Designing of Potential New Estrogen Antagonists for Treatment of Endometriosis: Designing of Ligands, Molecular Docking, Activity, ADME & Toxicity Prediction Study

Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 5, Suppl 3, 2013 Research Article DESIGNING OF POTENTIAL NEW ESTROGEN ANTAGONISTS FOR TREATMENT OF ENDOMETRIOSIS: DESIGNING OF LIGANDS, MOLECULAR DOCKING, ACTIVITY, ADME & TOXICITY PREDICTION STUDY ABHISHEK THAKUR Department of Pharmaceutical Sciences; Birla Institute of Technology, Mesra, Ranchi 835215, India. Email: thakurabhishek242@gmail.com Received: 03 Jun 2013, Revised and Accepted: 25 Jun 2013 ABSTRACT The main objective of this study is to get more potent estrogen antagonist with greater safety profile. As estrogen antagonists and aromatase inhibitors both are generally used to treat endometriosis. But the large side effect profile of aromatase inhibitor are like Osteoporosis, increased coronary heart disease, and more over cost of treatment is more as compared with the treatment of estrogen antagonist. Due to many other factors these drugs have decreased compliance in patient use. Among these estrogen antagonist like ethinylestradiol, in available preparations like Ovral-L and Novelon is used. Commonly used drugs in this patient subgroup are Clomifene and Zuclomifene, which have found greater acceptance. Clomifene and Zuclomifene are one of the widely used estrogen antagonists to control estrogen level during menstrual cycle. As endometriosis is a complex gynecological disorder that is characterized by the presence of endometrial tissues outside the uterine cavity, often in the peritoneal cavity. These endometrial cells respond to cyclic changes in hormonal milieu in a manner similar to the uterine uteral endometrial cell. Therefore we have designed some new estrogen antagonists by modification of Clomifene. In this study we have designed some new estrogen antagonist and reported them as new potential estrogen antagonist by using various insilico tools and molecular docking analysis taking Clomifene as a prototype. The modifications made in Clomifene have shown a better and safe profile as compared to Clomifene and it has been proved to have better binding affinity with estrogen receptor than Clomifene in docking study. Keywords: Estrogen antagonist; Clomifene; Endometriosis; Molecular Docking; Activity Prediction; ADME & Toxicity Prediction. INTRODUCTION Endometriosis is a disorder of estrogen production on a cellular level. It is a condition which generally occurs due to presence of endometrial tissue outside the uterine cavity and their response to cyclic changes in hormonal milieu in a manner similar to uterine endometrial cells during their reproductive age with a global impact of 6 to 10% of women annually[1,2,9]. It is the abnormal growth of cells (endometrial cells) similar to those that form the inside of the uterus, but in a location outside of the uterus. Endometrial cells are cells that are shed each month during menstruation. The cells of endometriosis attach themselves to tissue outside the uterus and are called endometriosis implants. These implants are most commonly found on the ovaries, the fallopian tubes, outer surfaces of the uterus or intestines, and on the surface lining of the pelvic cavity. They can also be found in the vagina, cervix, and bladder, although less commonly than other locations in the pelvis [3,4,5,9]. The main hormone responsible for this disease is estrogen and condition worsens with each menstrual cycle. Under the influence of menstrual female hormones each endometrial tissue grows and sheds blood at the time of menstrual cycle [9,10]. Estrogen is a female hormone synthesized in the theca interna of the female ovary and varies though the menstrual cycle. Estrogen is found to be at highest level at the end of follicular phase just before ovulation [16]. Estrogen promotes secondary sexual characters in female, like breast development. It also helps to maintain the thickening of endometrium and prepare uterus lining for implantation of fertilized ovary [17]. Highest level of estrogen is a major factor contributing to the growth and development of endometriosis. With the secretion of estrogen each endometrial tissue grows like cancer and cyst. These endometrial implants respond with great affinity to estrogen and they proliferate rapidly. This is due to a reason that endometrium implants produce a high level of estrogen and possesses high level of estrogen receptor. Endometriosis implants