Theoretical And Experimental Study On Mechanism Of Interaction Between Typical Pollutants And Target Proteins

Environmental pollutants and their metabolites in vivo may induce harmful biological effect through interrupting normal physiological process by binding specific target proteins. In fact, the recognition and interaction between pollutants and target proteins plays a predominant role in environmental toxicology. Recently, related study has been focused on the effect of pollutant structure on possible ecological hazard and health problem. However, complicated toxicity mechanism cannot be revealed without the necessary understanding in biotarget molecule itself. It may not only provide useful mechanism information to ensure environmental safety and human health, but also establish reliable toxicity prediction method for risk assessment practice by exploring the interaction mechanisms of pollutant vs. target protein using various computational toxicological methods. In the present study, both experimental and theoretical study was carried out to gain an insight into the possible mechanisms and resulting macro-effects for typical pollutant-target protein interactions. More attention have been paid to the structure plasticity and molecular characteristics of target proteins as well as its influence on the related endpoints.Multi-discipline theories and methodologies have been adopted to develop novel structure-activity relationship methods based on both receptor and ligand structure feature. The exploration in this study may provide theoretical foundation and methodology support to uncertainty reduction in both health risk assessment and ecological risk assessment. The work mainly consists of three parts:1.Role of Structure Plasticity in Target Protein Modulation by Pollutant BindingComputational toxicology methods such as molecular structure similarity analysis, molecular docking, molecular dynamics and quantitative structure-activity relationships have been integrated to explore the effect of target protein structure on the binding modes and the consequent biological effect of pollutants. Insight into the intrinsic linkage between pollutant binding iniatiated structure adjustment of target protein and the biological endpoint has been gained to elucidate the decisive role of protein plasticity in the biological effect at the different levels such as key residue orientation, change in secondary structure, and tertiary structure rearrangement.(1) Role of Pocket Flexibility in ERa Modulation by Arg394 Estradiol derivatives of similar structure as estradiol or estradiol metabolites have been recognized to have detrimental health effects on wildlife and humans. However, information about interactions of these compounds with biotargets is still lacking at molecular levels. Herein, a flexible docking approach was used to characterize the molecular interaction of nine estradiol derivatives with ERa, especially the role of pocket flexibility in ERα modulation by Arg394. All ligands were docked in the buried hydrophobic cavity of the hormone steroid pocket. In addition, the plasticity of ERa active site was identified by reversing amino acid Arg394 for better ligand-receptor binding affinity. Besides, bioassays data based on genetically modified yeast strains was used to validate the result of molecular simulation. The experimental findings about logarithm values of the median effective concentration value had a linear correlation with computational binding affinity from molecular docking (r2=0.9913), which described a uniform pattern of interaction between estradiol derivatives and ERa. The estrogenic activity of all compounds, although more or less lower than E2, was proved to possess high severe environmental risks. Considering the side chain flexibility in the ligand-binding domain, 17α-ethylestradiol-3-cyclopentylether was reported to highly significantly correlate with known induced fit conformational changes with the preservation of a strong salt bridge between Glu353 and Arg394.(2) Role of Leu300 in Open/Closed State Control of hALR2 Secondary Pocket Flavones are endocrine disrupting chemicals that occur ubiquitously in foods of plant origin, and some flavones exhibit strong inhibitory activity to the human aldose reductase (hALR2). In the present study, the molecular similarity analysis, flexible docking and molecular dynamic analysis was used to characterize the molecular interaction of 56 flavones with hALR2. The molecular similarity technique categorized these flavones into two groups. Furthermore, molecular docking was carried out to distinguish different binding modes, which were involved in protein flexibility of the hALR2. Forty nine flavones in group I exhibited a binding mode with the active binding site without secondary pocket. But seven flavones with bulky substituent in group II were proved to interact with open-stated secondary pocket by molecular dynamic analysis. The CoMFA and CoMSIA models were established to explore the structural specificity and predicted inhibitory activity of 56 selected flavones to hALR2. The result confirmed that the structure flexibility of hALR2 ligand binding domain controlled the opening of the second pocket through the key switch residue Leu300.