A MALDI-TOF-based method for studying the transport of BBB shuttles-enhancing sensitivity and versatility of cell-based in vitro transport models.

In recent decades, peptide blood-brain barrier shuttles have emerged as a promising solution for brain drugs that are not able to enter this organ. The research and development of these compounds involve the use of in vitro cell-based models of the BBB. Nevertheless, peptide transport quantification implies the use of large amounts of peptide (upper micromolar range for RP-HPLC-PDA) or of derivatives (e.g. fluorophore or quantum-dot attachment, radiolabeling) in the donor compartment in order to enhance the detection of these molecules in the acceptor well, although their structure is highly modified. Therefore, these methodologies either hamper the use of low peptide concentrations, thus hindering mechanistic studies, or do not allow the use of the unmodified peptide. Here we successfully applied a MALDI-TOF MS methodology for transport quantification in an in vitro BBB cell-based model. A light version of the acetylated peptide was evaluated, and the transport was subsequently quantified using a heavy internal standard (isotopically acetylated). We propose that this MALDI-TOF MS approach could also be applied to study the transport across other biological barriers using the appropriate in vitro transport models (e.g. Caco-2, PAMPA)

HAI Peptide and Backbone Analogs-Validation and Enhancement of Biostability and Bioactivity of BBB Shuttles

Low effectiveness and resistance to treatments are commonplace in disorders of the central nervous system (CNS). These issues concern mainly the blood-brain barrier (BBB), which preserves homeostasis in the brain and protects this organ from toxic molecules and biohazards by regulating transport through it. BBB shuttles-short peptides able to cross the BBB-are being developed to help therapeutics to cross this barrier. BBB shuttles can be discovered by massive exploration of chemical diversity (e.g. computational means, phage display) or rational design (e.g. derivatives from a known peptide/protein able to cross). Here we present the selection of a peptide shuttle (HAI) from several candidates and the subsequent in-depth in vitro and in vivo study of this molecule. In order to explore the chemical diversity of HAI and enhance its biostability, and thereby its bioactivity, we explored two new protease-resistant versions of HAI (i.e. the retro-D-version, and a version that was N-methylated at the most sensitive sites to enzymatic cleavage). Our results show that, while both versions of HAI are resistant to proteases, the retro-D-approach preserved better transport properties

Chemically synthesized peptide libraries as a new source of BBB-shuttles. Use of Mass Spectrometry for peptide identification and quantification

[cat] La barrera hematoencefàlica (BH) és una barrera biològica situada als capil.lars sanguinis del cervell que juga un paper crucial per a la regulació del flux d'entrada i sortida de molècules necessàries pel correcte funcionament d'aquest òrgan i per a la seva protecció davant d'agents neurotòxics. Aquesta forta regulació dificulta el pas a través de la BH dels fàrmacs dirigits al parènquima cerebral. Per superar aquest obstacle, s'han proposat estratègies com l'ús de llançadores de la BH. Aquestes es descriuen com a molècules tant capaços de travessar la BH com capaces d'assistir el pas d'una molècula incapaç de creuar la barrera enganxada a ella. Aquesta tesi es centra en l'estudi i la modificació de llançadores de la BH ja descrites per millorar la seva eficàcia, estabilitat i solubilitat i en la recerca de nous pèptids llançadora per mètodes de cribratge d'alt rendiment. Es va sintetitzar una biblioteca d'estereoisòmers basada en Ac-(N-MePhe)(4)-NH(2) per avaluar el paper de la quiralitat en la difusió passiva a través d'un assaig de transport in vitro: PAMPA. A més, es va dissenyar i sintetitzar una biblioteca de 21 anàlegs basats en Ac-HAIYPRH-NH(2) per millorar l'eficàcia, l'estabilitat i la solubilitat d'aquest pèptid a través d'un assaig in vitro de BH basat en un co-cultiu cel.lular. La tècnica de cribratge d'alt rendiment phoge disploy es va utilitzar per explorar unitats peptídiques mínimes capaços de dirigir-se preferentment al cervell. Es va sintetitzar una biblioteca de fags CX3C i es va assajar in vivo en ratolins BALB/c. Els fags CFLFC, CRWEC i CNSQC van ser seleccionats després d'un protocol de la panning compost de 3 rondes basades en enriquiment al cervell. S'ha desenvolupat una nova metodologia de selecció d'alt rendiment per buscar nous pèptids llançadora de la BH, en un espai químic no disponible amb la tècnica phoge disploy. Aquesta metodologia combina la química combinatòria per a la síntesi de biblioteques de pèptids, els models in vitro que mimetitzen la BH per a l'avaluació de les biblioteques i tècniques avançades d'espectrometria de masses per a la identificació dels pèptids capaços de travessar aquests assajos in vitro.[eng] The blood-brain barrier (888) is a biological barrier that plays a crucial role protecting the brain from neurotoxic agents and regulating the influx and efflux of molecules required for the correct function of this organ. This stringent regulation hampers the passage across the BBB of drugs targeting a variety of targets in the brain parenchyma. To overcome this hurdle, strategies such as usage of BBB-shutties had been proposed. BBB-shutties are described as molecules both able to cross the BBB and able to mediate the passage of a molecule unable to cross the barrier attached to it. This thesis focus on the study and tuning of already described peptide BBB-shutties to improve their efficiency, stability and solubility and on the search of new peptide BBB-shutties by highthroughput screening methods. First, the effect of stereochemistry on passive diffusion across the BBB was studied. A library composed by a complete set of Ac-{N-MePhekNH2 stereoisomers (16) was synthesized and evaluated with PAMPA assay. Homochiral versions, particularly all-O version, showed the best diffusion rates among library analogues. A degree of enantiomeric discrimination was observed. Second, to improve the features of Ac-HAIYPRH-NH2 (a peptide found by phage display described to interact with human transferrin receptor), a library of 21 analogues based on this peptide was designed, synthesized and assayed on an in vitro cell-based BBB model. The library broadens the screened chemical space by exploring o-peptide backbone and a variety of non-natural side chains. Transport results were assessed by two distinct quantification techniques: HPlC-UV and MALDI-TOF. For transport quantification using mass spectrometry techniques an internal standard is reqUired. Hence, a library of internal standards containing the isotopically labelled CD1llCO- tag was synthesized to spike the samples assayed on the in vitro cell-based BBB model. Third. the high-throughput screening technique phage display was used to explore minimalist moieties able to target the brain. A phage display CX3Clibrary was synthesized and assayed in vivo in BALB/c mice. Three panning rounds and a post-panning were performed and led to three lead sequences: CFlFC, CRWEC and CNSUe. Those sequences were studied individually to assess biodistribution on mice and immunofluorescence experiments to track the fate of these phages. We concluded that compared to a negative phage (with no peptide insert) all selected phage have an affinity of more than one fold to the brain. However, phages accumulate in greater or significant quantities in several other organs, especially in liver. In terms of localization, we hypothesize phages could target astrocytes, although e)(periments should be repeated to further confirm these results. Finally, a new high-throughput screening methodology was developed to seek for new peptide BBB-shutties in a chemical space not available with phage display. 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. The library was synthesized with the mix and split methodology to generate a library based on: Ac-o-Arg-XXXXX-NH;u where X were: D-Ala. D-Arg, D-lIe, D-Glu, D-Ser, D-Trp, D-Pro. The assays used were the in vitro cell based BBB assay (mimicking both active and passive transport) and PAMPA assay (only mimicking passive diffusion). From these BBB models two groups of candidates were postulated depending on the transport mechanisms used (passive diffusion vs other mechanisms of transport). The identification of candidates was determined in a two-steps mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Sequences within 13-A1-PI-R1 and 14-PI-R1 compositions were found as other mechanisms transport candidates while sequences within 13-P2-R1, P3-S2-R1 and A3-R2-P1 compositions were found for passive diffUSion transpart candidates

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 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 peptide

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 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 peptide