University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 1-1-2019 Cheminformatic Approach for Deconvolution of Active Compounds in a Complex Mixture - phytoserms in Licorice Manal Mohammad Alhusban Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Alhusban, Manal Mohammad, "Cheminformatic Approach for Deconvolution of Active Compounds in a Complex Mixture - phytoserms in Licorice" (2019). Electronic Theses and Dissertations. 1954. https://egrove.olemiss.edu/etd/1954 This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. CHEMINFORMATIC APPROACH FOR DECONVOLUTION OF ACTIVE COMPOUNDS IN A COMPLEX MIXTURE - PHYTOSERMS IN LICORICE A Dissertation Presented in partial fulfillment of requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences, Emphasis in Pharmacognosy The University of Mississippi Manal M. Alhusban August 2019 Copyright © 2019 by Manal M. Alhusban All rights reserved ABSTRACT The genus Glycyrrhiza, encompasses several species exhibiting complex structural diversity of secondary metabolites and hence biological activities. The intricate nature of botanical remedies, such as licorice, rendered them obsolete for scientific research or medical industry. Understanding and finding the mechanisms of efficacy or safety for a plant-based therapy is very challenging, yet it remains crucial and warranted. The licorice plant is known to have Selective Estrogen Receptor Modulatory effects (SERMs), with a spectrum of estrogenic and anti-estrogenic activities attributed to women’s health. On the contrary, licorice extract was shown to induce pregnane xenobiotic receptor (PXR), which may manifest as a potential route for deleterious effects such as herb-drug interaction (HDI). While many studies attributed these divergent activities to a few classes of compounds such as liquiritigenin (a weak estrogenic SERM) or glycyrrhizin (weak PXR agonist), no attempt was made to characterize the complete set of compounds responsible for these divergent activities. A plethora of licorice components is undermined, which might have the potential to be developed into novel phytoSERMS or to trigger undesirable adverse effects by altering drug metabolizing enzymes and thus pharmacokinetics. In this work, we explored the mechanism associated with the efficacy and safety of components reported in the licorice plant. We utilized smart screening techniques such as cheminformatics tools to reveal the high number of secondary metabolites produced by licorice, which are capable of interfering with the human Estrogen Receptors (hERs) and/or PXR or other ii vital cytochrome P450 enzymes. After the validation of our in silico models by using the previous knowledge in this area, the alerting phytochemicals from two Glycyrrhiza species (G. glabra and G. uralensis) were clustered. Exhaustive computational mining of licorice metabolome against selected endocrinal and metabolic targets led to the discovery of a unique class of compounds, which belong to the dihydrostilbenoids (DHS) class appended with prenyl groups at various positions. To the best of our knowledge, this interesting group of compounds has not been studied for their estrogenic activities or PXR activation. In addition, some of the bis-prenylated DHS have been reported to be present only in G. uralensis. Thus, we have ventured to synthesize a set of constitutional isomers of stilbenoids and DHS (archetypal of those found in licorice) with different prenylation patterns. Sixteen constitutional isomers of stilbenoids (M2-M10) and DHS (M12-M18) were successfully synthesized, of which six of them (M8, M9, M14, M15, M17, and M18) are synthesized for the first time to be further tested and validated with cell-based methods for their estrogenic activities. We have unveiled a novel class of compounds, which possess a strong PXR activation. These results, which were in accord with the in silico prediction, were observed for multiple synthesized prenylated stilbenoid and DHS by the luciferase reporter gene assay at µM concentrations. Moreover, this activation was further validated by the six-fold increase in mRNA expression of Cytochrome P450 3A4 (CYP3A4), where three representative compounds (M7, M10, and M15) exceeded the activation fold of the positive control. Another aspect of the current project was to predict the phase I primary metabolites of iii compounds found in both species of Glycyrrhiza and assess them with computational tools to predict their binding potential against both isoforms of hERs or drug metabolizing enzymes such as (CYP) inhibition models. Our investigations revealed estrogenic character for