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Development of Fluorescent Ligands for A1 Adenosine Receptor And Development of fluorescent ligands for A1 adenosine receptor and cannabinoid receptors A dissertation submitted for the degree of Doctor of Philosophy at the University of Otago, Dunedin, New Zealand Sameek Singh July 2018 Abstract Adenosine A1 receptor (A1AR), cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R) are class A G protein-coupled receptors (GPCRs) and play important roles in human pathophysiological conditions such as cardiovascular, neurological, metabolic and immunological disorders. Fluorescent ligands are powerful tools to investigate processes such as receptor expression, localisation, trafficking and receptor-protein interactions in the native cell environment. Fluorescent ligands can also be used as tracers in pharmacological assays, instead of the commonly used radioligands that carry inherent safety risks. The development of fluorescent ligands with high affinity, selectivity and suitable imaging properties for A1AR, CB1R and CB2R would greatly contribute to an increased understanding of receptor biology and thus facilitate the drug development process. Development of fluorescent ligands with sufficient polarity for cannabinoid receptors (CBRs), which have lipid-based endogenous ligands, is an especially challenging task. This thesis describes the development of small molecule- based fluorescent ligands for A1AR, CB1R and CB2R, via attachment of a linker and fluorophore to a ligand. (Benzimidazolyl)isoquinolinols, analogues of previously reported high affinity A1AR (benzimidazolyl)isoquinolines, were explored in chapter 2 for the development of A1AR fluorescent ligands. A procedure for 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) mediated aromatisation of tetrahydroisoquinolines and multistep synthesis for the preparation of (benzimidazolyl)isoquinolinols was developed. Based on the previously reported structure–activity relationship (SAR), linkers (and linker plus fluorophore conjugates) were introduced in the C-6 or C-7 position of (benzimidazolyl)isoquinolinols. Unfortunately, these fluorescent ligands did not exhibit any significant affinity for A1AR in a bioluminescence resonance energy transfer (BRET) assay using the NanoLuc luciferase and it was concluded that (benzimidazolyl)isoquinolinols might not be a suitable scaffold for the development of A1AR fluorescent ligands. NMR spectroscopy and reverse phase HPLC studies showed (benzimidazolyl)isoquinolinols exhibit tautomerism. Chromenopyrazoles, previously reported as high affinity CB1R ligands, were investigated for development into CB1R fluorescent ligands in chapter 3. Based on previous SAR, i linkers then fluorophores were introduced at six different chromenopyrazole positions. Disappointingly, fluorescent chromenopyrazoles did not exhibit high affinity for CB1R in a radioligand binding assay. However, several chromenopyrazoles including a linker conjugate 3.22 and a peptide linker conjugate 3.39 exhibited high affinity for CB2R that were promising candidates for development of CB2R fluorescent ligands. All of the chromenopyrazoles that were evaluated in a cyclic adenosine monophosphate (cAMP) functional assay behaved as agonists at CB2R. Docking studies were carried out using a CB2R homology model and showed that high affinity CB2R chromenopyrazoles with linkers attached likely exit via a cavity located between transmembrane helix (TM) 1 and TM7. In chapter 4, efforts were made to develop high affinity CB2R fluorescent ligands and more polar CB2R linker conjugates that built on the results of chapter 3. The highest affinity CB2R linker conjugate 3.22 (obtained in chapter 3) was conjugated to four different fluorophores (BODIPY-FL, Cy5, TAMRA, BODIPY-630/650) and two high affinity CB2R fluorescent ligands (4.01, 4.02) were obtained. The highest affinity CB2R fluorescent ligand 4.02 (Ki = 41.8 ± 4.5 nM at hCB2R; 5856 ± 1264 nM at hCB1R) behaved as an inverse agonist (4.02, EC50 = 142.0 ± 13.1 nM at hCB2R, 196.7 ± 9.11 % of forskolin response at hCB2R) in the cAMP functional assay and showed CB2R- specific-binding in widefield imaging experiments using CB2R expressing HEK-293 cells. Fluorescent ligand 4.02 exhibited higher affinity for CB2R than any other reported CB2R fluorescent ligands and is the first high affinity CB2R fluorescent ligand for which functional data has been reported (as of July 2018). Fluorescent ligand 4.02 possesses suitable CB2R imaging properties and will be a useful tool for researchers studying CB2R biology in techniques such as fluorescence-based assays, widefield microscopy and flow- cytometry and can be used in resonance energy transfer experiments with other fluorescent partners. In addition, three high-moderate affinity peptide linker conjugates (4.06, 4.07, 4.08) with considerably