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Blood-Brain Barrier Shuttles: from Design to Application

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Blood-Brain Barrier Shuttles: from Design to Application Blood-Brain Barrier Shuttles: From Design to Application Pol Arranz Gibert Aquesta tesi doctoral està subjecta a la llicència Reconeixement- NoComercial – SenseObraDerivada 3.0. Espanya de Creative Commons. Esta tesis doctoral está sujeta a la licencia Reconocimiento - NoComercial – SinObraDerivada 3.0. España de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution-NonCommercial- NoDerivs 3.0. Spain License. Blood-Brain Barrier Shuttles: From Design to Application Tesi doctoral Gibert Arranz Pol Pol Arranz Gibert 2016 Programa de doctorat de química orgànica Blood-Brain Barrier Shuttles: From Design to Application Pol Arranz Gibert Tesi doctoral dirigida per: Prof. Ernest Giralt Lledó Dra. Meritxell Teixidó Universitat de Barcelona IRB Barcelona Facultat de Química Programa de Química i Departament de Química Orgànica Farmacologia Molecular Barcelona, 2016. CONTENTS ABBREVIATIONS i INTRODUCTION 1 Origins of the Gatekeepers of the Brain—Evolution of Brain Barriers 3 Understanding the Door—Physiology of the Blood-Brain Barrier 8 Reasons to Look Inside—Physiology and Disease of the CNS: Social Impact 14 Devising a Key—Peptides as Therapeutics and for Drug Delivery to the Brain 16 OBJECTIVES 21 RESULTS AND DISCUSSION 25 Chapter 1: Study of Passive Diffusion BBB Shuttles 27 1.1. Peptide-Shuttle Design 32 1.2. Transport Ability of (PhPro)4 Shuttle Using the PAMPA Assay 33 1.3. Design and Synthesis of a 16-Steroisomer Library of (PhPro)4 37 1.4. Physicochemical Characterization of Pro4 and (PhPro)4 Shuttle 38 1.5. Passive Diffusion Transport Studies and Chiral Discrimination at the BBB 41 Chapter 2: Study of Actively-Transported BBB Shuttles through Receptor-Mediated Transcytosis 45 2.1. Previous Studies with HAI Peptide 48 2.2. Amino Acid Replacement Effect on Transport Study Using a Novel Method for Transport Quantification Based on MALDI-TOF MS 55 Chapter 3: Study of Immunogenic Responses to BBB Shuttle Peptides 71 Chapter 4: Attempts to Develop a Therapy for FRDA at the CNS 83 4.1. Protein Replacement Therapy for Friedreich’s Ataxia at the CNS —Chemistry with Proteins 92 4.2. Gene Therapy for Friedreich’s Ataxia at the CNS —Chemistry with Enveloped Viral Particles 101 CONCLUSIONS 115 EXPERIMENTAL SECTION 119 Materials and Methods 121 Solid-Phase Synthesis of Compounds 123 Peptide and Amino Acid Purification and Characterization 128 Structural Data 130 In Vitro Assays 132 Protein Expression, Purification, Bioconjugation and Characterization 137 HSV-1 Bioconjugation and Characterization 140 In Vivo Experiments 143 Product Characterization 145 Amino Acids 147 Peptides 148 Biologics 164 REFERENCES 167 SUMMARY IN CATALAN 187 ABBREVIATIONS Abbreviations AAA amino acid analysis AAV adeno-associated virus ABC ATP binding cassette Ac acetyl ACH α-cyano-4-hydroxycinnamic acid AcOH acetic acid AJs adherens junctions AM amino methyl AME adsorptive-mediated endocytosis AMT adsorptive-mediated transcytosis amu atomic mass unit AO aminooxy moiety, i.e. aminooxyacetyl aPhe 4-amino-ʟ-phenylalanine apoB100 apolipoprotein B100 apoE apolipoprotein E apoTf apotransferrin ASMS affinity selection of proteins coupled to MS AtFH Arabidopsis thaliana frataxin homolog ATP adenosine triphosphate AU arbitrary units AuNPs gold nanoparticles BAC bacterial artificial chromosome BBB blood-brain barrier BBECs bovine brain endothelial cells BBMVEC bovine brain microvascular endothelial cells BBs brain barriers BMECs brain microvasculature endothelial cells Boc tert-butoxycarbonyl bp base pair BPLE brain polar lipid extract BSA bovine serum albumin C. elegans Caenorhabditis elegans Caco-2 human colorectal adenocarcinoma cell line CBMSO centro de biología molecular Severo Ochoa CD circular dichroism CDX candoxin Cf 5(6)-carboxyfluorescein CFA complete Freund’s adjuvant Cl-HOBt 6-chloro-1-hydroxybenzotriazole CLSM confocal laser scanning microscopy CNS central nervous system COMU 1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino carbenium hexafluorophosphate Conc. concentration CoQ10 coenzyme Q10 COSY correlation spectroscopy CPP cell-penetrating peptide CPs choroid plexuses CSD chemical shift deviation CSF cerebrospinal fluid CyaY Escherichia coli frataxin CYPs cytochrome P450s DBU 1,8-diazabicyclo[5.4.0]undec-7-ene Dbz diaminobenzoic acid iii Abbreviations DCC N,N'-dicyclohexylcarbodiimide DCM dichloromethane De enantiomeric discrimination DHAP 2,6,-dihydroxyacetophenone DIPEA N,N-diisopropylethylamine DIPCDI N,N'-diisopropylcarbodiimide DLS dynamic light scattering DM-I diabetes mellitus type I DMAP 4-dimethylaminopyridine DMEM Dulbecco's modified Eagle medium DMF N,N-dimethylformamide DNA deoxyribonucleic