1. The role of endocrine disrupting chemicals(EDCs) on estrogen receptors(ERs) using molecular docking Progress Report of DST Project Presented by Dr. Biswanath Bhunia Assistant Professor Department of Bio Engineering NIT Agartala

2. Estrogen Estrone and estradiol are both natural estrogens. Estrogens are responsible for the normal development of the female genital tract, of the breast, and of the female secondary sex characteristics. Estrogen Receptor Mechanism of action of Estrogen hormone

3. The biochemical pathway in the conversion of cholesterol to: androgens (Dihydrotestosterone) and estrogens (Estradiol) Mechanism of action of steroid hormone  They penetrate into the cell and bind to a receptor.  Complex steroid/receptor translocates into the cell nucleus, which is the principal site of action and where RNA/protein synthesis occurs  Compounds that occupy the receptor without causing translocation into the nucleus act as Antagonists

4. Endocrine disrupting chemicals(EDCs) Any exogenous agent that causes adverse health effects in an intact organism, or its progeny, consequent to changes in endocrine function. Pesticides Herbicides Fungicides Plasticizers Surfactants Organometals Halogenated PAHs Phytoestrogens Chemicals: Wide Variety - 1. Serving as steroid receptor ligands. 2. Modifying steroid hormone-metabolizing enzymes. 3. Perturbing hypothalamic pituitary release of trophic hormones. 4. Miscellaneous or unknown.  Interactions with the functions of estrogens, androgens, and thyroid hormones have been the most highly studied. At Least 4 Modes of Action

5. Estrogen preparations Ethinylestradiol (t 1/2 13 h) is a synthetic agent of first choice for contraceptive use; it is effective by mouth. Estradiol and estriol are orally active mixed natural estrogens. It can be effective and convenient for women who dislike oral therapy. Conjugated estrogens ( Premarin ® ) are orally active mixed natural estrogens containing 50–65% estrone obtained from the urine of pregnant mares. Estrogen Antagonist preparations Tamoxifen citrate (e.g., Nolvadex®), raloxifene hydrochloride (Evista®) and toremifene (Fareston®) for the treatment of breast cancer

6. Objectives To screen the EDC as agonist / antagonist of ERs and their binding affinity in Ers To find out the binding pattern of receptor with different EDCs

7. Docking studies: Auto Dock 1.5.6 with MGL tools 1.5.6 Binding pattern: Discovery Studio 4.1 Ligand Structure Preparation: Chem Draw Ultra 15.0, Open Babel GUI, Online smiles translator, All the Ligand structures were saved in PDBQT file format, for input into AutoDock version 1.5.6 Protein Structure Preparation: For the molecular docking study, protein structure was obtained from protein data bank; the PDB ID was 2YJA and 2YJD for Estrogen α and β respectively. Software and Database Used Methodology

8. Downloaded the protein pdb in fasta file as shown in figure….

9. Now I need to download the ligand pdb files through pubchem….

10. As the file is in sdf file need to convert to pdb file…!!we can covert in online as well as offline through open babel software…

11. The inhibitors which we did not found can draw in chem draw by seeing the structure and save it in sdf file….. and convert in pdb format…

12. Through open babel convert sdf to pdb file….

13. Now you got the pdb files of both protein as well as ligand. Now we can start docking….. Open the fasta file of protein in “Notepad++” you can see data of protein…as shown…….!!

14. Before starting docking delete water molecules[HOH] in “notepad++”

15. Delete smallest chain through “notepad++”

16. Follow the steps

30. You can get the log file generated in your default drive after getting the result…open it in notepad++ can see the result….wisely do it for all the ligands…

31. LigandEstrogen Receptor (kcal/Mol) AlphaBeta 4-hydroxi tamoxifen-4.2-3.7 Clomiphene-4.0 Chlorotrianisene-4.9-4.0 DDT-4.3-4.4 Estradiol-4.6-4.0 Estriol-4.4-4.3 Estrone-5.0-4.2 Ethinylestradiol-4.6-4.3 Fulvestrant-3.0-2.6 Genistein-4.0-4.5 Quinestrol-5.1-4.8 Raloxifene-4.1-3.8 R-R-cis-diethyl-THC-4.7-4.2 Tamoxifen-3.7-4.7 Toremifene-3.6-3.5 Docking analysis for 40 Ligands ResultsObjective I Interaction energy between Ligand-receptor have been calculated with a free energy-based expression & the docking score were generated by Vina.

32. Protein Ligand Alpha (kcal/Mol) Beta (kcal/Mol) Chlormequat-2.2-1.6 CHLOROTHALONIL-3.1-3.4 Chlorpyrifos-2.8-3.2 Daminozide-2.1-2.2 DDE-3.7-3.8 Deltamethrin-3.5-2.5 Dichlorvos-2.2-2.3 Dieldrin-3.6-3.5 Dimethoate-2.3 Endosulfan-4.1-3.5 ETHEPHON-2.0 FENARIMOL-4.0-3.7 Fenpropathrin-4.6 IPRODIONE-3.3-2.7 MethiocarbSulfoxide-4.5-2.6 METHIOCARB-2.4-2.7 Methomyl-2.2-3.3 Paclobutrazol-2.8 phosetyl_aluminium-2.4-2.3 PIRIMICARB-3.3-3.5 PROCHLORAZ-3.0-2.3 PROPAMOCARB-2.6-2.3 TCDD-3.6-3.7 Tolclofos_methyl-3.4-3.3 TRIBENURON_METHYL-4.1-3.9 VINCLOZOLIN-3.5-3.6

33. Estradiol analogs Protein Ligand Alpha (kcal/Mol) Beta (kcal/Mol) Estradiol-4.6-4.0 Estriol-4.4-4.3 Estrone-5.0-4.2 Ethinylestradiol-4.6-4.3 Agonist I-5.1 Agonist II-5.1 Estradiol Agonist I Agonist II Estradiol was considered as common agonist for both estrogen receptors.

34. Docking Study of Agonist I Alpha Receptor Beta Receptor

35. Docking Study of Agonist II Alpha Receptor Beta Receptor

36. Protein Ligand Alpha (Kcal/Mol) Beta (Kcal/Mol) Raloxifene-4.1-3.8 Antagonist I-4.3-3.8 Antagonist II-4.5-4.1 Raloxifene analogs Raloxifene Antagonist I Antagonist II Raloxifen was considered as common antagonist for both estrogen receptors.

37. Alpha Receptor Docking Study of Antagonist I Beta Receptor

38. Docking Study of Antagonist II Alpha Receptor Beta Receptor

39. Results Objective II Active site of alpha receptor was analysed by discovery studio. Active site proposed from LEU 346 to LEU 525 for Alpha Estrogen Receptor Non bond interaction with Ligand for alpha estrogen receptor

40. Non bond interaction with Ligand for Beta estrogen receptor Active site of alpha receptor was analysed by discovery studio. Active site proposed from MET 295 to LEU 491 for Beta Estrogen Receptor

41. Salient features of the project Two analogs for estradiol and Raloxifen were found to most effective as agonist and antagonist for both estrogen receptor. Active site proposed from LEU 346 to LEU 525 for Alpha Estrogen Receptor Active site proposed from MET 295 to LEU 491 for Beta Estrogen Receptor

42. Acknowledgments would like to acknowledge the DST SERB, Government of India for Funding (SB/YS/LS-272/2013). would like to acknowledge the National Institute of Technology, Agartala, for administrative support