Substrate Selectivity Profiling of the Human Monoamine Transporters
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DISSERTATION Titel der Dissertation Substrate Selectivity Profiling of the Human Monoamine Transporters Verfasst von Amir Seddik, B.Sc., M.Sc. angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr. rer. nat.) Wien, 2015 Studienkennzahl lt. Studienblatt: A 796 610 449 Dissertationsgebiet lt. Studienblatt: Pharmazie, DK: Molecular Drug Targets Betreut von: Univ.-Prof. Mag. Dr. Gerhard F. Ecker A. Seddik - Substrate Selectivity Profiling of the Human Monoamine Transporters A. Seddik - Substrate Selectivity Profiling of the Human Monoamine Transporters Acknowledgement Hereby I would like to express my sincere gratitude to Prof. Gerhard F. Ecker, who has integrated me into the scientific community by letting me join his research group. I am thankful for his training during all these years, which has formed me into a very independent researcher. I thank him for his time and support and I admire his ambitions and interest for integrating students on European and international level. It has been an honor to work at the pharmaceutical department of the University of Vienna in this beautiful city. Gerhard, thank you for the great time. My gratitude goes out to Michael Freissmuth and my co-supervisor Harald H. Sitte with whom we had very successful collaborations and I thank them for giving me the opportunity to learn the experimental methods. I acknowledge the support from the MolTag program, to which I have applied for in the first place. The consortium has proven that collaboration between groups of different expertise is educative and beneficial to publish in world-class journals. I thank Steffen Hering, Gerhard Ecker, Marko Mihovilovic, Margot Ernst, Doris Stenitzer and Sophia Khom for devoting their time to the management of the project. I thank the scientific advisory board for the critical feedback during the retreats. My gratitude goes out to Thomas Stockner; the molecular dynamics simulations on the transporter systems would not have been possible without him. Many thanks go out to Nico Vermeulen and Daan Geerke in Amsterdam, for their hospitality during my internship. Foremost I am grateful to Daan for his patience and interest in my work, while giving me the opportunity to learn about the fundamentals of biomolecular simulation. I would like to thank Chris Oostenbrink for organizing the exciting ABC seminars and giving me the honor to present there, and thank Thierry Langer for the stimulating feedback on my presentation. Especially I will miss the warm-hearted Pharmacoinformatics colleagues: I thank Barbara Zdrazil for supervising and introducing me into the field of computational drug-design. Many thanks go out to so many people that I have worked together with and that were around: Lars, Nathan, Thomas Haschka, Marta, Melanie, Chonticha, René, Andreas, Freya, Daniela Digles, Yogesh, Ishrat, Andrea, Daria, Floriane, Doris, Eva, Eleni, Michael, Taymara, Alina, Theresa, Dennis, Alexandra, Bernhard, Sankalp, Priska, Lydia, Eugenia, Noor, Katharina Prokes, Katharina Bulyha, Marian, Victoria, Natesh, Petra, Anika, Katharina Krammler and Daniela Past. Thank you for the great time I had and for always being there for me. I wish everyone the best of luck in their career. Certainly I will miss the educational and fun time I had in Amsterdam, thanks to Luigi, Ruben, Marc, Koen and Rosa, who I wish great luck with their further projects. Of course I will miss the members of the Währingerstrasse, who devote their life to science and with whom I enjoyed the fruitful discussions, Walter, Sonja, Kusumika, Marion, Oliver, Felix, Ali, Tina, Thomas Steinkellner and many others I thank for the supervision and the great time in the wet-lab. I have learned a lot in a short amount of time thanks to them. My love goes out to my father who unfortunately passed away during my Master’s. I thank my mother, Selma and my family for always being there when I visited the Netherlands. My love goes out to Laura who has survived living with a PhD student. 3 A. Seddik - Substrate Selectivity Profiling of the Human Monoamine Transporters 4 A. Seddik - Substrate Selectivity Profiling of the Human Monoamine Transporters INDEX Acknowledgement 3 List of Abbreviations 6 Abstract 7 I Background 9 Motivation 10 A Biological Background 11 a Monoamine Transporter Function and Classification 11 b Monoamine Transporter Structure 12 c Pharmacochemistry of Monoamine Transporter substrates 14 B Methods in Computational Drug design 17 a Homology Modelling 17 b Docking 18 c Molecular Dynamics 21 d Free Energy Calculations 22 e Quantitative Structure-Activity Relationship (QSAR) studies 24 C Experimental Methods 26 a Site-directed mutagenesis 26 b Transient transfection 27 c Uptake inhibitory