Development And Application Of New Cysteine Selective Probes

Cysteine residue is the most important nucleophilic group in protein system.Its activity is closely related to the biochemical function of protein.Many clinical drugs,such as Osimertinib and Ibrutinib,produce relevant pharmacological and pharmacodynamic properties by covalently modifying the active cysteine of the target protein.Therefore,the development of cysteine modified groups is of great significance for drug discovery.At present,the main electrophilic group for cysteine is still α,β-unsaturated double bond.However,the target and cysteine sites found by the single structure of α,β-unsaturated double bonds are relatively limited,and there are still a large number of active cysteine sites to be identified in vivo.Therefore,we hope to develop new cysteine selective probes,combined with activity-directed protein profiling(ABPP),and find new modifiable cysteine sites and target proteins,so as to provide a basis for drug discovery.Tetrazole group has good hydrophobicity and electron absorption,and it is also an important structural segment of some clinical drugs.In the early stage,the research group carried out a series of research work on tetrazoles.In this paper,we have constructed a series of activity-directed molecular probes(Tz1-8)based on the tetrazole.We hope that the probes can modify the previously unknown active cysteine sites through the active substructure fragments of tetrazole.The obtained molecular probes showed good biological activity and labeling efficiency,and a series of cysteine sites were identified.The main work includes:1.A series of halomethyl-tetrazole probes were designed and synthesized,and the labeling efficiency of the probes was compared at the pure protein level and the cell level.Tz-1 was selected as the probe with the highest labeling efficiency and was used in the follow-up study.2.Protein labeling was carried out at the level of pure protein,cell lysate and living cell,which verified the concentration of the probe and the incubation time,and proved the good labeling efficiency of the molecular probe.At the same time,the selectivity of the probe was verified by adding competitive inhibitors such as IAA.3.We have confirmed that iodomethyltetrazole is more than 90% selective for cysteine at all levels by mass spectrometry,and found that it can mark the active center of many important proteins in the process of cell metabolism and proliferation or the cysteine in the area near the active center,such as Cys32 of glutathione S-transferase,Cys47 of thiol specific peroxidase and Cys310 near the phosphorylation sites of nerve growth factor receptor.It is of great significance for the follow-up study.In conclusion,we have successfully developed a highly reactive and selective group—iodomethyltetrazole.In the future,we hope that we can make a quantitative description of the activity of cysteine residues through more in-depth study,so as to find out more active sites of protein and provide valuable tools compound for disease treatment and chemical proteomics research.