acid Dpr diaminopropionic acid DSS sodium-3-(trimethylsilyl)propanesulfonate DTT dithiothreitol E. coli Escherichia coli ECL enhanced chemiluminescence ECM endothelial cell medium ECs endothelial cells EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide EDT 1,2-ethanedithiol EDTA ethylenediamine tetraacetic acid EFACTS European Friedreich’s Ataxia consortium for translational studies EGFR epidermal growth factor receptor ELISA enzyme-linked immunosorbent assay EPO erythropoietin eq. equivalent Eq. equation ESI-MS electrospray ionization-mass spectrometry FARA Friedreich’s Ataxia Research Alliance FDA food and drug administration FGE formylglycine-generating enzyme fGly formylglycine Fmoc 9-fluorenylmethoxycarbonyl FPhe 4-fluoro-ʟ-phenylalanine FPLC fast protein liquid chromatography FRDA Friedreich’s Ataxia FXN frataxin GABA γ-aminobutyric acid GFP green fluorescent protein GSH glutathione HBSS Hanks’ balanced salt solution HBTU O-(benzotriazol-l-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate hCha homocyclohexyl-ʟ-alanine HDAC histone deactylase HeNe helium-neon HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HFIP hexafluoro-2-propanol HIV-1 human immunodeficiency virus 1 HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole holoTf holotransferrin HPLC high-performance liquid chromatography HPLC-MS high-performance liquid chromatography coupled to mass spectrometry iv Abbreviations HRMS high-resolution mass spectrometry HRP horseradish peroxidase HsFtx human frataxin HSQC heteronuclear single quantum coherence HSV-1 herpes simplex virus type 1 hTf human transferrin IAA iodoacetic acid ICP-MS inductively coupled plasma mass spectrometry IDL intermediate-density lipoprotein IFA incomplete Freund’s adjuvant IFN-γ interferon gamma IGF-1 insulin/insulin-like growth factor 1 IgG immunoglobulin G IgM immunoglobulin M IMAC immobilized metal affinity chromatography INAA instrumental neutron activation analysis i.p. intraperitoneal iPSCs induced pluripotent stem cells ISCU iron-sulfur cluster assembly enzyme JAMs junctional adhesion molecules KLH keyhole limpet hemocyanin ʟ-DOPA ʟ-3,4-dihydroxyphenylalanine LDLR low-density lipoprotein receptor LOD limit of detection LOQ limit of quantification LY lucifer yellow lithium salt MAL maleimide MALDI matrix-assisted laser desorption/ionization MES 2-(N-morpholino)ethanesulfonic acid MetAPs methionine aminopeptidases MHC major histocompatibility complex MLS mitochondrial localization signal MPP mitochondrial processing peptidase MS mass spectrometry MTBE methyl tert-butyl ether MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MW molecular weight nAChR nicotinic acetylcholine receptor NADH nicotinamide adenine dinucleotide Nbz N-acyl-benzimidazolinone NCL native chemical ligation neg. negative NFS1•ISD11 sulfur donor complex NHS N-hydroxysuccinimide NIP (R)-piperidine-3-carboxylic acid, or nipecotic acid NMePhe N-methyl phenylalanine NMR nuclear magnetic resonance NOESY nuclear Overhauser spectroscopy NP nanoparticle o/n overnight Oxyma ethyl (hydroxyimino)cyanoacetate P-gp P-glycoprotein pAbs polyclonal antibodies PAMPA parallel artificial membrane permeability assay v Abbreviations pAb polyclonal antibody Papp apparent permeability Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PBS phosphate-buffered saline PC phosphatidylcholine PDA photodiode array detector PDB protein data bank Pe effective permeability PE phosphatidylethanolamine PEG polyethylene glycol PhPro cis-3-phenylpyrrolidine-2-carboxylic acid PI phosphatidylinositol Pip pipecolic acid PLP pyridoxal-5’-phosphate PMP pyridoxamine-5’-phosphate PPII polyproline II PS phosphatidylserine PUFA polyunsaturated fatty acid PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate QD quantum dot RC random coil ref. reference REMD replica exchange molecular dynamics rD retro-ᴅ-version/ peptide made of ᴅ-amino acids and with the reversed sequence of a parent peptide RME receptor-mediated endocytosis RMSD root-mean-square deviation RMT receptor-mediated transcytosis RNA ribonucleic acid RNAPII RNA polymerase II ROS reactive oxygen species RP-HPLC reversed phase-high-performance liquid chromatography RSV Rous sarcoma virus r.t. room temperature RVG rabies virus glycoprotein SD standard deviation SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEC size-exclusion chromatography SPPS solid-phase peptide synthesis stain. staining SUMO small ubiquitin-like modifier T transport TALE transcription activator-like effector TAT human immunodeficiency virus-1 trans-acting activator of transcription TBST tris-buffered saline with Tween 20 tBu tert-butyl TCEP tris(2-carboxyethyl)phosphine TEER transendothelial electrical resistance TEM transmission electron microscopy TFA trifluoroacetic acid Tf transferrin TfR transferrin receptor
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