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Design, Synthesis and Characterization of Galanin Receptor Selective Ligands

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!" #$"% "!&$$ ' () * ( +",-& . #/0!$ !$ 1 &2 . &2 "3 ( &. 4 . ( ( ( . &5 ( &6 ( ( 4 ( ( 4 & 0 & ( & 2 & " # (3 7#""8&9 (. : 4 4 & # 4 4 "& ( . : & ! # : & ; &7!/<8 4 & 9 ( ( & !" #$"% =00 && 0 > ? == = = "!/@@$ 9-/%@/"%,;/%#"$ 9-/%@/"%,;/%##% # $% # ("$,/" Design, Synthesis and Characterization of Galanin Receptor Selective Ligands Kristin Webling Abstract Galanin is a 29/30 amino acid long bioactive peptide discovered over 30 years ago when C-terminally amidated peptides were isolated from porcine intestines. The name galanin originates from a combination of the first and last amino acids - G from glycine and the rest from alanine. The first 15 amino acids are highly conserved throughout species, which indicates that the N-terminus is important for receptor recognition and binding. Galanin exerts its effects by binding to three different G protein-coupled receptors, which all differ according to regional distribution, the affinity for shortened galanin fragments, as well as the intracellular G-protein signaling cascade used. When first discovered, galanin was found to cause muscle contraction as well as hyperglycemia. Over the years, galanin has been reported to be involved in a wide variety of biological functions, for example food intake and neurogenesis, and pathological functions, for example epilepsy and de- pression. Determining the specific involvement of the three different galanin recep- tors in biological and pathological processes is limited by the small amount of galanin receptor selective/specific ligands available as research tools. Furthermore, the fast degradation of peptides limits the administration routes in animal studies. This thesis aims at developing new galanin receptor-selective ligands to help delineate the involvement of the three different galanin receptors. Paper 1 presents the shortest galanin fragment with a galanin receptor 2 specific binding preference where only a single amino acid substitution was made, Ala5Ser in galanin (2-11). In addition, G-protein coupled receptor signaling were evaluated through both a classical second messenger assay and a real-time label-free technique in cells overexpressing the receptor as well as low receptor expression. Paper 2 demonstrates that the neuroprotective effects of galanin in a kainic acid-induced excitotoxic animal model were mediated through galanin receptor 1. Furthermore, a new robust protocol for evaluating G- protein signaling using a label-free real time impedance technique was pre- sented and compared to two different classical second-messenger assays. Paper 3 presents a series of systemically active galanin receptor 2 selec- tive ligands subsequently evaluated in two different depression-like animal models. Paper 4 investigates a mutated form of human galanin which was found in epilepsy patients and binding and signaling properties of the mutated associ- ated ligand p.(A39E) was examined. In conclusion, this thesis presents the discovery of eight new galanin lig- ands, which can be used to evaluate the galaninergic system as well as to help investigate the possible use of peptides as pharmaceuticals in different diseases. Publications This thesis is based on the following publications 1) Webling, K., Runesson, J., Lang, A., Saar, I., Kofler, B., Langel, Ü., 5 2016. Ala -galanin(2-11) is a GAL2R specific galanin analogue. Neuropeptides, 60, 75-82. 2) Webling, K*., Groves-Chapman, J.L*., Runesson, J., Saar, I., Lang, A., Sillard, R., Jakovenko, E., Kofler, B., Holmes, P.V., Langel, Ü., 2015. Pharmacological stimulation of GAL1R but not GAL2R atten- uates kainic acid-induced neuronal cell death in the rat hippocampus. Neuropeptides, 58, 83-92. 3) Saar, I., Lahe, J., Langel, K., Runesson, J., Webling, K., Järv, J., Rytkönen, J., Närvänen, A., Bartfai, T., Kurrikoff, K., Langel, Ü., 2013. Novel systemically active galanin receptor 2 ligands in de- pression-like behavior. J. Neurochem. 127, 114-123. 4) Guipponi, M., Chentouf, A., Webling, K.E., Freimann, K., Crespel, A., Nobile, C., Lemke, J.R., Hansen, J., Dorn, T., Lesca, G., Ryvlin, P., Hirsch, E., Rudolf, G., Rosenberg, D.S., Weber, Y., Becker, F., Helbig, I., Muhle, H., Salzmann, A., Chaouch, M., Oubaiche, M.L., Ziglio, S., Gehrig, C., Santoni, F., Pizzato, M., Langel, Ü., Anto- narakis, S.E., 2015. Galanin pathogenic mutations in temporal lobe epilepsy. Hum. Mol. Genet. 24, 3082-3091. * These authors contributed equally to the project Additional publications Publications not included in this thesis • Webling, K., Langel, Ü., 2017. Novel peptide based galanin receptor 3 selective antagonists. (Manuscript) • Yu, X., Munoz-Alarcón, A., Ajayi, A., Webling, K.E., Steinhof, A., Langel, Ü., Ström, A.L., 2013. Inhibition of autophagy via p53-mediated disruption of ULK1 in a SCA7 polyglutamine disease model. J. Mol. Neurosci. 50, 586- 599. • Muñoz-Alarcón, A., Helmfors, H., Webling, K., Langel, Ü. 2013. Cell-Penetrating Peptide Fusion Proteins. In: Fusion Protein Technologies for Biopharmaceuticals, (Hoboken, NJ, USA: John Wiley & Sons, Inc.). 397-411. ISBN:978-0-470-64627-4 • Webling, K.E., Runesson, J., Bartfai, T., Langel, Ü., 2012. Galanin receptors and ligands. Front Endocrinol (Lau- sanne). 3:146 • Abela, D., Ritchie, H., Ababneh, D., Gavin, C., Nilsson, M.F., Khan, M. K., Carlsson, K., Webster, W.S. 2010. The effect of drugs with ion channel-blocking activity on the ear- ly embryonic rat heart. Birth Defects Res B Dev Reprod Tox- icol.89, 429-440. This PhD-thesis is an expansion of the work previously presented in my Licentiate thesis, ISBN: 978-91-7649-338-0, where Paper 2 and 3 were included. 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" # $ % < 2 3***. .& "# # 0 %!)= 2 3*********************************. 9 "!'%"!% 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444 @9 @ "#'0$(&!%# % !&&!! #1 %(!% 444444444444444444444 @A A !") !&% 4444444444444444444444444444444444444444444444444444444444444444444444444444444444 @C B !$!% 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444 A7 Abbreviations 5-HT Serotonin or 5-hydroxytryptamine AC Adenylyl cyclase ACh Acetylcholine ARC Hypothalamic arcuate nucleus BBB Blood-brain barrier BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate CHO Chinese hamster ovary CNS Central nervous system DIC Diisopropylcarbodiimide Dmt 2,6-dimethyl-L-tyrosine DR Dorsal raphe nucleus DRG Dorsal root ganglia ECL Extracellular loop EDTA Ethylenediaminetetraacetic acid EEG Electroencephalography EM Electron microscopy ER Endoplasmic reticulum ERE Estrogen responsive element ESI-MS Electrospray ionization mass spectrometry FAM 5(6)-carboxyfluorescein Fmoc 9-Fluorenylmethyloxycarbonyl GABA Gamma-aminobutyric acid GALP Galanin-like peptide GAL1R Galanin receptor 1 GAL2R Galanin receptor 2 GAL3R Galanin receptor 3 GIRK G protein-regulated inwardly rectifying K+ GPCR G-protein coupled receptor GTP Guanosine triphosphate HEK Human embryonic kidney HPLC High-performance liquid chromatography ICL Intracellular loop i.c.v. Intracerebroventricularly i.p. Intraperitoneal IP Inositol phosphate
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    The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal Venetia Zachariou*†, Darlene H. Brunzell*, Jessica Hawes*, Diann R. Stedman*, Tamas Bartfai‡, Robert A. Steiner§, David Wynick¶,U¨ lo Langelʈ, and Marina R. Picciotto*,** *Department of Psychiatry, Yale University School of Medicine, New Haven, CT 06508; †Faculty of Medicine, Department of Pharmacology, University of Crete, 711 10 Crete, Greece; ‡Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037; §Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195; ¶Department of Medicine, Bristol University, Bristol BS2 8HW, United Kingdom; and ʈDepartment of Neurochemistry and Neurotoxicology, Stockholm University, 106 91 Stockholm, Sweden Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved June 2, 2003 (received for review May 23, 2003) Much research has focused on pathways leading to opiate addic- galanin-mediated effects on morphine withdrawal were exam- tion. Pathways opposing addiction are more difficult to study but ined in transgenic mice that overexpress the peptide under the may be critical in developing interventions to combat drug depen- control of the dopamine-␤-hydroxylase (D␤H) promoter, which dence and withdrawal. Galanin decreases firing of locus coeruleus normally drives gene expression in noradrenergic neurons (20), neurons, an effect hypothesized to decrease signs of opiate with- and in studies of c-fos and tyrosine hydroxylase (TH) phosphor- drawal. The current study addresses whether galanin affects mor- ylation in the LC of wild-type mice administered galnon. phine withdrawal signs by using a galanin agonist, galnon, that crosses the blood–brain barrier, and mice genetically engineered Methods to under- or overexpress galanin peptide.
  • Identification of a Novel Synaptic G Protein-Coupled Receptor Controlling Nicotine Dependence and Withdrawal

