Fluorescent Probes for the Labelling of Cardiomyocyte and Mitochondrial Proteins. Thomas D. Z. Pearson
Total Page：16
File Type：pdf, Size：1020Kb
Fluorescent Probes for the Labelling of Cardiomyocyte and Mitochondrial Proteins. A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy. By Thomas D. Z. Pearson 2017 School of Science and Technology Nottingham Trent University Clifton Lane Nottingham NG11 8NS United Kingdom i Declaration I hereby certify that the work embodied in this thesis is the result of my own investigations except where reference has been made to published literature. ii Abstract Organophosphates are a known danger to human health with an array of biochemical targets and complex toxic effects. Phenyl saligenin phosphate (PSP) is an organophosphate and related phenoxy substituted organophosphates irreversibly bind to a variety of important enzymes causing the condition known as organophosphate induced delayed neuropathy (OPIDN). These compounds also potentially cause cardiac muscle damage but little is known about how this class of compounds effect cardiomyocytes. The identification of proteins targeted by these toxins is of interest. This study investigates the synthesis and application of novel fluorescently labelled organophosphates to investigate organophosphate interaction with H9c2 cardiomyoblasts. The synthesis of probes with BODIPY, pyrene and rhodamine fluorescent component is explored with success in preparation of pyrene and rhodamine probes. The fluorescent behaviour of three rhodamine labelled phenyl saligenin phosphates probes with alkyl and PEG linkers is examined and the cytotoxicity of the probes assessed by monitoring MTT reduction, and LDH release from exposed H9c2 cardiomyoblasts. All three analogues showed cytotoxicity (4 h exposure; 100 µM). These probes were found to bind to proteins within mitochondria and spots from 2D-gel electrophoresis experiments, visualised at 532 nm, which are undergoing MS analysis. This project also describes progress towards a photoreactive-fluorescent probe to identify the site of action of the potassium channel opener, diazoxide, which activates cardioprotective pathways. The drug is believed to target mitochondrial KATP channels, but the protein structure of these channels has yet to be elucidated. The fluorescence labelling of whole cardiac cells with a dansyl fluorescent - benzophnone-photoreactoive diazoxide probe has been demonstrated and the progress toward a rhodamine fluorescent analogue is presented. iii Acknowledgements I would like to thank all my friends and family for your support, love and kindness throughout this PhD. Particularly my grandparents and parents, brothers, sister and the current Mrs. Pearson… Rachel all of whom I love lots, and value way more than I probably show. I have immense gratitude for my Supervisors, Chris, John, and Alan who always made time for me when I needed help, thank you for your time and attention. I would like to thank Shatha, Nada, David, Clare, Quentin and Warren for the collaboratory work that made completion of thesis possible. Special thanks to James, Fal, Giulia, Alanood, Laurence, Matteo, Dan, Myles, Jonny, Hitten and Josh for all the time and banter we had in the lab! Finally, I would like to thank God, without whom, my very breath would not be possible. iv List of Acronyms, Abbreviations and Symbols Acronym, Abbreviation or Symbol Name ABC ATP-binding cassette Ac Acetyl ACh Acetylcholine AChE Acetylcholinesterase ADP Adenosine diphosphate ANT Adenine nucleotide translocator aq. Aqueous Ar Aryl / heteroaryl ATP Adenosine triphosphate BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid br Broad BSA Bovine serum albumin CBDP Cresyl saligenin phosphate CDI Carbonyldimidazole d Doublet DCE Dichloroethane DCM Dichloromethane dd Doublet of doublets DiAM Dialkynylimidazole DMAP 4-dimethylaminopyridine DMF N, N-dimethylformamide DMSO Dimethylsulfoxide ε Extinction coefficient eNOS Endothelial nitric oxide synthase eq. Equivalents Et Ethyl FBS Foetal bovine serum h Hour H2O2 Hydrogen peroxide HMQC Heteronuclear multiple quantum coherence HRMS High resolution mass spectrum HSP Heat shock proteins I/R Ischaemia/reperfusion IMS Intermediate syndrome iNOS inducible nitric oxide synthase IPC Ischaemia preconditioning Kca Calcium-activated potassium channel Kir Inwardly rectifying potassium channel subunit m multiplet M1 /M2 Transmembrane unit m. /min. Minutes mABC Mitochondrial ATP-binding cassette MAPK Mitogen-activated protein kinases Me Methyl MitoKATP Mitochondrial ATP-activated potassium channel MnSOD mitochondrial antioxidant enzyme superoxide dismutase mol Mole µM Micromolar Mp Melting point MS Mass spectrometry NADP nicotinamide adenine dinucleotide NBD Nucleotide binding domain NEt3 / TEA Triethylamine v NF-κB transcriptional nuclear factor