Cyclic peptides and small proteins in molecular recognition
[eng] The present thesis is based on the work developed about the use of peptides and small proteins in protein surface molecular recognition. Peptides present several advantages when compared with small molecules and biologics: (i) flexibility, which is translated into adaptability to large surface...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Recursos: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/107612 |
| Acesso em linha: | https://hdl.handle.net/2445/107612 http://hdl.handle.net/10803/400830 |
| Access Level: | acceso abierto |
| Palavra-chave: | Síntesi de pèptids Síntesis de péptidos Peptide synthesis Interacció cel·lular Interacción celular Cell interaction Proteïnes Proteins |
| Resumo: | [eng] The present thesis is based on the work developed about the use of peptides and small proteins in protein surface molecular recognition. Peptides present several advantages when compared with small molecules and biologics: (i) flexibility, which is translated into adaptability to large surfaces; (ii) easy modularity, which increases structural diversity and consequently allows higher selectivity and potency; (iii) size, which limits accumulation in tissue; and (iv) complete biocompatibility, which means low toxicity in humans. The last two features are highly desirable given the growing interest in PPIs as therapeutic targets. Moreover, non- natural building blocks and various chemical scaffolds can be incorporated into a peptide sequence to create a palette of modified peptides with a wide range of functionalities and chemical diversity. However, progress towards the development of therapeutic peptide PPI modulators is hindered by the following drawbacks of these molecules: low stability against degradation by proteolytic enzymes of the digestive system and blood plasma; rapid removal from the circulation; poor ability to cross physiological barriers; and potential immunogenicity. In spite of these limitations, the large number of successful peptide PPI modulators reported so far and the great effort to tackle the bottlenecks that impair their use as pharmaceutics are impressive. In addition to features that allow cell and tissue permeability, many chemical modifications and smart linker conjugations have been introduced into PPI modulators in order to reduce proteolytic degradation and improve bioavailability. In the first chapter of this theses we described the use of cyclic peptides able to bind VEGF. For this purpose we screened a library of cyclic hexapeptides against VEGF by NMR. We found a cyclic hexapeptide (c(EpWEpW)) that bound VEGF in the receptor binding epitope with a low affinity. In order to gain more insight into how VEGF binding was affected by different parameters such as: C2 symmetry, amino acid replacement and stereochemistry, we evaluated a set of derivatives of peptide c(EpWEpW). Unfortunately none of the evaluated peptides displayed a better affinity than the parent compound. Finally we observed that ring size expansion was key to improve VEGF-binding and selectivity. We found a dodecapeptide that binds VEGF in the receptor binding domain with µM affinity. In this chapter we also described the conformational study of a set of cyclic hexapeptides. In the second chapter we described the conformational analysis done with different apamin analogues in order to determine their conformational behavior to explain the differences observed in BBB- permeability when these peptides were tested in vitro. Finally, the last chapter describes the work that I did during my short stay in Prof. Imperiali’s lab at MIT, where I was working on the development of fluorescence-based biosensors for protein detection. In this sense I performed the development and optimization of a protocol for the expression, purification and labeling with 4-DMN derivatives of Sso7d-based cysteine mutants. Yeast surface display was also used in order to obtain Sso7d-based binders of a relevant target (hEGF). We obtained the first Sso7d-based hEGF binders that were also characterized in order to obtain their sequences. Nowadays, the conversion of these Sso7d-based hEGF binders into future biosensors is being done in order to detect hEGF in vivo. |
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