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Final Copy 2021 01 21 Algha This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Alghazwani, Yahia S A Title: Characterisation of Compounds as Potential Inhibitors of G Protein-Coupled Receptor Kinases General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Characterisation of Compounds as Potential Inhibitors of G Protein- Coupled Receptor Kinases Yahia Alghazwani BSc, MSc A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy in the Faculty of Life Sciences School of Physiology, Pharmacology and Neuroscience August, 2020 Word count: 33602 Abstract G-protein coupled receptor kinases (GRKs) are key regulators of GPCR signalling, and the search continues for potent and selective GRK inhibitors, for example, for the treatment of congestive heart failure. Compound101 (cmpd101), a GRK inhibitor, was developed by the Takeda Pharmaceuticals Company, and it exhibits some selectivity for the GRK2/3 subfamily. In this thesis, a number of novel cmpd101 analogues were investigated to determine their abilities to inhibit agonist-induced arrestin-3 recruitment to the µ opioid receptor (MOPr) and δ opioid receptor (DOPr) (as a proxy of GRK-induced phosphorylation of the receptor). In addition, the selectivity of cmpd101 and novel analogues towards different GRK isoforms was investigated following cellular overexpression of the GRK isoforms. To further explore the general selectivity of these novel analogues, their effects on agonist-induced Gi activation and MOPr internalisation were also explored. In DAMGO-stimulated cells, novel compounds BU14016, BU16005 and BU16007 showed significantly greater inhibition of arrestin-3 recruitment to MOPr than compound101, whilst compounds BU14013, BU14014 showed significantly less inhibition. In contrast, in cells treated with the lower efficacy MOPr agonist morphine, compound101 did not inhibit arrestin-3 recruitment, whilst only compounds BU16005 and BU14016 had any inhibitory capacity against morphine. Thus, the inhibitory effect of the novel analogues on GRK phosphorylation and following arrestin-3 recruitment to MOPr is apparently agonist-dependent. Interestingly, the novel analogues were not effective at reducing SNC80- or DADLE-induced arrestin-3 recruitment to DOPr. Thus, the effects of the novel analogues may also be GPCR subtype-dependent. Moreover, GRK isoform-overexpression studies with subsequent arrestin-3 recruitment measurements, suggested that the novel analogues show little or no selectivity between the isoforms, although this reflects the key role of GRK2 in promoting initial phosphorylation of MOPr. Overall, this thesis provides new evidence regarding the selectivity of cmpd101 and novel analogues for inhibition of GRK/arrestin function in HEK293 cells. ii Acknowledgement I cannot express enough thanks to my PhD supervisor, Prof. Eamonn Kelly, for his continuous support, encouragement, tireless guidance and patience. His guidance helped me throughout doing the research and writing of this thesis. Without his support, I would not have completed my PhD research. I am extremely grateful for what he has offered me. I could not have imagined having a better advisor for my PhD study. Also, I would like to thank Prof. Graeme Henderson for his motivation, enthusiasm and immense knowledge, and for his constructive comments and support. Special thanks to Dr. Alex Conibear for teaching me all the lab techniques, for the stimulating discussions and insightful comments. Thank you to Dr. Gerda and Dr. Katy Sutcliffe for being supportive and cooperative and for helping me getting the lab work started. Thank you Nokomis and all lab members for your kindness and support. To my caring, loving and supportive wife, Fatimah, thanks from my deepest heart for your encouragement during tough times and during writing my thesis. Thank you for your great and successful management of the house and taking care of the kids while completing my work. Special thanks to my little daughters, Raseel and Samaya, for loving, supporting and understanding of my work. I am extremely grateful to my mother, sisters and brothers for loving, caring, support and prayers along the way. Special thanks to King Khalid University for sponsoring my PhD research and supporting me in my journey towards this degree. iii This thesis is dedicated to ……. my Father’s Soul Yahia iv Author’s Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ............................................................. DATE:.......................... v Abbreviations AA Amino acid AC Adenylyl cyclase AMP Adenosine monophosphate ANOVA Analysis of variance AP2 Adaptor protein 2 β2AR Beta-2 adrenergic receptor AT1R Angiotensin II receptor type 1 ATP Adenosine triphosphate BRET Bioluminescence resonance energy transfer BSA Bovine serum albumin CaMKII Calcium/calmodulin-dependent protein kinase II CCP Clathrin-coated pits CCR5 C-C chemokine receptor type 5 CHO cells Chinese hamster ovary cell CNS Central nervous system CO2 Carbon dioxide CRISPR Clustered regularly interspaced short palindromic repeats DADLE [D-Ala2, D-Leu5]-enkephalin DAMGO [D-Ala2, N-mephe4, Gly-ol]-enkephalin DC assay Detergent compatible protein assay DFS Differential scanning fluorimetry DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethyl sulfoxide DNM Dominant-negative mutant DOPr Delta opioid receptor DPDPE [D-pen2,5]enkephalin EC50 Half maximal effective concentration ECL Extracellular loop EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay EM Cryogenic electron microscopy vi ERK Extracellular signal-regulated kinase FACS Fluorescence-activated cell sorting FBS Foetal bovine serum FDA Food and Drug Administration FITC Fluorescein isothiocyanate FRET Fluorescence resonance energy transfer FSC Forward scatter GABA Gamma-aminobutyric acid GDP Guanosine-5'-diphosphate GFP Green fluorescent protein GIRK G protein-coupled inwardly-rectifying potassium channel GLUT4 Glucose transporter protein type-4 GPCR G protein-coupled receptor GRK G protein-coupled receptor kinase GTP Guanosine-5'-triphosphate HA Human influenza hemagglutinin HDAC5 Class II histone deacetylase 5 HEK293 Human embryonic kidney 293 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HRP Horse-radish peroxidase IC50 He half maximal inhibitory concentration ICL Intracellular loop IRS1 Insulin receptor substrate 1 JNK C-Jun N-terminal kinase KOPr Kappa opioid receptor LB Lysogeny broth LC Locus coeruleus MAPK Insulin receptor substrate 1 ME Met-enkephalin MEF2 Myocyte enhancer factor 2 MOPr Mu opioid receptor MSK Mitogen- and stress-activated kinases NIH National Institute of Health NLS Nuclear localisation sequence U2OS CELL U2-osteosarcoma vii PBS Phosphate-buffered saline PDB Protein data bank PDGFR Platelet-derived growth factor receptor β PDVF Polyvinylidene fluoride PFA Paraformaldehyde PH Pleckstrin homology PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol 4,5, biphosphate PKA Protein kinase A PKC Protein kinase C PRK2 Protein kinase C-related protein kinase RH Regulator of G protein signalling homology RLuc2 Renilla luciferase ROCK1 Rho-associated protein kinase 1 RSK1 P90 ribosomal protein S6 kinase RVM Rostral ventromedial medulla SCG Superior cervical ganglion SDS Sodium dodecyl sulfate SELEX Systematic evolution of ligands by exponential enrichment SEM Standard error of the mean SERT Serotonin transporters SGK1 Serum and glucocorticoid-related kinase SK-N-BE cells Human neuroblastoma cell line SOC Super optimal broth with catabolite repression SRC Proto-oncogene tyrosine-protein kinase SSRI Selective serotonin reuptake inhibitor TBS Tris-buffered saline TBST Tris-buffered saline+Tween20 TM Transmembrane domain UTP Uridine triphosphate WB Western blot WT Wild type YFP Yellow fluorescent protein viii Table of Contents
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