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Novel Covalent Probes for Mapping Α7 Nicotinic Acetylcholine Receptor Allosteric Site/S

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Novel Covalent Probes for Mapping Α7 Nicotinic Acetylcholine Receptor Allosteric Site/S Novel Covalent Probes For Mapping α7 Nicotinic Acetylcholine Receptor Allosteric Site/s Master’s Thesis Research Defense by Vasantha Duggirala Advisor: Ganesh A. Thakur, Ph.D Department of Pharmaceutical Sciences Northeastern University November 2013 1 ACKNOWLEDGEMENTS I would like to express my deep appreciation to Dr. Ganesh Thakur for giving me a wonderful opportunity in his Drug Discovery lab and pursue Thesis under his guidance. I am grateful for the quality time he gave me to develop me as a person with character as well as teach science. I am obliged to sit through his medicinal chemistry classes for the strong concepts and realize real time drug discovery research process which I would not have had if I chose to graduate by just doing coursework. I thank him for having confidence in me and boosting my morale when the project was under stake. He is one teacher who not only wishes his students should develop into learned professionals but also into amazing individuals with self-esteem and personality. I would like to thank Dr. Purnima Mungalachetty for her willingness to be my Thesis committee member and spare her precious time out of a very busy schedule to review and guide me through Thesis. I am inspired by her dedication and perseverance to excel in this field and be a leader ultimately. I am very pleased to acknowledge Dr. John S. Gatley, my academic advisor and well-wisher from the bottom of my heart for his confidence in me and to his valuable criticism which helped me give my Thesis a good shape. I am thankful for his willingness to be my Thesis committee member and guide me in the whole process. I am thankful for his good will and time to allow me work with him in developing one of the probes in spite of his tight schedule. I am pleased to thank our post doctoral fellow, Dr. Pushkar Kulkarni who assisted me in various difficult situations like right from handling chemicals to troubleshooting reactions with his strong chemistry knowledge, command over literature and judgment. I would thank him for all the times he guided me during my thesis and provided his help in every aspect he can. I would like to extend my heartfelt gratitude to a senior, friend, mini-teacher, well wisher and project-peer 2 Abhijit Kulkarni for teaching me laboratory techniques, patiently answering my questions, kindly help me overcome my mistakes, helping me manage time while juggling between different activities at school and guiding through my coursework too. I am so happy to work with such an amazing person and thank him for the quality time he spent for me. I would like to thank other lab member and friend Ameya Ranade who shared the burden of course work and thesis all along and help me in whichever way he could. I extend my gratefulness to Prisca Mungalachetty, lab member, best friend, project-peer who stood by me and encouraged to never give up at a point when I thought my project would not go ahead. I would also thank her for assisting me in working with reactions. I am pleased to thank Dr. Roger Kautz for kindly giving me access to use NMR and guide me through basics and troubleshooting. I would like to thank Dr. Jim Glick and Dr. Michael Pollasri who gave me an opportunity to access MS and LC-MS instruments whenever required for my samples. My special thanks to Sarom Lay, administrative assistant who helped me a lot in all the formalities related to the proposal and defense. Finally, I would like to thank my loving parents and siblings for having faith in me to send me overseas to live my dreams and giving me the mental support to keep up high spirit and encourage me to keep going and finally accomplish what I for all throughout. Last but never least I would like to extend my heartfelt gratitude to all my friends who supported me in various ways and making me proud for what I am today. 3 CONTENTS 1. Abbreviation -------------------------------------------------------------------------------------------5 2. List of figures-------------------------------------------------------------------------------------------7 3. List of schemes-----------------------------------------------------------------------------------------7 4. List of tables--------------------------------------------------------------------------------------------8 5. Abstract--------------------------------------------------------------------------------------------------9 6. Goals----------------------------------------------------------------------------------------------------10 7. Introduction -------------------------------------------------------------------------------------------10 7.1 α7 nicotinic acetylcholine