Development Of Novel Fluorescent Probes Capable Of Selctive Labeling And Imaging Of Bruton’s Tyrosine Kinase In Living Cells

Bruton’s tyrosine kinase（BTK）is a key component of B cell receptor（BCR）signal transduction and an important regulator of cell proliferation and cell survival in various B cell malignancies,and has been validated as an effective therapeutic target in oncology.Up to now,a number of BTK inhibitors have been advanced into clinical drugs.For example,Ibrutinib is a marketed drug approved by the FDA in 2014 for the treatment of diseases such as mantle cell lymphoma（MCL）and chronic lymphocytic leukemia（CLL）.Thus,the development of new chemical reagents for the accurate detection of BTK expression and function is of great importance for the diagnosis and treatment of related diseases and the development of BTK targeted drugs.Activity-based proteome profiling（ABPP）and bioimaging have been powerful approaches for monitoring the function and expression of protein in native environments.A series of novel druggable targets and bioactive lead compounds have been discovered by these approaches.Recently,researchers have developed an activity-based probe and several fluorescence imaging probes based on Ibrutinib,these probes have been demonstrated to be suitable for target profiling,real-time fluorescence imaging living cells.These work provide strong support for the qualitative and quantitative analysis of BTK,the expression and function study of target protein,the study on the distribution and mechanism of Ibrutinib in vivo,and the screening of BTK inhibitors,providing useful tool probes for the treatment of related diseases and the discovery of innovative drugs.However,these probes still need to be improved in the following aspects:1)none of them is suitable for simultaneous no-wash imaging and chemoproteomics studies;2)strong washing conditions are required prior to imaging studies and the signal-to-background ratios need to be improved;3)Most fluorescent probes showed weak inhibitory activity against target proteins.In this project,to alleviate these shortages,we have developed a fluorescent probe IB-4 based on the photoinduced electron transfer（PET）effect by incorporating a maleimide–coumarin into Ibrutinib.The resulting probes IB-1/2/3/4 showed very weak fluorescence,but a strong turn-on fluorescence can be observed upon reaction with Cys 481 of the active site of BTK.Through a series of comparative experiments,the dual-purpose probe,IB-4,was developed with excellent inhibitory activity(IC₅₀=35nM),and has been demonstrated to be suitable for simultaneous imaging endogenous BTK activity and studying its target engagement in live cells.The unique design endows the probe with excellent sensitivity and selectivity under both in vitro and in situ conditions,and can be further extended to other irreversible inhibitors for protein characterization,quantification and inhibition studies.