Design,Synthesis And Biological Evaluation Of PROTACs Targeting Bcl-2 Family Proteins

Proteolysis Targeting Chimeras?PROTACs?comprise three components,the ligand of target protein,linker and ligand targeting E3 ligase.PROTACs recruit an E3 ligase to the target protein and utilizes ubiquitin-proteasome system to degrade oncogene proteins.Upon the target protein being degraded,PROTACs will be released and degrade target proteins again,exhibiting super-stoichiometry manner,solving the problems meeting by traditional occupancy-based strategies and offering a new pharmacological strategy.Protein-protein interactions?PPIs?are involved in most cellular processes including apoptosis.For example,Bcl-2 family proteins balance cell homeostasis and apoptosis by PPIs,and anti-apoptotic proteins,such as Mcl-1 and Bcl-2,are considered as“key nodes”for cancer treatment.However,as PPIs interfaces are flat,shallow,it is challenging to design inhibitors exhibiting great affinity toward Bcl-2 and Mcl-1.What's worse,as the structures of Bcl-2family proteins share great similarity,it is difficult to design selective Mcl-1 or Bcl-2 inhibitors.In this dissertation,it is reasonable to design PROTACs targeting Bcl-2 family proteins,compensating the low affinity by catalyzing the ubiquitination of target proteins.At the same time,by forming stable ternary among PROTACs,target protein and E3 ligase,PROTACs could degrade Mcl-1/Bcl-2 selectively.We constructed two series of PROTACs,namely,H1-H5 and C1-C5,by introducing the E3 ligase cereblon?CRBN?-binding ligand pomalidomide to Bcl-2/Mcl-1 dual inhibitors S1-6and Nap-1 via alkyl and PEG linkers.Among them,PROTACs C3 and C5 were observed to potently and selectively induce the ubiquitination and proteasomal degradation of Mcl-1 and Bcl-2 in vivo,with half maximal degradation concentration(DC₅₀)values at 0.7 and 3.0?M,respectively.With binding affinity to Mcl-1 in the?M range,C3 induced super-stoichiometric ubiquitination of Mcl-1 that showed>10-fold stoichiometry during 4 h in vitro,which translated into much more lethality in Mcl-1-dependent H23 cells than the most potent Mcl-1occupancy-based inhibitor A-1210477,which has a nM range binding affinity.Moreover,structure-activity relationship?SAR?analysis and molecular dynamic?MD?simulations discovered the structural basis for turning nonselective or promiscuous Bcl-2 family ligands into selective PROTACs by changing the length and conformation of the linkers connecting the CRBN ligands.To our knowledge,this work represents the first application of PROTACs to protein-protein interactions?PPIs?targets as well as the first selective PROTACs against Mcl-1and Bcl-2.This study also provides the structural basis and principle for the design of the selective PROTACs.