Estrogen Receptor-based Multi-residue Screening of Bisphenol Compounds and Structure-activity Relationships Studies
Author:Guan Tian Zuo
Supervisor:sun yong hai
Endocrine disrupt chemicals(EDCs),are compounds that affect the normal regulation of the endocrine system,and interfere with the synthesis,transport,release and metabolism of hormones in the humans,which are characterized by concealment,migration,mobility and persistence.Among these EDCs,bisphenol compounds(BPs)such as BPA,BPAF and TBBPA are all typical representatives.Because of their extensive distribution in food and environment and potential role in inducing the transcriptional activity of estrogen receptor(ER)?these compounds are also known as environmental estrogens.With the rapid development of urbanization and industrialization,concerns of food safety and environmental pollution are becoming increasingly prominent.In addition,due to the complexity of environmental system and food supply chain system,as well as the interplay between two systems,BPs will become more diverse and the interactions will become more complex,posing a serious threat to human health.Therefore,it is necessary to develop a broad-spectrum,sensitive and easy-to-use detection method to monitor BPs in the environment and food samples.The main works and innovations are as follows:(1)Preparation of soluble ERα-LBD recombinant proteinHuman estrogen receptor a ligand binding domain(ERa-LBD)was expressed as a glutathione S-transferase(GST)-tagged recombinant protein.Expression of fusion protein was induced with 0.5 mM IPTG at 20℃.The target protein was provided as recognized elements for the following step.(2)Establishment of ERa-LBD based fluorescence polarization methodThe work confirms that ERa-LBD and coumestrol(CS)are recognition element and tracer that can be used to monitor multi-residue of BPs by fluorescence polarization assay.Firstly,10 nM CS and 250 nM ERa-LBD were selected for the detection system.And competitive binding curves of BPs with ICso values were ranging from 0.11-123.70 μM.Recovery experiments were conducted for three types of substrates including soil,canned drinks and urine,and the results show that the precision and accuracy of this method are good.According to the optimized sample pretreatment procedure,the detection limit of BPA in soil by this method is 0.05 mg/kg,lower than the European Union standard(0.6mg/kg);the detection limits of single bisphenol-diglycidyl ether in canned drinks are all lower than the EU standard(1mg/kg),which is applicable to the detection of actual samples.Finally,the detection results of fluorescence polarization method were compared with those of LC-MS/MS,and the two methods were highly correlated,proving that the detection results of fluorescence polarization method were true and reliable.(3)Broad-spectrum study of ERa-LBD based detection methodThe systematic research on broad-spectrum phenomenon is of great significance since broad-spectrum is an important evaluation index in the biological multi-residue detection.In combination with molecular surface electrostatic potential(ESP)analysis method and two pharmacophore modeling methods,this chapter illustrates the internal effects of broad-spectrum in receptor-based method.Firstly,by observing the molecular structures and the ESP,it is found that BPs not only own the common structural framework of connecting two hydroxyphenyl groups by carbon or sulfur atoms,but also own a regular change in the electrostatic potential on the molecular surface due to the electron absorption effect of halogens.Based on this observation,it was furtherly found in HipHop and Receptor-ligand pharmacophore generation models that,apart from the the hydroxyl groups of BPs are superimposed with HBA,PI and HY also be obtained based on the receptor structure.In conclusion,hydrogen bonding,hydrophobic interaction and electrostatic interaction are all affect the interaction between BPs and ERa-LBD,and contribute to the broad-spectrum of ERa-LBD based receptor method.(4)Structure-activity relationship study of BPs and ERa-LBDIn order to further elucidate the intrinsic influence factors of BPs and ERa-LBD binding mode,the binding activity set of BPs and ERa-LBD was taken as the original data to construct a 3D-QSAR model.The results showed that the linear relation between predicted value and experiment value of training set was Y=0.9939X+0.0309,R2 was 0.9939,and the linear equation of test set compound was Y=0.9342X+0.1953,R2 was 0.7565,it indicated that the 3D-QSAR model had good predictive ability.In addition,molecular docking was also used in this study,all BPs could be roughly divided into two types of binding modes by induction.Firstly,the type of binding mode was represented by BPA,TCBPA and BPS,which similar to the agonist E2.The compounds could mainly form a hydrogen bond with amino acids of ERa-LBD such as Glu353,Gly521 and His524,so as to be stably anchored in the hydrophobic binding cavity.The second type of binding mode was represented by HPTE,BPAF and BADGE·2H2O,which was similar to the antagonist OHT.One of side chain such compounds extended into the channel of helix H3 and helix H11 to form hydrogen bond with THR347 and/or ASP351.To summarize,a receptor-based fluorescence polarization assay for the determination of BPs in different matrices has been developed,which was confirmed by recovery test and LC-MS/MS validation.Moreover,such as molecular docking and pharmacophore model approaches were performed to explore the probable binding mode between BPs and ERa-LBD.This study provides a new way and reliable theoretical basis for BPs high-throughput screening test,lays a good foundation for the development and promotion of BPs multi-residue detection kit,and provides theoretical support and helpful reference for the further optimization and innovation with subsequent methods.