The Use Of MD In The Study Of Interactions Between Typical Endocrine Disrupting Chemicals And Key Nuclear Receptors

There are more than 120 million chemicals registered in Chemical Abstract Service(CAS),and this number is still increasing rapidly.Many of these chemicals have been proven to be endocrine disrupting chemicals(EDCs),however,till nowadays,a large number of chemicals with potential endocrine disrupting effects has not been detected.From a technical perspective,the traditional laboratory testing is limited by high-expense and low speed,which cannot be used to detect the activities of large numbers of EDCs.Moreover,it has been found that although the quantitative structure-activity relationship(QSAR)techniques have been developed in recent year,but use these methods along could result in "false positive" phenomenon,which hinder their use in real practice.However,the inner defects of QSAR provide a clue to solve it:if the activeness of chemicals could be identified by a classification model at first,then apply QSAR methods to predict these "known-active" chemicals,the activities of all chemicals could be predicted preciously.The most important factor of building classification model is to confirm the key factor in the process of producing endocrine disrupting effects.As the classic adverse outcome pathways(AOP)describes,in the process of inducing activity and effect,a chemical should firstly interact with receptors after exposure,and then induce cellular response,then induce effects in organ and population level.Thus,the interactions between chemical and receptors are the key factor in exhibiting activity and inducing effects.For nuclear receptors(NRs),the interactions between ligands and receptors include four steps:1)ligand docking with NR and form ligand-receptor complex;2)the conformation of complex subsequent change by the interaction between ligands and receptors;3)binding of NRs to each other to form homo-or hetero-dimers;4)recognition by homo-or hetero-dimers of specific sequences of genomic DNA through the binding of various co-regulators(including co-activators and co-repressors)related to transcriptional activation/repression to mediate the transcription of target genes.Among these steps,ligand interacts with receptor and form a stable complex is the key process by which the activeness could be identified.Moreover,the process of forming homo-or hetero-dimers and the influences of co-regulators are important factor by which the endocrine disrupting effect and key nuclear receptors in the endocrine disrupting regulation pathways could be identified.Among the computer simulation methods,molecular dynamics simulation(MD)can describe the motion trajectory of compound in space and simulate the molecular microcosmic behavior,which can be used to evaluate the interactions between ligands with biomacromolecule and predict the active conformations of ligands.Therefore,this study intends to use the MD characteristics,to analyze the relationship between chemicals and receptors to achieve two objectives:(1)build qualitative models that can discriminate the activeness of endocrine disrupting chemicals of flavonoids and polybrominated diphenyl ethers(PBDEs),and combine with QSAR to construct a predictive systemic method that has the ability to precisely discriminate and predict the activities of unknown compounds;(2)by simulating the interactions between TCEP and zebrafish NRs,together with the gene activation of nine NRs and 70 genes in low exposure level,as well as the changes in morphology and morality in high exposure level,mechanisms of interactions between different receptors,the discordant of gene expression could be analyzed and the key nuclear receptors that could induce the final adverse outcomes were distinguished.In addition,the methods of identifying anti-androgenic chemicals are expended to build a classification model in identifying anti-glucocorticoid activity of PBDEs.(1)To build a new predictive system combining qualitative and quantitative simulation.MD simulation was applied in the second and third chapter to simulate and analyze ligand "escaping","mouse trap" mechanism,hydrogen bonds,the stability of ligands and receptors in the process of interactions between chemicals and ligands to form stable complex.In the second chapter,the results showed that "escaping" and"mouse trap" phenomenon was found in 3-hydroxy-6-methoxyflavone and 6-methoxyflavanone,respectively,the chemicals with methoxy group in the A-ring while forming little or no hydrogen bonds with key residues tend to have no anti-androgenic activity,94.1%of active flavonoids and 79.3%of inactive flavonoids could be distinguished by analyzing the stability of ligand RMSD,and the activeness of 85.4%of ligands could be identified by analyzing the stable of receptors.In order to play full advantages of a single method,a combination classification method including these findings was built,with the total accuracy 85.4%and inactive fliting ability of over 90%.In the third chapter,a novel predictive system was built to predict anti-androgenic activities of PBDEs.Different from flavonoids,PBDEs cannot form hydrogen bonds with AR and no "escaping" or "mousertrap mechanism" was found,thus the ligand-receptor complex could reach stable status in a short time.A qualitative classification model was built by using principal component analysis(PCA)analysis of the stabilities of ligands and the H12 of complexes,and the accuracy reached 90.5%.The classification models built in this study did not rely on the laboratory testing,which means it had good applicability and high accuracy.Finally,a new predictive systemic method could be built combining the qualitative identification model and traditional QSAR,which could greatly reduce the lab-intense and expense.In order to investigate whether the separation methods built in the second and third chapter could be used by other nuclear receptors,a qualitative identification model of anti-glucocorticoid activities of 16(OH/MeO)-PBDEs was established in the fourth chapter.In this chapter,the fluctuation of RMSD of all ligands were small,indicating all chemicals were potentially active;the separation accuracy of identification model based on the stabilities of complexes was 88.2%.However,unlike the results of the second and third chapter,the active chemicals in the separation model were concentrated in the middle,which might be related to the indirect effect of anti-GR transcription,which worth further research.(2)Analyze the discordant of gene expression induced by TCEP and identify key nuclear receptors inducing adverse outcomes.In the fifth chapter,MD was used to analyze influences of TCEP toward the regulation pathways of zebrafish,and identify the key nuclear receptors producing discordant of gene expression and adverse effects.Firstly,the endocrine disrupting effects of TCEP toward zebrafish embryos/larvae were determined by water exposure experiments,and the effects of TCEP on the expression of seventy receptor-related genes were determined,which confirm the endocrine disrupting effects in cellar and organ level.Secondly,MD was used to simulate the interactions between TCEP and 9 nuclear receptors to analyze the stabilities of ligand-receptor complexes and confirm whether TCEP had potential activities toward these receptors.Based on these analysis,together with different features of transcriptional responses and the interactions of different receptors,as well as the roles of co-regulators,it was found:(1)For these ligand-mediated receptors,such as androgen receptor(AR),estrogen receptor(ER),glucocorticoid receptor(GR),mineralocorticoid receptor(MR),pregnane X receptor(PXR),which only form homo-dimers,the stable complexes indicating the transcriptional responses could be induced by TCEP;(2)retinoid X receptors(RXRs)were important because they can affect the transcriptional expression of thyroid hormone receptors(ThR),retinoic acid receptor(RAR),peroxisome proliferator activated receptor(PPAR)by forming heterodimers with them.Among them,TCEP-raraa/rxrab complexes could not reach stable status,suggesting TCEP could not induce raraa/rxrab transcriptional responses.Moreover,although raraa could form stable complex with TCEP,this receptor could not form,hetero-dimer with rxraa,resulting no transcriptional response;(3)ThR and PPAR could either form homo-dimer or hetero-dimer with RXR,and in simulation process these receptors could form stable complexes with TCEP,thus they exhibit transcriptional response;(4)Co-regulators(including co-activator and co-repressors)were important factors affecting transcriptional expression,and since almost all co-activators were ER downstream genes,all co-suppressors were ThR downstream genes,thus ER and ThR receptors are important receptors.In short,ER,ThR and RXRs are key nuclear receptors that influence the endocrine disrupting effects and discordant of gene expression induced by TCEP.