Activity-Based Proteome Profiling Of Tumor Target Proteins(ABPP)-Design And Synthesis Of Activity-Based Probes And Four-Function Reporting Groups

Programmed cell death,also known as apoptosis,was regulated by Bcl-2 family proteins,which were overexpressed in tumor cells and were important target proteins.The activity-based proteome analysis(Activity-Based Proteome Profiling,ABPP)technique was used to study the tumor target protein Bcl-2 family proteins.By designing an activity-based molecular probe(Activity-Based Probes,ABPs),the non-covalently bound Bcl-2 inhibitor was linked to the photocrosslinking group and the "click chemistry" group,and then the non-covalently bound Bcl-2 protein was covalently bound to the target protein through the photocrosslinking reaction of alkynyl groups.Using fluorescein or biotin as reporting group can accurately reflect the labeling of ABPs to protein,which can realize in situ monitoring,tracking target protein,identifying Bcl-2 protein and analyzing the functional state of target protein.It has the advantages of in situ,real-time and visual research at proteome level.However,there were still great challenges in using ABPP technology to study Bcl-2 family proteins.Firstly,most of the active molecules that target Bcl-2 family proteins have low affinity,which is not suitable for constructing ABPs for ABPP technology;while a few active molecules with higher affinity,such as ABT-199,only maintain high activity on a single target protein,and due to the extremely complex synthetic route,it was currently difficult to construct ABT-199 ABPs.Secondly,the existing biotin reporter group used in ABPP technology had the defects that the conditions required to release the target protein are extremely harsh and the target protein cannot be fluorescently labeled.All these great challenges to the in-situ monitoring,tracking of Bcl-2 family proteins,target confirmation and active site identification using ABPP technology.Therefore,the development of broad-spectrum,high-affinity ABPs targeting Bcl-2 family proteins and a new multifunctional biotin reporter group are critical scientific issues to be solved.In this study,based on the original Bcl-2 protein and Mcl-1 protein dual inhibitor S1-B of this research group and the "minimalist linker" which integrates an alkynyl group and a photo-crosslinking group into one,we synthesized the first ABP: S1-B-minimalist linker,which could covalently bound to Bcl-2 protein and Mcl-1 protein.The recognition and labeling of Bcl-2 protein and Mcl-1 protein expressed and purified in vitro were realized,which proves that the Bcl-2/Mcl-1 protein inhibitor of this molecular skeleton can be used to further study tumor target proteins by ABPP technology,which provides an important experimental reference for the monitoring,tracking and target identification of Bcl-2 family proteins at proteome level.Since then,in order to obtain higher efficiency ABPs,this study continued to optimize the affinity of S1-B derivatives for target proteins.A series of new active molecules targeting Bcl-2 protein and Mcl-1 protein were synthesized by structural analysis of the p2 and p4 hydrophobic pockets of Bcl-2 protein and Mcl-1 protein.An active molecule,B4,with higher affinity was screened.The Ki values of B4 for Bcl-2 and Mcl-1 proteins were 0.31 μM and 0.16 μM,respectively.Compared with S1-B,its affinity is increased by 6 times.At the same time,the ability of B4 to induce Bcl-2 and Mcl-1 protein dual-dependent tumor cells is superior to the commercial Bcl-2 protein and Mcl-1 protein dual inhibitor(-)-Gossypol.In addition,B4 has good selectivity for inducing apoptosis,and it has almost no toxic and side effects on normal cells.Therefore,the construction of ABPs based on B4 provided a new molecular tool for the in-situ study of Bcl-2 family proteins by ABPP technology.