measurements 27 Bibliography 28 II Results and Discussion 31 A Probing the Selectivity of Monoamine Transporter Substrates by Means of 32 Molecular Modeling B Combined Hansch analysis and Docking of Cathinone Designer Drug Analogs in 38 the Serotonin Transporter C Molecular Dynamics studies on (4-iodo)-methcathinone binding to DAT and 52 ‘SERT’-ized DAT D ‘Second-Generation’ Mephedrone Analogs, 4-MEC and 4-MePPP, Differentially 65 Affect Monoamine Transporter Function E Aminorex, a metabolite of the cocaine adulterant levamisole, exerts 77 amphetamine like actions at monoamine transporters F Synthesis and in Silico Evaluation of Novel Compounds for PET-Based 88 Investigations of the Norepinephrine Transporter Concluding Remarks 108 Appendix 110 Curriculum Vitae 115 5 A. Seddik - Substrate Selectivity Profiling of the Human Monoamine Transporters List of Abbreviations 4-MEC 4-Methyl-N-ethylcathinone MOE Molecular Operating Environment + 4-MePPP 4'-Methyl-α-pyrrolidinopropiophenone MPP Methylpyridinium 5-HT 5-hydroxytryptamine MR Molar refractivity 5-HTR 5-hydroxytryptamine receptor MSA Multiple Sequence Alignment + Aa Aquifex aeolicus Na Sodium ion ADHD Attention-Deficit Hyperactivity Disorder NaOH Sodium Hydroxide APC Amino-acid/Polyamine/Organocation NCBI National Center for Biotechnology Information Arg Arginine NE Norepinephrine Asp Aspartate NET Norepinephrine Transporter BAT Biogenic Amine Transporter NMR Nuclear Magnetic Resonance BBB Blood-Brain-Barrier NSS Neurotransmitter/Sodium Symporter BLAST Basic Local Alignment Search Tool OG Octyl glucoside CDCl 3 Deuterated Chloroform OPLS Optimized Potentials for Liquid Simulations Cl- Chloride PEA Phenylethylamine COPII Coat Protein II PB Poisson-Boltzmann COS CV-1 Origin SV40 PCR Polymerase-chain reaction DA Dopamine PDB Protein Data Bank DAT Dopamine Transporter PDL poly-d-lysine DMEM Dulbecco's Modified Eagle's medium PET Positron Emission Tomography DMF Dimethylformamide PLP Piecewise Linear Potential DMSO Dimethyl Sulphoxide PLS Partial-Least Squares DNA Deoxyribonucleic Acid PME Particle Mesh Ewald summation dNTP Deoxyribonucleophosphate POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine DOPE Discrete Optimized Protein Energy POVME Pocket Volume Measurer Dpn Diplococcus pneumoniae PP Phenylpiperazine EBI European Bioinformatics Institute QM Quantum mechanics EL Extracellular loop QQ Electrostatics GA Genetic Algorithm QSAR Quantitative Structure-Activity Relationship GAT Gamma-amino-butyric acid Transporter RCSB Research Collaboratory for Structural Bioinformatics GB Generalized Born RMSD Root-mean-square Deviation Glu Glutamate SA Surface area GlyT Glycine Transporter SAR Structure-Activity Relationship GOLD Genetic Optimization for Ligand Docking SDS Sodium Dodecyl sulfate GRIND Grid Independent Descriptors SERT Serotonin Transporter HB Hydrogen Bond SFF (S)-fenfluramine HCl Hydrogen Chloride SLC Solute Carrier HEK Human Embryonic Kidney SMILES Simplified Molecular-input line-Entry System HeLa Henrietta Lacks SNRI Selective Norepinephrine Reuptake Inhibitor HEPES 2-[4-(2-OH-Et)piperazin-1-yl]ethanesulfonic acid SSRI Selective Serotonin Reuptake Inhibitor IFD Induced-Fit Docking SPC Single point charge IMAP 2-(methylaminol-l-(4-iodophenyl)propan-l-one SVL Scientific Vector Language KCl Potassium Chloride TCA Tricyclic Antidepressant keV kilo electron volt TI Thermodyna mic Integration LeuT Leucine Transporter Tris tris(hydroxymethyl)aminomethane LB Lysogeny Broth vdW Van der Waals LC-MS Liquid Chromatography/Mass VMD Visual Molecular Dynamics LINCS LinearSpectrometr Constraint Solver VSA van der Waals Surface Area LJ Lennard-Jones WT Wild-type Lys Lysine MAT Monoamine Transporter MCAT Methcathinone MD Molecular Dynamics MDMA 3,4-methylenedioxymethamphetamine MEPH Mephedrone MLR Multiple Linear Regression MM Molecular mechanics 6 A. Seddik - Substrate Selectivity Profiling of the Human Monoamine Transporters Abstract The serotonin, dopamine and norepinephrine transporter proteins (SERT, DAT, NET, respectively) are collectively named as the monoamine transporters (MATs) and are involved in a variety of psychiatric disorders such as depression, anxiety, addiction and attention-deficit hyperactivity disorder. The high sequence identity and functional similarity to each other, despite their involvement in different behaviors and disorders, have made them a central topic in life sciences research during the last decades. Small molecules that bind to the MATs can be divided into inhibitors and substrates, whose selectivity for the latter has been thoroughly studied during this thesis, using computational and biochemical methods. The binding modes of endogenous substrates such as dopamine, norepinephrine and serotonin have been suggested previously, but the binding and selectivity of exogenous compounds not extensively.