Study On The Focal Adhesion Kinase Inhibitor And Environmental Pollutants Interaction With Target Proteins By Electrospray Ionization Mass Spectrometry

The interactions between proteins and small molecules are very common in organism,and the formation of protein complexes plays a key role in biological processes.Study on the interactions between small molecules and target protein helps to reveal toxicity mechanism of environmental pollutants and the pharmacological effects of drug molecules to targeting protein.Focal adhesion kinase(FAK)is a key of regulating cell adhesion and translocation.It is a non-receptor protein tyrosine kinase that can be proliferation and survival in multiple types of cells.It can promote tumor growth and metastasis as well.Due to the expression of FAK and its activation in tumor cell survival,migration and angiogenesis plays an important role,so that FAK is regarded as a potential cancer target.In this paper electrospray mass spectrometry(ESI-MS)is used to study the interactions between Focal adhesion kinase(FAK)and FAK inhibitors as well as the interactions between perfluorooctanoic acid(PFOA),perfluorooctane sulfonate(PFOS),4'-OH-BDE-201 and transthyretin(TTR).Two domains of FAK that are FAK31-405 and FAK411-686 are detected and analyzed.The interactions between seven FAK inhibitors(PF-573228,PF-562271,PF-431396,PF-03814735,TAE-226,PND-1186 and Defactinib)and the peptide of FAK 411-686 are studied,and their binding abilities are calculated.Meanwhile the effects of different functional group of different inhibitors on binding ability are analyzed.ESI-MS was used to analyze two domains of FAK which are according to the FAK FERM domain(FAK31-405)and kinase domain(FAK411-686)amino acid sequence in expression and purification.The results indicate that the expressed FERM domain is instability,while the kinase domain is stability.Only four inhibitors can form the protein complexes with FAK411-686 at 1:1 stoichiometry ratio.The highest binding constant of inhibitors is PF-573228,the rest are Defactinib,PF-431396,PF-562271 respectively.The modification of FAK411-686 can effect on the interaction between inhibitors and FAK.According to binding properties of the 7 inhibitors,their structure are analyzed and compared.The functional groups that lead to increase the possibility of inhibitor interacting with FAK411-686 are discussed.With respect to the interaction of inhibitors with FAK411-686,the results as follows: if the inhibitors contain active group such as N-methylbenzamide,indolin-2-one,(methylsulfonyl)benzene,3,4-dihydroquinolin-2(1H)-one,N-methyl-N-(3-methylpyridin-2-yl)methanesulfonamide,and others similar functional groups could increase the binding between inhibitor and FAK411-686.PFOA,PFOS and 4'-OH-BDE-201 can form the protein coplexes with TTR at 1:1 and 1:2 stoichiometric ratio.Binding abilities of PFOA and PFOS are greater than that of 4'-OH-BDE-201.In this paper,the experimental result helps to reveal inhibitor drugs' inhibitive effect process and mechanism of FAK at the molecular level.It helps further research for searching the binding site between FAK and its inhibitors.It can also provide experiment theory basis and ideas for new anti-cancer drugs design.Meanwhile,it provides a experimental evidence reveals the toxicity mechanism of perfluoroalkyl acids(PFAAs)and hydroxylation poly brominated diphenyl ethers(OH-PBDEs).