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)

Immunosilencing peptides by stereochemical inversion and sequence reversal: retro-D-peptides

Peptides are experiencing a new era in medical research, finding applications ranging from therapeutics to vaccines. In spite of the promising properties of peptide pharmaceuticals, their development continues to be hindered by three weaknesses intrinsic to their structure, namely protease sensitivity, clearance through the kidneys, and immune system activation. Here we report on two retro-D-peptides (H2N-hrpyiah-CONH2 and H2N-pwvpswmpprht-CONH2), which are protease-resistant and retain the original BBB shuttle activity of the parent peptide but are much less immunogenic than the parent peptide. Hence, we envisage that retro-D-peptides, which display a similar topological arrangement as their parent peptides, will expand drug design and help to overcome factors that lead to the failure of peptide pharmaceuticals in pre- and clinical trials. Furthermore, we reveal requirements to avoid or elicit specific humoral responses to therapeutic peptides, which might have a strong impact in both vaccine design and peptide therapeutic agents

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

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 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.[spa] Los péptidos presentan varias ventajas sobre las pequeñas moléculas y las proteínas: (i) flexibilidad, (ii) fácil modularidad, (iii) tamaño reducido, y (iv) completa biocompatibilidad. Sin embargo, el progreso hacia el desarrollo de péptidos terapéuticos para modular interacciones proteicas se ve obstaculizado por los siguientes inconvenientes de estas moléculas: baja estabilidad frente a la degradación por encimas proteolíticas del sistema digestivo y del plasma sanguíneo; rápida eliminación del sistema circulatorio; dificultad para atravesar barreras fisiológicas; y potencial inmunogenicidad. A pesar de estas limitaciones, un gran número de péptidos han llegado a ser exitosos moduladores de interacciones proteína-proteína y el gran esfuerzo realizado para abordar este cuello de botella que perjudica su uso como fármacos es impresionante. En el primer capítulo se describe el uso de péptidos cíclicos capaces de unir a VEGF. Para ello primero se realizó un cribado de la librería EXORIS frente a VEGF que dio lugar a la obtención de un hexapéptido cíclico (c(EpWEpW)) capaz de unir a VEGF en su región de unión a receptores con una afinidad baja. Mediante la evaluación de varios parámetros tales como: simetría C2, sustitución de aminoácidos, o la estereoquímica de este tipo de péptidos cíclicos, se examinaron varios derivados del péptido c(EpWEpW) pero ninguno de ellos dio mejores resultados en cuanto a afinidad. Finalmente se observó que la ampliación del tamaño del péptido pasando de hexapéptido a dodecapéptidos daba lugar a una mejora significativa de la afinidad así como la selectividad de unión a VEGF. Se obtuvo así un péptido cíclico capaz de unir a VEGF. En este capítulo se realizó también un estudio conformacional de un set representativo de hexapéptido cíclicos. En el segundo capítulo se describe el estudio realizado para determinar la conformación de unos análogos de apamina que presentaron distintas permeabilidades frente a la barrera hematoencefálica. Finalmente en el último capítulo de la tesis se describe el trabajo realizado durante mi estancia en el MIT, donde llevé a cabo el desarrollo y optimización de un protocolo para la expresión, purificación y marcaje de derivados de la proteína Ss07d para ser usados como biosensores capaces de detectar proteínas in vivo. En este capítulo se llevó a cabo también el desarrollo de nuevos “scaffold” basados en Sso7d capaces de unir a hEGF mediante la técnica de YSD (“yeast surface display”)

‘À La Carte’ Cyclic Hexapeptides: Fine Tuning Conformational Diversity while Preserving the Peptide Scaffold

Cyclic peptides have recently emerged as promising modulators of challenging protein‐protein interactions. Here we report on the design, synthesis and conformational behavior of a small library composed of C2 symmetric cyclic hexapeptides of type c(Xaa‐D‐Pro‐Yaa)2, where Xaa and Yaa are chosen from alanine, isoleucine, serine, glutamic acid, arginine and tryptophan due to the favorable properties of the side chains of these residues to recognize complex protein surfaces. We used a combination of nuclear magnetic resonance and molecular dynamic simulations to perform an extensive conformational analysis of a representative set of cyclic hexapeptides. Our results indicated that both the chemical nature and the chirality of the variable Xaa and Yaa positions play an important role in the cis/trans configuration of the Xaa‐D‐Pro bonds and in the conformational preferences of this family of peptides. This structural tuning can be exploited in design strategies seeking to optimize the binding efficiency and selectivity of cyclic hexapeptides towards protein surfaces

‘À La Carte’ Cyclic Hexapeptides: Fine Tuning Conformational Diversity while Preserving the Peptide Scaffold

Cyclic peptides have recently emerged as promising modulators of challenging protein‐protein interactions. Here we report on the design, synthesis and conformational behavior of a small library composed of C2 symmetric cyclic hexapeptides of type c(Xaa‐D‐Pro‐Yaa)2, where Xaa and Yaa are chosen from alanine, isoleucine, serine, glutamic acid, arginine and tryptophan due to the favorable properties of the side chains of these residues to recognize complex protein surfaces. We used a combination of nuclear magnetic resonance and molecular dynamic simulations to perform an extensive conformational analysis of a representative set of cyclic hexapeptides. Our results indicated that both the chemical nature and the chirality of the variable Xaa and Yaa positions play an important role in the cis/trans configuration of the Xaa‐D‐Pro bonds and in the conformational preferences of this family of peptides. This structural tuning can be exploited in design strategies seeking to optimize the binding efficiency and selectivity of cyclic hexapeptides towards protein surfaces

MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery

Drug delivery across the blood-brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non-invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam-bridged peptidomimetic MiniAp-4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human-cell-based BBB model. Furthermore, MiniAp-4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice