The Mutli-target ?-Helical Mimetics And Their Target Identification

Apoptotic pathways are regulated by a multi-level,redundancy,connectivity and compensatory molecular networks of protein-protein interactions(PPIs).The p53/MDM2 interaction and Bcl-2 family interactions are both central nodes in apoptosis,and they both feature a-helical segments at their interfaces.In these a-helices,a small subset of the residues(hotspots)contributes most of the free energy of binding.As such,a-helical mimetics that reproduce the arrangement of the hotspots in these a-helices can serve as not only therapeutic agents but also research tools to understand PPIs.Until now,a-helical mimetics can only non-covalently inhibit single target or targets in one class.However,advances in systems biology suggest that complex diseases like cancer may not be effectively treatable by interventions at single nodes.Then,non-covalent PPI inhibitors have much lower binding affinity than kinases inhibitors,requires improved photo-crosslinkers for establishing activity-based probes(ABPs)to enable proteome-wide profiling of unknown Bcl-2-like proteins under native cellular settings.Finally,a-helical mimetics cannot targeting protein destruction through recruitment to the cellular quality control machinery.So there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition,which may bring potential off-target side effects.These three limitations significantly limit the utility of the ?-helical mimetic as therapeutic agents or probes to understand PPIs.As such,developing PPIs multi-target drugs,ABPs for PPI targets and dual-functional inhibitors degrading PPI targets are key issues of this field.In this study,we compared the crystallographic results of the p53/MDM2,Bim/Mcl-1 and Bim/Bcl-2 complexes and then constructed a novel semi-rigid pyramid-like scaffold B,which could adopt the different dihedral angles of the hotspots of p53 and BH3-only peptides.Based on this universal scaffold B,a series of substituent groups were installed to capture the hotspots of both p53TAD and BimBH3 and balance the differences of the bulks between them.The Mcl-1,Bcl-2 and MDM2 binding features of these compounds were tested by FP,ITC,and NMR spectroscopy.A compound B5 that directly binds to Mcl-1,Bcl-2,and MDM2 with balanced sub-?M affinities was obtained.1H-15N HSQC,FP,ITC,molecular docking and cellular experiments confirmed B5 could effectively disrupt Bax/Bcl-2,Bak/Mcl-1,and p53/MDM2 in cancer cells and induce cell cycle arrest in addition to apoptosis through Bcl-2/MDM2 dual inhibition simultaneously.Then,by means of limited proteolysis assay,three-dimensional NMR,X-ray crystallography and alanine mutations,a dynamic region at the Q221R222N223 motif of Mcl-1 is identified as a "conformational switch" which controls Mcl-1 ubiquitination.A dual-functional Mcl-1 inhibitor,E5,which not only interferes with the pro-apoptotic partners,but also facilitates Mcl-1 ubiquitination in living cells,is described.3D-NMR experiment showed that E5 forms hydrogen bond with His224 to drive a helical QRN conformation.As a result,E5 manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other Mcl-1 inhibitors.Finally,a "branched" photo-crosslinker L1 was designed to recapitulate non-covalent protein-small molecule interactions in situ.By incorporating L1 with a fluorescent Bcl-2 inhibitor C1,the resulting trifunctional probe C2 can recapitulate,enrich and visualize Bcl-2 family proteins in vitro and in situ,showing improved outcomes over previous probes with the"minimalist" linker in recapitulating PPI targets.Tubulin was validated as a cellular target of the Bcl-2-labeling probe C2,and the BH3-containing property of tubulin was preliminarily illustrated,as some well-known Bcl-2 inhibitors can block the labelling of tubulin by C2.