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Identification and Structure Activity Relationship of Small Molecule Antagonists

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Identification and Structure Activity Relationship of Small Molecule Antagonists Identification and structure activity relationship of small molecule antagonists of the human P2X4 receptor. Izzuddin Bin Ahmad A Thesis Presented for the Degree of Doctor of Philosophy to the University of East Anglia Faculty of Science, School of Biological Science © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. 1 Handy note 2 Declaration I hereby declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently submitted for any other degree at University of East Anglia or at any other institution. This thesis contains 55217 words including appendices. 3 Abstract P2X4 is a purinergic receptor distributed all over the body with various roles. Among them, it was reportedly overexpressed in several neuronal and immune cell types following peripheral nerve injury and its activation leads to neuropathic pain. Several compounds were found to block P2X4 but none has gone into a clinical stage, probably due to insufficient information about the compound itself and P2X4 in general. One of such compounds, 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4- diazepin-2-one (5-BDBD) is known to be widely used in P2X4-related studies despite its limited information. Therefore, this study aimed to (i) find a novel compound that can block the activation of P2X4 through high throughput screening and characterise it, and (ii) study structural-activity relationship between 5-BDBD and P2X4. Human P2X4 receptor was stably expressed in human 1321N1 astrocytoma cells and 1710 compounds from National Cancer Institute were screened for their activity at P2X4. Extensive tests led to identifying a natural product (thaspine) as the most potent inhibitor at reducing P2X4 activation. Further characterisation experiments revealed that thaspine had an IC50 value of 3.8 ± 0.2 µM and showed an allosteric mode, time- dependent and irreversible inhibition. Thaspine was similarly potent at mouse P2X4, but not effective at human P2X2, P2X2/3 and P2X7. It was also inactive at human P2Y2 and P2Y6 at concentrations below 10 µM and 30 µM respectively. In primary microglial cell model (BV2), it inhibited ivermectin-potentiated responses but not normal ATP-evoked responses. Meanwhile, 5-BDBD was found to be inhibiting P2X4 receptor competitively and diazepinone was a pivotal group of the structure to cause inhibition. The binding pocket of 5-BDBD at P2X4 was also predicted using molecular docking. In this study, a novel compound thaspine, has been shown to be effective at inhibiting human P2X4 and thus may have potential therapeutic applications while novel information about 5-BDBD was also acquired. 4 Table of Contents Handy note ................................................................................................................... 2 Declaration ................................................................................................................... 3 Abstract ........................................................................................................................ 4 List of Figures .............................................................................................................. 9 List of Tables.............................................................................................................. 12 List of Schemes .......................................................................................................... 13 Abbreviations ............................................................................................................. 14 Conference Abstract ................................................................................................... 17 Acknowledgement...................................................................................................... 18 Dedication .................................................................................................................. 19 CHAPTER 1: INTRODUCTION .............................................................................. 20 1.1 Purinergic signalling .................................................................................. 20 1.1.1 P1 receptors ......................................................................................... 21 1.1.2 P2Y receptors (P2YR) ......................................................................... 21 1.1.3 P2X receptors (P2XR) ......................................................................... 24 1.2 P2X4 receptor ............................................................................................. 27 1.2.1 Molecular structure of P2X4 ................................................................ 27 1.2.2 Polymorphisms in P2X4 ...................................................................... 35 1.2.3 Roles of P2X4 ...................................................................................... 36 1.2.3.1 Roles in the cardiovascular system ............................................... 36 1.2.3.2 Roles in the respiratory system ..................................................... 37 1.2.3.3 Roles in the nervous system .......................................................... 38 1.2.3.4 Roles in the immune system ......................................................... 39 1.2.3.5 Roles in the endocrine system ...................................................... 40 1.2.4 P2X4 and tactile allodynia ................................................................... 40 1.2.4.1 Signals which upregulate P2X4 ..................................................... 41 1.2.4.2 Signalling downstream of P2X4 .................................................... 44 1.2.4.3 Summary of tactile allodynia signalling cascade .......................... 46 1.2.5 P2X4 subcellular localisation and trafficking ...................................... 49 1.2.6 Pharmacology ...................................................................................... 50 1.2.6.1 Agonists ........................................................................................ 50 1.2.6.2 Positive modulators ....................................................................... 50 1.2.6.3 Non-selective antagonists of P2X4 ............................................... 53 1.2.6.3.1 Broad-spectrum antagonists ................................................... 53 1.2.6.3.2 Divalent cations and pH ......................................................... 56 1.2.6.3.3 Antidepressants ...................................................................... 56 1.2.6.3.4 Statins and cholesterol depleting agents ................................ 57 1.2.6.4 Selective antagonists ..................................................................... 57 1.2.6.4.1 Carbamazepine derivatives .................................................... 57 1.2.6.4.2 N-substituted phenoxazine ..................................................... 57 5 1.2.6.4.3 BX430 .................................................................................... 58 1.2.6.4.4 NP-1815-PX ........................................................................... 58 1.2.6.4.5 Duloxetine .............................................................................. 58 1.2.6.4.6 5-BDBD ................................................................................. 59 1.2.7 Structure activity relationship study .................................................... 61 1.3 Objectives................................................................................................... 61 CHAPTER 2: MATERIALS AND METHODS........................................................ 63 2.1 Materials..................................................................................................... 63 2.2 Methods ...................................................................................................... 67 2.2.1 Cell culture .......................................................................................... 67 2.2.1.1 Activation of BV2 cells ................................................................ 68 2.2.2 Generation of DNA recombinant plasmid ........................................... 68 2.2.2.1 Restriction analysis of P2X4 DNA ................................................ 71 2.2.2.2 Insertion of DNA into selected vector .......................................... 71 2.2.2.3 Transformation of recombinant plasmid ....................................... 72 2.2.2.4 Purification and confirmation of recombinant plasmid ................ 72 2.2.2.5 LB broth and agar preparation ...................................................... 73 2.2.2.6 Gel electrophoresis ....................................................................... 73 2.2.3 Generation of P2X4 overexpressing cell lines ..................................... 74 2.2.3.1 Production of lentiviral particles ................................................... 74 2.2.3.2 p24 ELISA and determination of viral titre .................................. 75 2.2.3.3 Transfection into human astrocytoma cells .................................. 76 2.2.3.4 Sorting the P2X4 highly expressing cells ...................................... 77 2.2.4 Measurement of intracellular
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