Investigation On The Interaction Between Organotin Antitumor Compounds With Human Cytochrome P450 3A4 Protease

Objective:To investigate the relationship between CYP3A4 protease and four organotin compounds including [ bis(2,6-difluoro)substitutive dibutyltin(DBDFT),bis(2,4-difluoro)substitutive diphenyltin(DPDFT),bis(2,4-dichloro)substitutive diphenyltin(DPDCT)and bis(4-chloro)substitutive dibutyltin(DBDCT)],The methods of spectroscopy,molecular docking and drug affinity responsive target stability techniques were used to compare the interaction between the organotin compounds with CYP3A4 protease and investigate the changes of the secondary structure of CYP3A4.Methods:(1)The complexations of organotin compounds with CYP3A4 were studied by UV-vis absorption spectroscopy.The quenching mechanism and the interactional binding parameters were investigated by fluorescence quenching technique.The interaction between four organotin compounds and CYP3A4 metabolic enzymes were found by comparing the binding force between the structure and the CYP3A4 metabolic enzymes.The energy transfers between organotin compounds with CYP3A4 protein were studied by the combination of UV-vis absorption spectroscopy and fluorescence spectrometry.(2)The methods of synchronous fluorescence,three-dimensional fluorescence,CD spectroscopy and molecular docking were used to study the conformation influences of organotin compounds with CYP3A4 protein,which included the microenvironment of tryptophan and tyrosine,the microenvironment of peptides,content of α-helix and hydrogen bonding position.(3)The resistances of digestive enzyme of CYP3A4 protein with organotin compounds were investigated by DARTS technique.The CYP3A4 proteins were protected by the complexes of organotin compounds with CYP3A4.Results:(1)The complexations of organotin compounds with CYP3A4 had different binding force,interaction pattern and the energy transfer.1)The absorbance of DBDFT-CYP3A4,DPDFT-CYP3A4,DPDCT-CYP3A4 and DBDCT-CYP3A4 increased by 1.62,3.28,2.53 and 1.56 compared with the absorbance of CYP3A4 at 280 nm respectively.The complexations were formed by the organotin compounds and CYP3A4;2)The Kq of complexations had surpassed 2.0×1010 L·mol-1·s-1,the fluorescence quenching of complexations were the static quenching mechanism.In the 298 K,the Kb of DBDFT-CYP3A4,DPDFT-CYP3A4,DPDCT-CYP3A4 and DBDCT-CYP3A4 were 3.67×105,2.51×107,3.46×104 and 3.63×106 L·mol-1 respectively.In the 310 K,the Kb of complexations were 6.92×104,6.76×105,7.59×103 and 3.09×105 L·mol-1 respectively.The orders of the binding force of the organotin compounds and CYP3A4 were DPDFT > DBDCT > DBDFT > DPDCT;3)Both the ΔH and ΔS are negative,hydrogen bond and van der Waals forces played the major role in the interactions of the organotin compounds with CYP3A4 protein;4)The energy transfers were occurred between the organotin compounds and CYP3A4.The distances of the DBDFT,DPDFT,DPDCT and DBDCT with tryptophan were 4.34,3.72,5.10 and 4.36 nm,respectively.(2)The conformations were changed by the organotin compounds with CYP3A4 protein and the organotin compounds bound the active regions of the CYP3A4 protein.1)The microenvironments of tryptophan and tyrosine were changed by the organotin compounds.The maximum emission wavelength of tryptophan and tyrosine were red-shifted by DBDFT,DPDCT and DBDCT;2)The microenvironments of peptide chain were changed by the organotin compounds.The maximum emission wavelength of the characteristic peaks were red-shifted;3)The contents of alpha helix were reduced by the organotin compounds.The reduction values of alpha helix of DBDFT-CYP3A4,DPDFT-CYP3A4,DPDCT-CYP3A4 and DBDCT-CYP3A4 were 18.46%,20.08%,14.70% and 19.14%,respectively;4)Docking scores were above 4 of the complexations of organotin compounds with CYP3A4.The results showed the stable complexations of organotin compounds with CYP3A4 were formed.The results indicated that organotin compounds interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bonds.(3)The higher binding forces of the organotin compounds and CYP3A4,the higer resistances of digestive enzyme.DPDFT,DBDFT and DBDCT protected the dissociation of the CYP3A4 protein.Every concentration of DPDCT had no protection of the digestive protein,which illustrated the weaker binding force of the DPDCT with CYP3A4.The results were agreed with the fluorescence quenching study.Conclusions:The conformations are formed by the organotin compounds and CYP3A4 and the organotin compounds bind the active regions of the CYP3A4 protein.The combinations of organotin compounds and CYP3A4 enzyme may affect the metabolic progress of the organotin compounds.It could be speculated that the binding of the organotin compounds and CYP3A4 enzyme lead to the inhibition of the CYP3A4 enzyme activity for degradation of organotin compounds,so that the anticancer activity of the organotin compounds is increased.