(3) Theoretical Study on the Effect of Structure Plasticity on ER Agonism/Antagonism of Selected GinsenosidesA protein-protein docking methodology was applied to characterize the binding mode of Ginsenosides/ERa complex with coactivator PGC-la to revealing the molecular mechanism of ERa agonism/antagonism. It has been found that cofactor PGC-la exhibited strong binding affinity for agonistic conformation of ER. Two clamp residues, Leu143 and Leu147, were deeply embedded in the hydrophobic surface groove of ERa and formed H-bonds with Leu 143 and Va1376 of ERa. Meanwhile, key residues Ser140 and Leul46 of PGC-la formed H-bonds with G1u542 and Lys362 in ERa, respectively. However, the antagonistic conformation of ER had no fixed binding site for PGC-1α because H12 has already occupied the binding site of the coactivator. Protopanaxatriol was proved to be an agonist of ERa, while the intestinal ginsenoside metabolite Ml was an antagonist of ERa.2. Theoretical and Experimental Study on Enantioselectivity of Acetyl Cholinesterase to Chiral Organophosphorus PesticidesChirality is a common feature of many environmental pollutants. However, it is frequently overlooked when considering their biological effects and toxicities. Acetyl cholinesterase (AChE) is a well-known biological target for organophosphorus pesticides (OPs), and stereoselectivity of AChE to chiral OPs is essential for understanding their toxicity mechanism. In the present study, molecular docking approaches were used to discover molecular interactions between six pairs of chiral OPs and AChE. Then a pharmacophore model based on bioactive conformations of OPs was established for high-throughput virtual screening, and 24 potential AChE inhibitors were obtained from two hundred thousand compounds in the National Cancer Institute Database. At last, bioassays based on AChE competitive inhibition and enantiospecific fluorescence measurement were carried out to test the quality of virtual screening. Three compounds, ethyll p-nitrophenyl phenylphosphonate, 1-naphthaleneacetic anhydride and N,4-dimethyl-N-phenyl-benzenesulfonamide, were demonstrated to possess inhibitory activity to human AChE. Furthermore, S-isomer of ethyll p-nitrophenyl phenylphosphonate was proved to exhibit much stronger AChE inhibition activity than R-isomer using fluorescence identification and flexible docking method. The molecular mechanism of enantioselectivity of acetyl cholinesterase to ethyl p-nitrophenyl phenylphosphonate was discussed.3. Theoretical Study on Multi-Target Composite Effect of Selected ChemicalsEmphases have been put on the development of new three dimensional quantitative structure-activity relationship method by taking both target and organic pollutants structure into consideration. A structure-activity relationship based scenario was proposed to explore the target proteins and the mode of action for pollutants, and the molecular basis for multiple-target selectivity and the formation mechanism of non-monotonic dose-response curve was analyzed.(1) Possible Mechanism for Non-Monotonic Dose-Response Curve of PCBs in Chicken Embryo Hepatocype BioassayPolychlorinated biphenyls congeners (PCBs), as the typical endocrine disrupting chemicals, exhibit unique non-monotonic dose-response curve when inducing chicken embryo hepatocype EROD activities. A flexible docking approach was used to discover molecular interactions between 39 PCBs and aryl hydrocarbon receptor. Different binding modes for PCBs have been distinguished not only from aligned conformation but also from free binding energy. QSAR models were established for low and high doses ranges, separately, indicating that non-monotonic dose-response curve of PCBs in chicken embryo hepatocype bioassay may result from diverse mechanisms functioning at different dose ranges. Receptor binding may predominate in the interference of physiological function of cytochrome P4501A-P4501A in low dose range. But with the higher doses range, the EROD suppression might be related to acute toxicity owing to molecular polarity or distribution of charges and consequently damage structure and function of chicken embryo hepatocyte.(2) Theoretical Study on Multiple-Target Effect for Selected OH-PBDEs Close attention have been paid to polybrominated diphenyl ethers (PBDEs) in recent years, and their metabolites, hydroxyl polybrominated diphenyl ethers (OH-PBDEs), can interfere with normal regulation of human thyroid hormone by competitive binding to thyroxine transport proteins. In the present study, CoMFA and CoMSIA models were constructed to predict the binding affinities between OH-PBDEs and thyroid-binding globulin (TBG)/transthyretin (TTR). Molecular docking technique was also utilized to find the multi-target binding modes of OH-PBDEs vs. TBG/TTR. Fourteen OH-PBDEs were docked into the active pocket of TBG and TTR, forming hydrogen bonds with residue Lys270 of TBG and residue Leu110, Ser117 of TTR, separately.(3) Theoretical Study on Multiple-Target Effect for Selected Ginsenosides Natural Chinese medicine ginsenosides have an anti-tumor effect, but endocrine disrupting effects of their metabolites is commonly overlooked. Herein molecular simulation was applied to discover molecular interactions between ginsenosides and two target proteins, vascular endothelial growth factor receptor and estrogen receptor, aiming at investigating anti-tumor mechanism and endocrine disrupting effects of their metabolites. It has been found that ginsenosides, as an inhibitor of vascular endothelial growth factor receptor, can trigger ischemia of tumor cells and protopanaxatriol saponin have stronger anti-tumor activity than protopanaxdiol ones. Meanwhile, ginsenosides does not exhibit significant endocrine disrupting effects, but their metabolites, protopanaxdiol and protopanaxatriol, shows relative high endocrine disrupting effect.