Characterisation of the α1B-Adrenoceptor by Modeling, Dynamics and Virtual Screening Kapil Jain B.Pharm, M.S.(Pharm.) A Thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2018 Institute for Molecular Bioscience 0 Abstract G protein-coupled receptors (GPCRs) are the largest druggable class of proteins yet relatively little is known about the mechanism by which agonist binding induces the conformational changes necessary for G protein activation and intracellular signaling. Recently, the Kobilka group has shown that agonists, neutral antagonists and inverse agonists stabilise distinct extracellular surface (ECS) conformations of the β2-adrenergic receptor (AR) opening up new possibilities for allosteric drug targeting at GPCRs. The goal of this project is to extend these studies to define how the ECS conformation of the α1B-AR changes during agonist binding and develop an understanding of ligand entry and exit mechanisms that may help in the design of specific ligands with higher selectivity, efficacy and longer duration of action. Two parallel approaches were initiated to identify likely functional residues. The role of residues lining the primary binding site were predicted by online web server (Q-Site Finder) while secondary binding sites residues were predicted from molecular dynamics (MD) simulations. Predicted functionally significant residues were mutated and their function was established using FLIPR, radioligand and saturation binding assays. Despite the α1B-AR being pursued as a drug target for over last few decades, few specific agonists and antagonists are known to date. In an attempt to address this gap, we pursued ligand-based approach to find potential new leads. The outcomes of this research will help to understand the GPCR activation process. This research may open the door to the rational development of new modulators that can recognise the ECS of GPCRs in distinct conformational states. Chapter 1 provides an overview of the GPCR including the structure, function, pharmacology and regulation of Class A GPCRs with special reference to the α1-AR. The present work is an effort to address the issues pertaining to the activation of the α1-AR and how the binding of the known ligands affect the ECS of the receptor and stabilise specific conformations. The ligand- based In Silico drug discovery approach is implemented to address rational development of leads which could be subtype specific. The use of computational tools has been shown to study the receptor for drug discovery and development. Chapter2 describes research to characterise the egress pathway of norepinephrine (NE), the endogenous α1-AR agonist. Using a homology model of the α1B-AR built from turkey-β1-AR as template, we performed molecular docking of NE at the orthosteric binding site. Validation of the docking model was performed using mutants generated from site directed mutagenesis and 1 testing those using radiolabelled, functional and binding studies to gain insights into the role played by the residues lining the egress pathway. Residues Trp121, Cys195, Tyr203 and Ser207 were identified to be particularly involved and effect the simulations at two distinct positions which may probably regulate the access and egress of NE to- and from- the orthosteric binding site to the ECS. This study revealed that the negatively charged residues lining the extracellular loop 2 (ECL2) may also provide an allosteric site for the positively charged ligands by retaining them for some time and stabilising the ECS as inferred from our MD studies. Chapter 3 describes research to understand the activation process of the apo and NE-bound α1B- AR using accelerated molecular dynamics (aMD). The receptors in both states were energy minimised, equilibrated and subjected to classical MD for a specific time frame. Residues known from the experimental studies to be involved in receptor activation were mutated individually along with additional double mutants to further characterise their role in activation process. This study identified single (W184A) and double mutant (Y223F-Y348F) in both the apo and NE- bound α1B-AR that preferentially moved the receptor towards the active state. Chapter 4 describes virtual screening (VS) for novel modulators of the α1-AR. Using Rapid Overlay of Chemical Structures (ROCS), 17.9 million drug-like compounds were screened from the ZINC database. The initial results identified 80 “hits” which were confirmed by flexible docking to likely bind either at orthosteric or adjacent allosteric sites along the ligand binding/unbinding pathway. To validate the potential of this set of ligands to target the α1-AR, 12 hits were selected for pharmacological evaluation across the three α1-AR subtypes. The results of our initial characterisation revealed that several ligands that docked in the orthosteric site of the α1B-AR activated all the three α1-AR subtypes confirming the potential of this approach to identify new α1-AR modulators. Chapter 5 provides an overall conclusions drawn from the above work done and future prospects. In summary, this study has revealed the critical involvement of residues in TM3, TM5, TM7, ECL1 and ECL2. This study identified two distinct positions; auxiliary site 1 and auxiliary site 2 which could act as allosteric sites and might be subtype specific. aMD led to identification of single (W184A) and double mutant (Y223F-Y348F) that were close to receptor activation while VS identified leads which activated all the three α1-AR subtypes confirming the potential of this approach to identify new α1-AR modulators. 2 Declaration by the author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. 3 Publications included in this thesis No publications included. Other publications during candidature No manuscripts submitted for publication. Other publications during candidature Peer-reviewed article 1. Brust, Andreas; Croker, Daniel; Colless, Barbara; Ragnarsson, Lotten; Andersson, Asa; Jain, Kapil; Caraballo, Sonia; Castro, Joel; Brierley, Stuart; Alewood, Paul; Lewis, Richard: Conopeptide-derived κ-opioid agonists (conorphins): potent, selective and metabolic stable dynorphinA mimetics with anti-nociceptive properties. Journal of medicinal chemistry 2016, 59(6):2381-2395. Conference abstract 1. Jain K, Ragnarsson L, Lewis RJ (2014) “Identification of functionally active residues in α1B-AR by computational approaches” at 10th International Conference on Chemical structures-German Conference on Chemoinformatics-2014, The Netherlands. 4 Contributions by others to the thesis Invaluable input in establishing research projects, designing experiments and interpreting results, proof reading the thesis has been given by my advisory team Professor Richard Lewis, Dr Lotten Ragnarsson and Asa Andersson. Dr. Lotten Ragnarsson and Asa Andersson assisted me with molecular biology experiments like cell culture, FLIPR assay, radioligand binding assay and saturation binding assay along with data analysis and plotting graphs for Chapter 2 and Chapter 4. Initial molecular dynamics set up was carried out with the help of Dr. Grischa Meyer. Results in chapter 2 and chapter 3 include data from published mutants. I acknowledge the support and co-operation provided to cross-validate the results from steered and accelerated molecular dynamics with the experimental results from Dr. Lotten Ragnarsson work. Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis. Research Involving Human or Animal Subjects No animal or human participants were involved in this research. 5 Acknowledgements I would like to acknowledge my supervisor Prof. Richard Lewis for guiding and giving me the opportunity to complete my PhD in this field of research that I am passionate about. Richard‟s suggestions in presentations, designing experiments, interpreting