title: Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.
abstract: The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028, Barcelona, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Guixer
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028, Barcelona, Spain.|Intelligent Pharma, Baldiri Reixac 4, 08028, Barcelona, Spain.
authors.forename: X
authors.initials: X
authors.lastname: Arroyo
authors.affiliation: Intelligent Pharma, Baldiri Reixac 4, 08028, Barcelona, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Belda
authors.affiliation: Proteomics Unit, Centre de Regulació Genòmica (CRG), Dr. Aiguader 88, 08003, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Sabidó
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028, Barcelona, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Teixidó
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028, Barcelona, Spain.|Department of Organic Chemistry, University of Barcelona, Martí Franquès 1-11, 08028, Barcelona, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Giralt
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D018920:Coculture Techniques; D020650:Combinatorial Chemistry Techniques; D015194:Diffusion Chambers, Culture; D004337:Drug Carriers / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008954:Models, Biological; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D010539:Permeability; D061251:Primary Cell Culture; D055550:Protein Stability; D051381:Rats
chemical_list: D002352:Carrier Proteins; D004337:Drug Carriers; D008567:Membranes, Artificial; D019151:Peptide Library; D010455:Peptides
keywords: LTQ-Orbitrap; Q-trap; blood-brain barrier (BBB); in vitro cell-based BBB model; mass spectrometry (MS); mix-and-split; parallel artificial membrane permeability assay (PAMPA); single reaction monitoring (SRM); solid-phase peptide synthesis (SPPS)