produce high level of estrogen as they contain an enzyme named aromatase which is found in endometriosis implants which converts the precursor of estrogen into estrogen. These increased levels of estrogen contribute towards a growing factor of endometrium implants [18]. It has become a chronic disease nowadays and it can be controlled by estrogen antagonist as estrogen antagonist inhibits the action of estrogen and proliferation of endometrial cell is stopped [6, 13, 18]. The discovery of the first design of Estrogen antagonist drug has inspired a number of continuing efforts for the discovery of next generation drugs for endometriosis. It was found that treatment of infertile women with Clomifene leads to congenital malformation; congenital heart disease creates a big problem in treatment [15]. In this study we have designed some new estrogen antagonists by modification of Clomifene which has been proved to possess better binding affinity with estrogen receptor than Clomifene in docking study. MATERIALS & METHODS Designing of ligands: Four ligands have been designed by structural modification of Clomifene which are then judged by docking study to predict their estrogen antagonist activity similar to that of Clomifene. Ligand name: AT-1 Ligand name: AT-2 Juvekar et al. Int J Pharm Pharm Sci, Vol 5, Suppl 3, 451-455 Ligand name: AT-3 Ligand name: AT-4 Fig. 1: 2-D images of the designed ligands Fig. 2: Comparison of Modification done on AT-1 with that of Clomifene Fig. 3: Comparison of Modification done on AT-2 with that of Clomifene Fig. 4: Comparison of Modification done on AT-3 with that of Clomifene 452 Juvekar et al. Int J Pharm Pharm Sci, Vol 5, Suppl 3, 451-455 Fig. 5: Comparison of Modification done on AT-4 with that of Clomifene [ Molecular Docking Analysis Molecular Property Prediction 1-click docking [19] web based server has been used to dock the designed ligands with human estrogen receptor (PDB ID: 1R5K) [20]. 1-Click docking uses Vina filter and dock multiple ligands into a single target. Default binding site centre specification of 1R5K in 1-Click Docking Molecular Property such as hydrogen bond donor (Donor HB) and hydrogen bond acceptor (Acceptor HB) has been predicted using MolSoft (http://www.molsoft.com/mprop/) [23] as molecular properties are essential for every stage of drug development from design to synthesis. Hence prediction of these parameters is important from the drug development point of view. Default binding site center X: 9.17 RESULTS AND DISCUSSION Default binding site center Y: 56.604 Docking Analysis Default binding site center Z: 325.1457 The molecular docking study of the designed ligands with human estrogen receptor (PDB ID: 1R5K) shows that all the designed ligands are showing better docking score than that of prototype Clomifene which predicts that the designed ligands have the better binding affinity with the estrogen receptor than Clomifene (Table 1). This prediction leads us to believe that the designed ligands will possibly be suitable for treatment of endometriosis. Activity Prediction The activity of the designed ligands and Clomifene has been predicted using PASS Server [21] to confirm its Anti-fertility (female) and estrogen antagonist activity. ADME and Toxicity prediction The AMDE/T properties of drugs, together with its pharmacological properties are conventionally viewed as a part of drug development. The best ligands after docking analysis were subjected to predict the pharmacokinetic properties using Pre-ADMET online server [22]. Structures with unfavorable absorption, distribution, metabolism and elimination have been identified as a major cause of failure of candidate molecule in drug development. Therefore early prediction of ADME properties has been done with the objective of increasing the success rate of the designed ligands in future development processes. Table 1: Docking score of the Designed Ligands and Clomifene with human estrogen receptor (PDB ID: 1R5K) S. No. 1 2 3 4 5 Ligand name AT-1 AT-2 AT-3 AT-4 Clomifene AT-1 binding with 1R5K AT-2 binding with 1R5K AT-3 binding with 1R5K AT-4 binding with 1R5K Docking score -11.6 -11.3 -11.3 -11.1 -10.2 453 Juvekar et al. Int J Pharm Pharm Sci, Vol 5, Suppl 3, 451-455 Clomifene Fig. 6: The binding of the designed ligands and Clomifene with human estrogen receptor (PDB ID: 1R5K) visualized with GL mol Viewer after docking analysis with 1-click docking Activity Prediction The activity prediction of all the designed ligands has been done