higher polarity compared to commonly used cannabinoid receptor ligand CP55,940 were obtained, all of which behaved as CB2R agonists. Development of high affinity CB1R ligands based on a reported pyridyl scaffold was explored in chapter 5. Fluorescent ligands were designed using previously reported SAR coupled with results obtained from CB1R docking using the reported CB1R crystal ii structure. O-Linker pyridyl-2-carboxamides and corresponding fluorescent conjugates were prepared via a multistep synthesis. Unfortunately, none of the fluorescent ligands exhibited any significant affinity for CB1R, however, three moderate affinity CB1R linker conjugates (5.33, 5.34, 5.35) were obtained. It was therefore concluded that optimised derivatives of pyridyl-2-carboxamide with different O-linkers or with linkers conjugated at different positions of the pyridine could be another strategy for the development of CB1R fluorescent ligands. iii Acknowledgements First, I would like to say a huge thank you to my primary supervisor Dr Andrea Vernall for her astute guidance, tremendous support and endless patience throughout my PhD. Thank you for providing me the opportunity to work under your supervision, for teaching me a lot of things, and providing guidance with my scientific writing. I would also like to express my sincere gratitude to my co-supervisor Associate Professor Joel Tyndall for his support throughout the PhD and for teaching me the computational studies. My sincere appreciation goes to the many lovely people in the medicinal chemistry group (now the expanded group), who have been part of this journey. Anna – you have been a great friend. It was awesome to have you as my partner in computational studies, in review writing, and in the fume-hood; I am sure you are going to impress everyone at Heptares. Sara you and I started together and you have been very supportive. Thanks for taking care of the lab and tolerating me. Deji, we have a great chemistry between us, the random talks, and the after hour jokes, which I am going to miss. Siddharth and Sumit, the members of after hour club, it has been a pleasure working with you guys. Jess, you are a fellow pharmacist, and it has been a pleasure seeing you transit into a medicinal chemist. The new members of the group – Daniel, Shivaji (part of the badminton club), Fiona, Jasmine, Lohitha, Yasmin, Dhanya – it was real fun working with you guys. I would also like to thank my good friend – Basanth (especially for cooking those delicious dishes), Sassi, Arnold (for the video gaming sessions) and members of the badminton club. I am very grateful to the School of Pharmacy, University of Otago for providing scholarship for my studies throughout the PhD and the Otago medical research foundation for the travel grants. I am thankful to members of Professor Stephen hill’s research group at the University of Nottingham, United Kingdom for carrying out biological studies for the compounds described in chapter 2. I would like to express my gratitude to Professor Michelle glass at the University of Auckland for providing me the opportunity to learn and carry out radioligand binding assays and functional assays for compounds described in chapter 3, 4, and 5. Special thanks go to Christa MacDonald for providing me with the training in radioligand assays, functional assays, and culturing cells for my functional iv studies, and Dr Natasha Grimsey, Caitlin Oyagawa for carrying out widefield imaging experiments. This journey would have been impossible without the support of my family. My elder sister Anu, my younger sister Tanu (for sending me rakhi throughout my time here in New Zealand); my mum, my dad (for all the sacrifices), Manu bhai, and all the young champions of the family. v Table of contents Abstract ............................................................................................................................. i Acknowledgements ......................................................................................................... iv Abbreviations .................................................................................................................. xi Single and three letter amino acid codes ..................................................................... xv Publications ................................................................................................................... xvi Chapter 1 Introduction ......................................................................................... 1 1.1 G protein-coupled receptors ........................................................................... 1 1.2 Chemical tools to study Class A GPCRs ........................................................ 4 1.2.1 Radioligands ........................................................................................
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