receptors--------------------------------------------------------------10 7.2 Localization and neuronal expression of α7 nAChRs in human brain-----------------------12 7.3 α7 nAChRs- targets for treating cognition impairment-----------------------------------------13 7.4 Agonists and Antagonists--------------------------------------------------------------------------14 7.5 Properties of α7 nAChRs---------------------------------------------------------------------------16 7.6 First generation allosteric modulators of a7 nAChRs------------------------------------------17 7.7 Second generation allosteric modulators of a7 nAChRs---------------------------------------18 7.8 Chimeric subunit studies and site-directed mutagenesis---------------------------------------22 7.9 Molecular docking simulations--------------------------------------------------------------------23 7.10 Importance of recently discovered allosteric agonist and PAM, 4BP-TQS---------------24 8. Significance--------------------------------------------------------------------------------------------26 8.1 Ligand binding studies using covalent probes---------------------------------------------------26 8.2 Covalent probe approach---------------------------------------------------------------------------27 8.3 Study of radio-labelled ligand---------------------------------------------------------------------31 9. Schemes and chemistry -----------------------------------------------------------------------------32 4 10. Results and discussion-------------------------------------------------------------------------------38 11. Conclusion--------------------------------------------------------------------------------------------42 12. Experimental------------------------------------------------------------------------------------------42 13. References---------------------------------------------------------------------------------------------56 1. ABBREVIATIONS: ACh– Acetylcholine nAChRs – Nicotinic acetylcholine receptors AD - Alzheimer’s disease ADHD - Attention-deficit-hyperactivity disorders MS – Mass spectrometry NAM – Negative Allosteric Modulator PAM – Positive Allosteric Modulator Ago-PAM – allosteric agonist positive allosteric modulator TQS – 4-Naphthalene-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide 4BP-TQS – 4-(4-bromophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide 4IP-TQS – 4-(4-iodophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide α-BTX – α-Bungarotoxin MLA – Methylcaconitine ECD – Extracellular domain TM – Transmembrane TMD – Transmembrane domain CD – Cytoplasmic domain ERK – Extra cellular signal regulated kinase 5 CREB - cAMP response element binding AChBP - Acetylcholine binding protein 5-HI - 5-Hydroxy indole EC50 – Measure of the potency of a [compound] uPAR - Urokinase plasminogen activator receptor BSA - Bovine serum albumin h- Hour RT – Room temperature 5-HT – 5- Hydroxytryptamine GABA – γ Aminobutyric acid UV – Ultraviolet THF – Tetrahydro furan PCC – Pyridinium Chlorochromate EtOAc – Ethyl acetate DPT – Di-2-pyidyl thionocarbonate DCM – Dichloro methane EtOH - Ethanol CPM – Counts per minute mg – Milli gram mmol – Milli moles µCi – Micro curie nCi – Nano curie equiv – equivalents 6 WR – working reagent BCA – bicinchonic acid 2. LIST OF FIGURES: Figure 1: Schematic representation of homomeric and heteromeric nicotinic acetylcholine receptors Figure 2: Ternary model of each unit of nicotinic acetylcholine receptors Figure 3: Alpha-7 nicotinic acetylcholine receptor agonists Figure 4: Alpha-7 nicotinic acetylcholine receptor antagonists Figure 5: First generation PAMs of α7 nAChRs Figure 6: Second generation Type I PAMs of α7 nAChRs Figure 7: Second generation Type II PAMs of α7 nAChRs Figure 8: Intermediate PAMs of α7 nAChRs Figure 9: Comparison of peak responses of TQS and 4BP-TQS when co-applied with ACh Figure 10: Photolysis of phenyl azide used as covalent probe Figure 11: Photolysis of phenyl diazirine used as covalent probe Figure 12: Isothiocyanate used as an electrophilic affinity covalent probe Figure 13: Brain image of mice after autoradiography 3. LIST OF SCHEMES: Scheme 1: General scheme for the synthesis of 4BP-TQS analogs by microwave-assisted Povarov reaction Scheme 2: Synthesis of 4-azidobenzaldehyde 7 Scheme 3: Synthesis of 4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H- cyclopenta[c]quinoline-8-sulfonamide from 4-(4-amino butyloxy carbonyl phenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide Scheme 4: Synthesis of 4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H- cyclopenta[c]quinoline-8-sulfonamide from 4-(4-nitro phenyl)-3a,4,5,9b tetrahydro-3H- cyclopenta[c]quinoline-8-sulfonamide Scheme 5: Synthesis of 4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H- cyclopenta[c]quinoline-8-sulfonamide from 4-(4-azido phenyl)-3a,4,5,9b tetrahydro-3H- cyclopenta[c]quinoline-8-sulfonamide
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