using PASS server and compared with Clomifene. It was found that designed ligands as well Clomifene when uploaded in the server as MOL format shows the significant properties like Antifertility (female) and estrogen antagonist which predicts that the designed ligands will probably be effective to treat endometriosis as that of Clomifene. been predicted using Pre-ADMET server and compared with the prototype molecule Clomifene which has been shown in Table 2 and Table 3 Permissible ranges ADME and Toxicity Prediction Mol wt.: (130–725); Donor hb: (0.0–6.0); Accept hb: (2.0–20.0); %human intestinal absorption: 70-100 % well absorbed, 20-70% moderately absorbed, 0-20 poorly absorbed, %plasma protein binding: >90 strongly bound, <90% weakly bound The ADME and Toxicity parameters of the designed ligands have *predicted using http://www.molsoft.com/mprop/ Table 2: Activity prediction of the designed ligands and Clomifene with PASS server* Sl. No. 1 2 3 4 5 Ligand name Antifertility in female Pa 0,971 0,974 0,362 0,976 0,988 AT-1 AT-2 AT-3 AT-4 Clomifene Pi 0,001 0,001 0,027 0,001 0,001 Estrogen antagonist Pa 0,683 0,546 0,633 0,660 0,851 Pi 0,004 0,004 0,004 0,004 0,003 *Pa= Probability of Active, Pi=Probability of Inactive, Pa>Pi confirms significant activity Table 3: AMDE and Drug Likeness properties of designed ligand molecule and prototype Clomifene S. No. 1 2 3 4 5 Ligand Name AT-1 AT-2 AT-3 AT-4 Clomifene Donor HB* 1 1 1 0 0 Acceptor HB* 3 7 6 4 2 Mol. Wt (gm/mol) %human intestine absorption %Plasma protein binding 413.12 472.13 528.09 443.14 405.19 96.988196 96.828827 97.240841 97.792734 100.000000 100.000000 95..861243 95.318652 92.909888 92.964233 Table 4: Toxicity Parameters of Designed Ligands and Prototype Clomifene calculated using preADMET server S. No. 1 2 3 4 5 Ligand name AT-1 AT-2 AT-3 AT-4 Clomifene Ames test Mutagen Mutagen Mutagen Mutagen Mutagen Carcinogenicity(Mouse) Negative Negative Negative Negative Positive As per the ADME prediction data it has been predicted that all the designed ligands possibly be well absorbed through intestine and have similar type of plasma protein binding profile that of Clomifene which shows that the designed ligands are as per with the prototype Clomifene in terms of ADME parameters and can be used in future as estrogen antagonist in treatment of endometriosis. Toxicity of all designed molecules have been predicted using PreADMET server and compared with the prototype drug Clomifene (Table 4). It is observed that in Ames test of all designed molecules as well as Clomifene shows mutagenic property. However Clomifene shows positive result in carcinogenicity prediction in mouse and Carcinogenicity(Rat) Positive Negative Positive Negative Negative negative result in carcinogenicity prediction in rat whereas the designed molecule AT-1 and AT-3 showed negative result in carcinogenicity prediction in mouse and positive result in carcinogenicity prediction in rat. Both of the above parameters are negative with the designed molecules AT-2 and AT-4. This predicts that AT-2 and AT-4 will have lower carcinogenic risk than that of prototype Clomifene. CONCLUSION Clomifene is a potent estrogen antagonist which is being effectively used to treat Endometriosis. Our approach was to design the 454 Juvekar et al. Int J Pharm Pharm Sci, Vol 5, Suppl 3, 451-455 molecules which is similar to that of Clomifene and which binds with more competence to the binding site of similar to Clomifene. Our study has given four molecules which demonstrate the better result in in-silico analysis with better binding efficiency (in terms of docking score) towards estrogen receptor than that of Clomifene. The docking result of AT-1,AT-2,AT-3 and AT-4 shows better docking score than that of Clomifene and AT-2 and AT-4 shows no trace of carcinogenic risk in toxicity prediction hence it has been predicted that all our designed ligands can possibly act as new leads for the treatment of endometriosis as they posses estrogen antagonistic activity. ACKNOWLEDGEMENT I would like to extend my sincere thanks to my professor Dr. Venketasan J and to my senior Soumendranath Bhakat. Without them it was impossible for me to complete my project. 9. 10. 11. 12. 13. 14. 15. 16. REFERENCES 17. 1. 2. 3. 4. 5. 6. 7. 8. Bulletti,C.; Coccia,EA.; Battistoni,S.; Borini,A.; Journal of Assisted Reproduction and Geneics; 2010; 27; 8; 441–447. Giudice, L.C.; The New England Journal of Medicine; 2010; 362; 2389-2398. 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