Author : Cristina Díaz Perlas
Publisher :
ISBN 13 :
Total Pages : 253 pages
Book Rating : 4.:/5 (15 download)
Book Synopsis From Phage Display and Venoms to Protease-resistant Peptides: Design of BBB-shuttles and Peptides Targeting EGF. by : Cristina Díaz Perlas
Download or read book From Phage Display and Venoms to Protease-resistant Peptides: Design of BBB-shuttles and Peptides Targeting EGF. written by Cristina Díaz Perlas and published by . This book was released on 2018 with total page 253 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
