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

Blood-Brain Barrier Shuttles: From Design to Application

[eng] The work of this thesis is based on research on peptides able to cross the blood-brain barrier and their use as tools to enable the delivery of drugs into the brain. The blood-brain barrier (BBB) is a permeable but selective barrier that tightly regulates the transport into the central nervous system (CNS). In this regard, therapeutic treatments at the CNS are hampered by the presence of this barrier (BBB). Thus, diverse strategies have been developed to overcome it. Blood-brain barrier shuttles are peptides able to cross this barrier and deliver drugs into the brain. Peptides are privileged structures from the therapeutic point of view, they share properties from small organic molecules and large biologics: the synthesis through solid-phase peptide synthesis (SPPS) enables a straightforward method to obtain them with high purity and at the same time they can be purified and characterized like small organic compound. In addition, their structure is present in nature and thus the risk of toxicity is lower or more predictable compared with organic compounds, and their larger structure enables to obtain more selective and stronger interactions with targets. In addition, peptides have been shown to cross the BBB by diverse transport mechanisms and thus enabling to select the best one for each therapy and drug. In this thesis a family of BBB shuttles crossing by passive diffusion (based on phenylproline) have been improved from a parent peptide shuttle (based on N-methyl-phenylalanine). The solubility was three orders of magnitude superior and the transport capacity was maintained upon cargo attachment. In addition, the role of stereochemistry in passive diffusion in biological membranes was demonstrated. A method which combined the use of MALDI-TOF MS and in vitro cell-based models of the BBB enabled the increase in sensitivity for transport quantification of three orders of magnitude compared to RP-HPLC-PDA. Additionally, a BBB shuttle library was evaluated and quantified by this novel methodology. Two new analogs showed better performance when evaluated in these in vitro cell-based models. Immunogenicity of BBB shuttle peptides made by L- or D-amino acids was evaluated and compared. Both peptide shuttles showed low immunogenic response in mice, however, the response to those made with D- amino acids was lower. Finally, the applicability of these peptide shuttles for a therapeutic use was considered for Friedreich’s Ataxia, a monogenic recessive disease. Both a protein replacement therapy and a gene therapy for the central nervous system were attempted by coupling covalently BBB shuttles to the affected protein or viruses, respectively. The protein replacement therapy was impeded by the high rate of proteolysis of the protein used. On the other hand, novel methods of conjugation of BBB shuttles into enveloped viruses (Herpes simplex Virus type 1; HSV- 1) were developed. These modified viral particles were subsequently characterized through a range of methods comprising molecular biology tools (SDS-PAGE, western blots), proteomics (mass spectrometry) and biophysical tools (dynamic light scattering and z-potential).[cat] La barrera hematoencefàlica (BHE) actua com a protecció del sistema nerviós central (SNC) regulant el transport de molècules d’una manera selectiva. Això dificulta el tractament de malalties que afecten al SNC, ja que la BHE també evita que fàrmacs que serien efectius no siguin transportats al cervell. Per això, s’estan desenvolupant mètodes que permetin enviar selectivament fàrmacs a través de la BHE. És el cas dels pèptids llançadora. Aquests es poden dissenyar per creuar per algun dels mecanismes de transport existents en la BHE. En aquesta tesi es desenvolupen uns pèptids que creuen per difusió passiva (basats en fenilprolines), que respecte al disseny anterior (basats en N‐ metilfenilalanines) milloren la solubilitat en aigua en tres ordres de magnitud i al transport un cop s’hi enganxa el fàrmac. Per una altra banda, es desenvolupa una metodologia per a la quantificació del transport basada en la combinació d’espectrometria de masses MALDI‐TOF amb models de BHE in vitro (cel∙lulars), millorant la sensibilitat respecte a la detecció per RP‐HPLC‐PDA en tres ordres de magnitud. L’avaluació d’una peptidoteca derivada d’un pèptid llançadora mitjançant aquesta metodologia permet el descobriment de dos anàlegs del pèptid original que milloren el transport. Addicionalment, s’estudia la immunogenicitat de pèptids llançadora formats per aminoàcids L o D. S’observa que encara que ambdós mostren una baixa immunogenicitat, la resposta dels pèptids amb aminoàcids D és encara menor. Finalment, s’estudia de forma preliminar la possibilitat de desenvolupar una teràpia de reemplaçament proteic i una teràpia gènica per atàxia de Friedreich al SNC mitjançant l’ús de pèptids llançadora

The Role of Orthogonality in Genetic Code Expansion

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code

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