From phage display and venoms to protease-resistant peptides: Design of BBB-shuttles and peptides targeting EGF

Autor: Díaz Perlas, Cristina
Přispěvatelé: Giralt Lledó, Ernest, Teixidó Turà, Meritxell, Universitat de Barcelona. Departament de Química Orgànica
Jazyk: angličtina
Rok vydání: 2018
Předmět:
Zdroj: TDX (Tesis Doctorals en Xarxa)
TDR. Tesis Doctorales en Red
instname
Dipòsit Digital de la UB
Universidad de Barcelona
Popis: [eng] Peptides play a critical role in human physiology and harbour a huge potential as therapeutic agents. In this thesis, new peptides have been discovered as ligands for the epidermal growth factor (EGF) and as new BBB-shuttles, using phage display and chemical synthesis of peptides and proteins. EGF is overexpressed in several cancers, inducing the proliferation and survival of these cells. By inhibiting EGF, we will prevent the activation of the receptor and, consequently, its negative effects. Moreover, phage display is a powerful tool to identify peptide ligands. However, only L-peptides can be displayed, which are protease unstable. Hence, mirror image phage display was developed to identify D-peptides, more stable in front of proteases. In this methodology, the selection is carried out against the mirror image of the original target and, after the panning selection and the synthesis of the enantiomer of the ligand, D-peptides are obtained. In this regard, the enantiomer of EGF was synthesised using a combination of solid-phase peptide synthesis and native chemical ligation. After the panning of two phage display peptide libraries against the immobilised protein, nine sequences were selected and synthesised with D-amino acids. Three of these peptides have high affinities for EGF and are stable in serum proteases for more than 24h. Most potential drugs for the treatment of central nervous system disorders (such as brain cancer and Alzheimer’s disease) do not cross the blood-brain barrier (BBB). Much effort has been devoted to the discovery of BBB-shuttle peptides – entities that have the capacity to carry cargoes across the BBB. The sources of BBB-shuttle peptides are diverse, ranging from the mimicry of endogenous proteins to the use of phage display. Phage display has been applied against a human BBB cellular model which consists in the co-culture of human brain capillary endothelial cells and bovine pericytes. From the screening of a phage display library containing random 12-amino acid sequences, SGVYKVAYDWQH (SGV) was selected. Validation studies were performed confirming that SGV is able to increase the uptake of a model protein in endothelial cells. When a BBB-shuttle is conjugated to a cargo, the ratio BBB-shuttle:cargo can range from 1:1 to 400:1, depending on the cargo. However, there are cases where the modification of the cargo with one copy of the BBB-shuttle is not sufficient to promote its passage through the BBB. More copies can be introduced, but a mixture of ratios of BBB-shuttle:cargo conjugates may be obtained. In those cases, it may be interesting to use multivalent BBB-shuttles, where more than one copy of the shuttle is attached to a core, which is linked to the cargo at one position. In this regard, branched dimerisation of a known BBB-shuttle was explored to enhance BBB permeability of model proteins. The results obtained with THRre peptide as the BBB-shuttle suggest that the mild improvement in permeability may not compensate the increased synthetic effort that these constructs represent. Moreover, chlorotoxin (CTX), a disulphide-rich peptide found in the venom of a scorpion, is reported to be able to enter the brain and bind specifically to tumour tissue. However, CTX is a relatively large peptide (36 amino acids) to be used as a BBB-shuttle, where we may need to scale it up or modify it. In this regard, we designed minimised versions of CTX (MiniCTXs) and evaluated their BBB properties, as well as their toxicity and stability. The results from these experiments produced two peptides as good BBB-shuttles, with similar stability, cellular uptake and BBB permeability than the best BBB-shuttles.
[cat] En aquesta tesi doctoral, s’han descobert nous pèptids com a lligands del factor de creixement endotelial (EGF) i com a noves llançadores peptídiques, usant les tècniques de phage display i la síntesi química de pèptids. EGF es troba sobre-expressat en diversos càncers i, mitjançant la seva inhibició, la proliferació d’aquestes cèl·lules es podria reduir. Amb phage display es poden trobar aquests lligands, però els fags només poden expressar pèptids L, els quals són degradats per proteases. En el mirror image phage display, la selecció es duu a terme contra la imatge especular de la diana original i, després de la selecció i la síntesi dels enantiòmers dels millors candidats, s’obtenen pèptids D com a lligands. En aquest context, l’enantiòmer de EGF s’ha sintetitzat usant una combinació de síntesi en fase sòlida i lligació química nativa. Després del procés de selecció amb dos biblioteques de fags contra la proteïna immobilitzada, nou seqüencies s’han seleccionat i sintetitzat amb aminoàcids D. Tres d’aquests pèptids presenten altes estabilitats i afinitats per EGF. La majoria de fàrmacs pel tractament de malalties del sistema nerviós central no creuen la barrera hematoencefàlica. Una alternativa és l’ús de llançadores, que tenen la capacitat de transportar fàrmacs al cervell. La metodologia de phage display s’ha aplicat en un model humà de la barrera hematoencefàlica i, després del cribratge, la seqüència SGVYKVAYDWQH (SGV) s’ha seleccionat. Estudis de validació s’han dut a terme confirmant que SGV és capaç d’incrementar el transport d’una proteïna en cèl·lules endotelials. Hi ha casos en que la modificació del fàrmac amb una sola llançadora no és suficient per creuar la barrera hematoencefàlica. En aquest sentit, s’ha explorat la dimerització d’una llançadora (THRre) per incrementar el transport de proteïnes. Els resultats obtinguts suggereixen un moderat increment en el transport, el qual pot no compensar l’esforç sintètic requerit. La clorotoxina (CTX), pèptid trobat en el verí d’un escorpí, i versions reduïdes d’aquesta (MiniCTXs), s’han estudiat com a nova font de llançadores. Les propietats d’aquests pèptids de creuar la barrera hematoencefàlica han estat avaluades. Al final d’aquest procés, dos MiniCTXs han estat seleccionades, amb uns valors de transport semblants a les millors llançadores.
Databáze: OpenAIRE
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