    Identification of a Novel Synaptic G Protein-Coupled Receptor Controlling Nicotine Dependence and Withdrawal

    Identification of a novel synaptic G protein-coupled receptor controlling nicotine dependence and withdrawal Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin by Beatriz Antolin Fontes from Girona, Spain Berlin, March 2014 This work was carried out in the period from June 2010 until March 2014 under the supervision of Dr. Inés Ibañez-Tallon and Prof. Dr. Constance Scharff at the Max- Delbrück-Center for Molecular Medicine (MDC) in Berlin and at The Rockefeller University in New York. 1st Reviewer: Dr. Inés Ibañez-Tallon 2nd Reviewer: Prof. Dr. Constance Scharff Date of defense: 18.06.2014 Scientific Acknowledgments I would like to express my sincere gratitude to all the people who made this thesis possible: - My supervisor Dr. Inés Ibañez-Tallon: For your advice, support and supervision throughout the years. Thank you for believing in me from the first moment, for giving me the opportunity to do research in different outstanding environments and specially, for transmitting always motivation and inspiration. I could not wish for a better supervisor. - My supervisor Prof. Dr. Constance Scharff from the Freie Universität Berlin: For your supervision and advice. - Prof. Dr. Nathaniel Heintz: For your valuable support and for so many useful and constructive recommendations on this project. - My fellow lab members, both current and past: Dr. Silke Frahm-Barske, Dr. Marta Slimak, Dr. Jessica Ables, Dr. Andreas Görlich, Dr. Sebastian Auer, Branka Kampfrath, Cuidong Wang, Syed Shehab, Dr. Martin Laqua, Dr. Julio Santos-Torres, Susanne Wojtke, Monika Schwarz-Harsi, and all Prof.