Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification
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...
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Published in | Journal of peptide science Vol. 22; no. 9; pp. 577 - 591 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Blackwell Publishing Ltd
01.09.2016
Wiley Subscription Services, Inc John Wiley & Sons |
Subjects | |
Online Access | Get full text |
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Summary: | 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.
Description of a new high‐throughput screening methodology to search for new protease‐resistant BBB shuttle 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 BBB assays. |
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Bibliography: | Generalitat de Catalunya istex:370D6EEDFD62F0DC91B0758A50E07D649BC009F4 Gate2Brain-RecerCaixa 2014 ark:/67375/WNG-N7GK0WZ4-1 MINECO-FEDER - No. BIO2013-40716-R; No. CTQ2013-49462-EXP; No. 2014-SGR-521 ArticleID:PSC2900 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1075-2617 1099-1387 |
DOI: | 10.1002/psc.2900 |