most of the predicted metabolites and have confirmed earlier reports of potential CYP3A4 and CYP1A2 inhibition. Compilation of such data is essential to gain a better understanding of the efficacy/safety of licorice extracts used in various botanical formularies. This approach with the involved cheminformatic tools has proven effective to yield rich information to support our understanding of traditional practices. It also can expand the role of botanical drugs for introducing new chemical entities (NCEs) and/or uncovering their liabilities at early stages. iv DEDICATION My Mother Fawzeya Who taught me how to dream, imagine and work hard My Father Mohammad Who taught me how to concentrate on the goals, not obstacles My Husband Ali Who taught me that there are no limits except the limits in our minds My Kids Ghaith, Ra’ad and Salma Who suffered with me the most v LIST OF ABBREVIATIONS AND SYMBOLS μL Microliter μM Micromolar AF-2 Active Functional Region 2 AKT Protein Kinase B Al2O3 Alumina CAR Constitutive Androstane Receptor CART Classification and Regression Tree Algorithm CERAPP Collaborative Estrogen Receptor Activity Prediction database CYP3A4 Cytochrome P450 3A4 DBD DNA-Binding Domain DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCM Dichloromethane DHS Dihydrostilbenoids DMF Dimethylformamide EC50 The concentration at the half maximum efficacy EtOAc Ethyl Acetate EtOH Ethanol US FDA US Food and Drug Administration GST Glutathione S-Transferase vi HDI Herb-Drug Interaction hERs Human Estrogen Receptors Hex Hexanes HMBC Heteronuclear Multiple-Bond Correlation Spectroscopy hrs. Hours LBD Ligand-Binding Domain m/z Mass-to-Charge Ratio MDR1 Phase III P-Glycoprotein or Multidrug Resistance-Associated Protein 1 MEMCl 2-Methoxyethoxymethyl Chloride MeOH Methanol mL Milliliter MOMCl Methoxymethyl Chloride NaH Sodium Hydride nBuLi n-Butyl Lithium NCEs New Chemical Entities NFkβ Nuclear Factor kappa-Light-Chain-Enhancer of Activated B Cells NOESY Nuclear Overhauser Effect Spectroscopy NMR Nuclear Magnetic Resonance NR1I2 Nuclear Receptor Subfamily 1 Group I OATP2 Organic Anion Transporting Polypeptide 2 vii OCHEM Online Chemical Environment PDB Protein Data Bank PXR Pregnane Xenobiotic Receptor phytoSERMs Phytochemicals that act as Selective Estrogen Receptor Modulators QSAR Quantitative Structure-Activity Relationships RBA Relative Binding Affinity RMSD Root-Mean-Square Deviation r.t. Room Temperature SDF Spatial Data Files SERMs Selective Estrogen Receptor Modulators Sult Sulfotransferases TEA Triethyl Amine THF Tetrahydrofuran TLC Thin Layer Chromatography UGTs Uridine Diphosphate (UDP)-Glucuronosyltransferases VDR Vitamin D Receptor VEGA Virtual Models for Property Evaluation of Chemicals Within a Global Architecture viii ACKNOWLEDGMENTS I would like to express my deep gratitude for all the people who helped and supported me throughout my Ph.D. at Ole Miss. Special thanks for my advisor Dr. Ikhlas Khan, who accepted me as a member of his group and supported me and my family. He was always cautious to offer us the best environment to learn and succeed. The guidance, supervision, honesty and critique of Dr. Amar Chittiboyina, my co-advisor, and mentor made me a better scientist. His passion for exploration and discovery is contagious. Although he had a tight schedule, he never skipped a chance for discussion and explanation. When things become impossible in the lab, he would always come and teach us how to troubleshoot. I will always be grateful for him, for his concern to give me the opportunity to engage in diverse projects and for his constant encouragement, kindness, and inspiration. I would also express my sincere thanks to Dr. Robert Doerksen, for his candid lectures and training, help, and guidance throughout the years. I am greatly indebted to him for his relentless support, mentoring and critiques, which greatly affected my academic development. I would like to thank Dr. Samir Ross for his support, advice, and kindness. To Dr. Cole Stevens for his support, effort and time for revising my ORP and dissertation. My special thanks for to Dr. John Rimoldi and Dr. Daneel Ferreira for their great lectures. I am also thankful to all the people in NCNPR who supported me, especially, Dr. Junaid Rehman, I will always be grateful for him and his family for their continuous support and help for my family. For Dr. Ali Zulfiqar, for making NMR easily understandable. I would also thank ix Dr. Shabana Khan, Dr. Jon Parcher, Dr. Bharathi Avula, and Dr. Yan-Hong Wang for their collaboration and advice, and for the post docs who trained me well in the labs, Dr.