Investigating the pharmacology of novel 5-HT3 receptor ligands; with the potential to treat neuropsychiatric and gastrointestinal disorders by Alexander Roberts A thesis submitted to the University of Birmingham for the Degree of Doctor of Philosophy Institute of Clinical Sciences College of Medical and Dental Sciences University of Birmingham February 2020 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The 5-hydroxytryptamine (5-HT; serotonin) 5-HT3 receptor is an excitatory ligand- gated ion channel expressed in for example the brain and the gastrointestinal tract. Two major subtypes of the receptor have been studied in the most detail; the homomeric 5-HT3A receptor and the heteromeric 5-HT3AB receptor. 5-HT3 receptor antagonists are used clinically to treat chemotherapy induced and post-operative nausea and vomiting, and demonstrate symptomatic relief in diarrhoea-predominant irritable bowel syndrome (IBS-d); but unfortunately, these medications cause adverse effects such as constipation or rarely ischemic colitis in the latter condition. This study has characterised the pharmacology of two structurally distinct 5-HT3 receptor partial agonists (vortioxetine and CSTI-300); and identified the unique binding properties of the cryptic orthosteric modulator 5-chloroindole (Cl-indole) for the human (h) 5-HT3 receptor. Vortioxetine is a multi-modal antidepressant, which displays affinity for the 5-HT transporter as well as a multitude of 5-HT receptors, including the 5-HT3 receptor. The first part of this study has identified vortioxetine as a relatively high affinity, competitive 5-HT3 receptor partial agonist with an intrinsic efficacy of approximately 40-50% that of 5-HT. Given the safety of vortioxetine in patients, it has the potential to be trialled in other conditions for which 5-HT3 receptor antagonists demonstrate efficacy but cause adverse effects, such as IBS-d or even schizophrenia. The second section of this study has identified CSTI-300 as a selective, high affinity 5-HT3 receptor partial agonist (intrinsic activity 30-40% that of 5-HT). Moreover, in a rodent model of IBS-d, CSTI-300 demonstrated comparable efficacy to alosetron, a 5-HT3 receptor antagonist with established efficacy in treating IBS-d. CSTI-300 is predicted to be able to relieve symptoms of IBS-d in patients without eliciting the side effect profile associated with 5-HT3 receptor antagonism. The final part of this study has demonstrated that the binding mechanism of Cl-indole for the 5-HT3A receptor is modulated by 5-HT3 receptor agonists, but not 5-HT3 receptor antagonists. This could have implications in designing 5-HT3 receptor allosteric ligands for potentially treating conditions such as IBS-d. Acknowledgements I would firstly like to thank Professor Nicholas Barnes for the opportunity to work for Celentyx and in the academic lab. I am grateful for his knowledge, experience and advice during my time here. I would also like to thank Dr Andy Powel for his advice and help. Furthermore, I am also grateful to Dr Gillian Grafton for all her help and expertise. I wish to give many thanks to Dr Fay Stewart, Lindsay Bentley, Tina Tang and Gillian Mackie, for all the fun nights out and adventures. To my wife Jess, for all your support and patience, and for organising our wedding. To my son Evan, I hope I can make you proud. “You don’t expect to be at the top of the mountain the day you start climbing” Ron Dennis Table of Contents 1 Introduction ............................................................................................................ 1 1.1 Discovery of 5-HT .............................................................................................. 1 1.2 Localisation of 5-HT ........................................................................................... 2 1.3 5-HT receptors ................................................................................................... 3 1.3.1 5-HT1A receptor ................................................................................................. 5 1.3.2 5-HT1B receptor ................................................................................................. 6 1.3.3 5-HT1D receptor ................................................................................................. 6 1.3.4 5-ht1e receptors ................................................................................................. 6 1.3.5 5-HT1F receptors ................................................................................................ 7 1.3.6 5-HT1p receptors ................................................................................................ 7 1.3.67 5-HT2A receptor ................................................................................................ 7 1.3.8 5-HT2B receptor ................................................................................................. 8 1.3.9 5-HT2C receptor ................................................................................................. 8 1.3.10 5-HT4 receptor ................................................................................................. 8 1.3.11 5-HT5A receptors ............................................................................................. 9 1.3.12 5-ht5b receptors ............................................................................................... 9 1.3.13 5-HT6 receptor ............................................................................................... 10 1.3.14 5-HT7 receptor ............................................................................................... 10 1.3.15 5-HT3 receptor ............................................................................................... 10 1.4 Cys-loop receptors ............................................................................................. 11 1.5 5-HT3 receptor structure ................................................................................... 11 1.6 Subunits of the 5-HT3 receptor ........................................................................... 16 1.7 5-HT3 receptor localisation ................................................................................. 19 1.8 5-HT3 receptor pharmacology ............................................................................. 23 1.9 Introduction to allosteric modulation ................................................................... 24 1.10 5-HT3 receptor allosteric modulation ................................................................ 26 1.11 Irritable bowel syndrome ................................................................................. 31 1.12 Vortioxetine ...................................................................................................... 37 1.13 Aims ................................................................................................................. 42 1.14 Hypotheses ...................................................................................................... 43 2 Methods ............................................................................................................... 44 2.1 Cell culture ......................................................................................................... 44 2.2 Preparing whole cell lysates ............................................................................... 45 2.3 Bradford assay ................................................................................................... 46 2.4 Intracellular calcium assays ............................................................................... 46 2.4.1 Agonist experiments ........................................................................................ 46 2.4.2 Antagonist studies ........................................................................................... 47 2.5 Radioligand binding ............................................................................................ 48 2.5.1 Competition radioligand binding ...................................................................... 48 2.5.2 Saturation radioligand binding ......................................................................... 49 2.5.3 Scintillation Proximity Assay ............................................................................ 50 2.5.4 Electrophysiology ........................................................................................... 50 2.5.5 Rat Bezold-Jarisch reflex ............................................................................... 51 2.5.6 Rat colon distention model ............................................................................. 52 2.5.7 Dog emesis and behavioural studies .............................................................. 53 2.5.8 Pharmacokinetic studies ................................................................................ 55 2.6 Data analysis .....................................................................................................