AN IN SILICO STUDY OF THE LIGAND BINDING TO HUMAN CYTOCHROME P450 2D6 Sui-Lin Mo (Doctor of Philosophy) Discipline of Chinese Medicine School of Health Sciences RMIT University, Victoria, Australia January 2011 i Declaration I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at RMIT university or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at RMIT university or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project‘s design and conception or in style, presentation and linguistic expression is acknowledged. PhD Candidate: Sui-Lin Mo Date: January 2011 ii Acknowledgements I would like to take this opportunity to express my gratitude to my supervisor, Professor Shu-Feng Zhou, for his excellent supervision. I thank him for his kindness, encouragement, patience, enthusiasm, ideas, and comments and for the opportunity that he has given me. I thank my co-supervisor, A/Prof. Chun-Guang Li, for his valuable support, suggestions, comments, which have contributed towards the success of this thesis. I express my great respect to Prof. Min Huang, Dean of School of Pharmaceutical Sciences at Sun Yat-sen University in P.R.China, for his valuable support. I especially thank A/Prof. Hai-Bin Luo at School of Pharmaceutical Sciences, Sun Yat-sen University, for the opportunity that he provided for me to access to the software used in the present project. I am also thankful to Dr. Wei-Feng Liu in Hospital of Orthopedics and Traumatology in Yuexiu District of Guangzhou, China, for his kind and excellent technical assistance. I also thank Dr. Shi-Hui Liang in Hospital of Orthopedics and Traumatology in Yuexiu District of Guangzhou, China, for her patient help in the construction of databases. I would like to express especially to my wife Mrs. Yu-Ling Chen, PhD candidate at University of New South Wales, Australia, who provided academic advices and spiritual support for my study. I thank my wonderful family for their loving kindness and continuous prayers and spiritual support. iii Summary of Thesis Cytochrome P450 (CYP) 2D6 is an important enzyme, since it can metabolize about 25% of clinical drugs and is subject to inhibition and polymorphism with significant clinical consequences. The elucidation of its crystal structure has provided very useful information on how ligands (e.g., substrates or inhibitors) interact with this enzyme and how CYP2D6 determine its substrate specificity and inhibitor selectivity. However, the resolved structure of CYP2D6 (Protein database (PDB) Code: 2F9Q) is ligand-free, and thus how and whether ligand binding induces conformational changes of the active site are unknown. Although there are reports on the binding of ligands to CYP2D6 using pharmacophore, homology and quantitative structure-activity relationship (QSAR) approaches, the molecular factors affecting the binding of synthetic or herbal compounds to CYP2D6 are not fully elucidated. Herbal medicines are becoming popular all over the world, which may result in adverse effects or potential adverse herb-drug interactions when used in combination with conventional drugs. However, because of their complicated chemical composition and limited in vivo and in vitro approaches, there are limited data on the binding affinities and binding mechanisms of herbal compounds with CYP2D6. In this regard, we hypothesize that a number of physicochemical factors determine the binding of compounds to human CYP2D6, and that a number of compounds share similar structural features, and therefore binding features as CYP2D6 substrates/inhibitors. Thus, the objectives of the current project are: (1) to further explore the molecular factors determining the binding of a compound to native and mutated CYP2D6; (2) to develop a QSAR model for the prediction of binding strength of existing or new compounds to CYP2D6; and (3) to examine the binding of Chinese herbal compounds from Huangqin (S. baicalensis) & Fangjifuling decoction with CYP2D6. In this project, the following work has been done: (1) a set of libraries of CYP2D6 substrates/inhibitors, and identified compounds from a single herb (S. baicalensis) and a herbal formula (Fangjifuling decoction) have been generated; (2) an array of pharmacophore and QSAR models of CYP2D6 substrates/inhibitor have been constructed and validated for the prediction of interaction between compounds and CYP2D6 via in silico approaches; (3) a large number of CYP2D6 substrates and inhibitors have been successfully subject to docking study iv to explore their binding mechanisms to CYP2D6 with/without mutation; and (4) the resultant models have been successfully applied to predict the interaction between herbal compounds and CYP2D6 with/without mutation. In this study, we have developed and validated pharmacophore models for the CYP2D6 substrates/inhibitors. The model for 2D6 substrates consisted of two hydrophobic features and one hydrogen bond acceptor (HBA) feature, giving a relevance ratio (RR) of 76% when a validation set of substrates was tested. Similarly, the pharmacophore model for CYP2D6 inhibitors also consisted of two hydrophobic features and one HBA, with an RR of 78.8%. There were longer distances between feature groups in the model for inhibitors than those of the model for substrates. We also have constructed and validated QSAR models for the prediction of binding affinity of existing or new compounds to CYP2D6. Unfortunately, when the training set was randomly chosen, models generated from 24 CYP2D6 substrates/inhibitors gave regressive equation of y=0.085x+82.824 with R2=0.085 for substrates and y=0.320x+9.879 with R2=0.320 for inhibitors, which all showed poor prediction accuracy. However, two QSAR models from selected 6 substrates and 9 inhibitors presented relatively high R2, in which equation of y=0.980x+2.829 with R2=0.980 was for substrates and y=0.948x+1.051 with R2=0.948 for inhibitors. The relatively high R2 values gave rise to a linear relationship and a better prediction. We have further validated and applied CDOCKER to explore the molecular factors determining the binding of a compound to native and mutated CYP2D6. A large number of CYP2D6 substrates (n=120) and inhibitors (n=33) were subject to molecular docking to the active site of wild-type CYP2D6. Our docking study demonstrated that 117 out of 120 substrates (97.50%) and 30 out of 33 inhibitors (90.91%) could be docked into the active site of CYP2D6. We have demonstrated that 11 residues for substrates and 8 residues for inhibitors played an important role in their binding and consequently determining the metabolic activity towards the substrate and inhibitor selectivity. In present study, the CDOCKER algorithm was also applied to study the impact of mutations of 28 active site residues (mostly non-conserved) in CYP2D6 on substrate/inhibitor binding modes using five probe substrates (bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol) and four known inhibitors (quinidine, pimozide, fluoxetine, and halofantrine). v Apparent changes of the binding modes have been observed with Phe120Ile, Glu216Asp, Asp301Glu mutations for these substrates and inhibitors. To examine the binding of active components from Chinese herbal medicines with CYP2D6, libraries of compounds from Huangqin & Fangjifuling decoction were constructed and subsequently subject to the docking. Overall, 18 out of 40 compounds from Huangqin and 60 out of 130 compounds from Fangjifuling decoction were mapped with our optimized pharmacophore models. Among them, 100% compounds from Huangqin and 90% from Fangjifuling decoction could be docked into the active site of the wild-type CYP2D6, which suggested that they may be substrates and/or inhibitors of CYP2D6. The role of Phe120, Glu216, Asp301, Ser304 for herbal compound binding to CYP2D6 has been further supported by our docking studies. In conclusion, our project has documented the main molecular factors determining the binding of a compound to native and mutated CYP2D6, which allow us to predict and understand the interaction between molecules and CYP2D6. Our study has demonstrated the use of an effective and efficient computational approach to studying the molecular mechanisms of interaction of herbal compounds and functionally important proteins. vi Table of Contents AN IN SILICO STUDY OF THE LIGAND BINDING TO HUMAN CYTOCHROME P450 2D6 .................................................................................................................. i Declaration ....................................................................................................................... ii Acknowledgements......................................................................................................... iii Summary of Thesis…………………………………………………………………….iv Table of Contents ........................................................................................................... vii Table of Figures ............................................................................................................... x Table of Tables…………………………………………………………………………xi Publications..................................................................................................................