κB NMR Nuclear magnetic resonance NTE Neuropathy target esterase Nu Nucleophile o Ortho OPIDN Organophosphate induced delayed neuropathy PAGE Polyacrylamide gel electrophoresis PAL Photoaffinity labelling PEG Polyethyl glycol Ph Phenyl PIC Mitochondrial phosphate carrier PKC Protein kinase C PPC Pharmacological preconditioning ppm Parts per million PSP Phenylsaligenin phosphate φ Quantum yield q Quartet Quin quintet Rf Retention factor Rho Rhodamine B ROMK renal outer medullary potassium channel ROS Reactive oxygen species s Singlet Structure and Reactivty SAR SCOTP Saligenin cyclic ortho-tolylphosphate SDH Succinate dehydrogenase SDS Sodium dodecyl sulphate SOD superoxide dismutases SUR Sulfonylurea receptor t Triplet T3P tripropylphosphinic anhydride tBu Tert-butyl TGase Transglutaminase THF Tetrahydrofuran TK Tyrosine kinase TLC Thin layer chromatography TMD Transmembrane domain TOCP Tri-ortho-cresyl phosphate UV Ultra-violet vi Contents Declaration .............................................................................................................................................. ii Abstract .................................................................................................................................................. iii Acknowledgements ................................................................................................................................ iv List of Acronyms, Abbreviations and Symbols ........................................................................................ v 1 Introduction ....................................................................................................................................... 15 1.2 Proteomics ...................................................................................................................................... 15 1.3 Functional Probes ........................................................................................................................... 15 1.4 Organophosphates .......................................................................................................................... 18 1.5 Mechanism of Toxicity .................................................................................................................... 21 1.6 Organophosphates’ Mechanism of Covalent Binding .................................................................... 23 1.7 Hydrolysis Products (Aging) ............................................................................................................ 25 1.8 Interesting targets for organophosphates ...................................................................................... 26 1.9 Focus of Study – Myocardial Cells ................................................................................................... 28 1.10 The Potential Use of an Organophosphate Probe in Protein Identification ................................. 30 1.10.1 Previous work ............................................................................................................................ 30 1.10.2 The synthetic route of an organophosphate probe ................................................................... 32 2 Cardiovascular Disease ...................................................................................................................... 35 2.1 Background ..................................................................................................................................... 35 2.2 Ischaemia-reperfusion injury .......................................................................................................... 36 2.3 Preconditioning ............................................................................................................................... 36 2.4 Diazoxide – Pharmacological Preconditioning ................................................................................ 38 7 2.5 Potassium Channels ........................................................................................................................ 40 2.5.1 Types of potassium channel......................................................................................................... 40 2.5.2 Sarcolemma KATP structure ........................................................................................................... 42 2.5.3 Mitochondrial KATP structure ........................................................................................................ 43 2.6 Mitochondrial Role in Cardiovascular Disease ................................................................................ 44 2.7 Structure and Reactivity of Diazoxide ............................................................................................. 45 2.7.1 Cardioprotective derivatives