteins through BH3 domain.The 1H-15N HSQC-TROSY NMR spectrum identified a new binding site of BimBH3 and Hsp70 NBD.Bim activated ATPase activity of Hsp70 through allosteric mechanism and positively regulated the chaperone function of Hsp70.Thirdly,specific inhibitors were used as a tool to prove that Hsp70/Bim dimer is the specific drug target in chronic myelogenous leukemia(CML)and BCR-ABL-independent TKIs resistant CML.First,we have screened the first Hsp70/Bim dimer inhibitor,namely Slg-2(methyl 3-((2-cyano-6-(cyclohexylthio)-1-oxo-1 H-phenalen-3-yl)amino)propanoate),from the existing Bcl-2 family inhibitors based on the structural characteristics of Hsp70 containing BH3 domain.Fluorescence polarization(FP),Isothermal titration calorimetry(ITC),Nuclear magnetic resonance spectroscopy(1H-15N HSQC-TROSY),Coimmunoprecipitation(co-IP)and other methods proved that Slg-2 is a specific Hsp70/Bim inhibitor in vitro and in cells.The affinity of Slg-2 to dissociate Hsp70/Bim PPI(Ki=0.2μM)was 10 times higher than that of Bcl-2/Bim(Ki=2.8μm).It has been proved that Hsp70/Bim dimer is the specific drug target in CML and BCR-ABL-independent TKIs resistance CML using Slg-2 as a tool and then the drug target significance and molecular mechanism of Hsp70/Bim dimer were revealed.An original anticancer prodrug with new chemical structure,new binding site,and new anticancer molecular mechanism was obtained.Then,gene transfection and RNA interference methods proved that BCR-ABL fusion protein expression promoted the formation of Hsp70/Bim dimer,and stabilized clients such as Raf-1,AKT,eIF4A,eIF4E,RPS16,etc.iTRAQ-based proteomics analysis,IP A and other methods revealed that Hsp70/Bim specifically regulated the eIF2 signaling pathway,the regulation of eIF4E and p70S6K signaling,and the mTOR signaling pathway in CML.In BCR-ABLindependent TKIs resistant CML cells,with the increasing of TKIs resistance index,the activation of BCR-ABL downstream kinase AKT and Raf-1 became more and more dependent on Hsp70/Bim and the apoptotic induction effect of Slg-2 increased step by step.The results of this thesis showed that different Bcl-2-like proteins of the canonical Bcl-2 family have different mechanisms of action(MoA)of drug targets.Furthermore,a new noncanonical Bcl-2 family member,Hsp70,was also discovered.As a Bcl-2-like protein,Hsp70 interacts with Bim and participates in the pan-Bcl-2 family PPIs network.Hsp70/Bim dimer has the significance of specific tumor target.