Toward an Optimal Blood−Brain Barrier Shuttle by Synthesis and Evaluation of Peptide Libraries

Several peptide families containing N-methylated amino acids were designed and synthesized using solid-phase peptide synthesis (SPPS). The permeability and phospholipophilicity of these compounds were studied by parallel artificial membrane permeability assay (PAMPA) and immobilized artificial membrane chromatography (IAMC) to select the best peptides in terms of length, terminal groups, and amino acid replacement to be used as carriers that pass through a model of the blood−brain barrier (BBB) by passive diffusion. Furthermore, the enzymatic stability of these peptides in human serum and their cell viability by MTT assay were tested. These peptide families showed great stability and nontoxicity. The three peptides that showed the greatest permeability were coupled to levodopa (a nonpassive permeating drug) and assessed. These peptides effectively transferred levodopa through an artificial membrane by means of passive diffusion

<i>N</i>-Methyl Phenylalanine-Rich Peptides as Highly Versatile Blood−Brain Barrier Shuttles

Here we studied the capacity of N-MePhe-(N-MePhe)3-CONH2, Cha-(N-MePhe)3-CONH2, and 2Nal-(N-MePhe)3-CONH2 to carry various drugs (cargos) in in vitro blood−brain barrier (BBB) models in order to determine the versatility of these peptides as BBB-shuttles for drug delivery to the brain. Using SPPS, the peptides were coupled to GABA, Nip, and ALA to examine their passive BBB permeation by means of PAMPA and their lipophilicity by IAMC. Unaided, these nonpermeating drugs alone did not cross the PAMPA barrier and the BBB passively; however, the peptides tested as potential BBB shuttles transferred them by passive transfer through the PAMPA phospholipid. The permeability of peptides that showed the highest permeability in PAMPA, and Ac-N-MePhe-(N-MePhe)3-CONH2 as the parent peptide was also examined in bovine brain microvessel endothelial cells (BBMECs). These peptide-based BBB shuttles open up the possibility to overcome the formidable obstacle of the BBB, thereby achieving drug delivery to the brain

Diketopiperazines as a Tool for the Study of Transport across the Blood−Brain Barrier (BBB) and Their Potential Use as BBB-Shuttles

Here we prepared and evaluated two libraries of mono-N-methylated and di-N-methylated diketopiperazines (DKPs) by parallel artificial membrane permeability assay and immobilized artificial membrane chromatography in order to obtain information on the features that govern the passage of peptidic molecules across the blood−brain barrier (BBB) by passive diffusion. On the basis of the results from these two libraries, we prepared and evaluated several DKP−baicalin and DKP−dopamine constructs. The DKPs or cyclic dipeptide scaffolds can be considered a novel family of brain delivery systems (BBB-shuttles) to transport to the brain drugs and other cargos that cannot cross the BBB unaided

The Use of Chimeric Vimentin Citrullinated Peptides for the Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and, in many cases, destruction of the joints. To prevent progressive and irreversible structural damage, early diagnosis of RA is of paramount importance. The present study addresses the search of new RA citrullinated antigens that could supplement or complement diagnostic/prognostic existing tests. With this aim, the epitope anticitrullinated vimentin antibody response was mapped using synthetic peptides. To improve the sensitivity/specificity balance, a vimentin peptide that was selected, and its cyclic analogue, were combined with fibrin- and filaggrin-related peptides to render chimeric peptides. Our findings highlight the putative application of these chimeric peptides for the design of RA diagnosis systems and imply that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported here (fibrin, vimentin, filaggrin) has a specific utility in the identification of a particular subset of RA patients

Lipid Bilayer CrossingThe Gate of Symmetry. Water-Soluble Phenylproline-Based Blood-Brain Barrier Shuttles

Drug delivery to the brain can be achieved by various means, including blood-brain barrier (BBB) disruption, neurosurgical-based approaches, and molecular design. Recently, passive diffusion BBB shuttles have been developed to transport low-molecular-weight drug candidates to the brain which would not be able to cross unaided. The low water solubility of these BBB shuttles has, however, prevented them from becoming a mainstream tool to deliver cargos across membranes. Here, we describe the design, synthesis, physicochemical characterization, and BBB-transport properties of phenylproline tetrapeptides, (PhPro)₄, an improved class of BBB shuttles that operates via passive diffusion. These PhPro-based BBB shuttles showed 3 orders of magnitude improvement in water solubility compared to the gold-standard (<i>N</i>-MePhe)₄, while retaining very high transport values. Transport capacity was confirmed when two therapeutically relevant cargos, nipecotic acid and l-3,4-dihydroxyphenylalanine (i.e., l-DOPA), were attached to the shuttle. Additionally, we used the unique chiral and conformationally restricted character of the (PhPro)₄ shuttle to probe its chiral interactions with the lipid bilayer of the BBB. We studied the transport properties of 16 (PhPro)₄ stereoisomers using the parallel artificial membrane permeability assay and looked at differences in secondary structure. Most stereoisomers displayed excellent transport values, yet this study also revealed pairs of enantiomers with high enantiomeric discrimination and different secondary structure, where one enantiomer maintained its high transport values while the other had significantly lower values, thereby confirming that stereochemistry plays a significant role in passive diffusion. This could open the door to the design of chiral and membrane-specific shuttles with potential applications in cell labeling and oncology

Uncovering the Selection Criteria for the Emergence of Multi-Building-Block Replicators from Dynamic Combinatorial Libraries

A family of self-replicating macrocycles was developed using dynamic combinatorial chemistry. Replication is driven by self-assembly of the replicators into fibrils and relies critically on mechanically induced fibril fragmentation. Analysis of separate dynamic combinatorial libraries made from one of six peptide-functionalized building blocks of different hydrophobicity revealed two selection criteria that govern the emergence of replicators from these systems. First, the replicators need to have a critical macrocycle size that endows them with sufficient multivalency to enable their self-assembly into fibrils. Second, efficient replication occurs only for library members that are of low abundance in the absence of a replication pathway. This work has led to spontaneous emergence of replicators with unrivalled structural complexity, being built from up to eight identical subunits and reaching a MW of up to 5.6 kDa. The insights obtained in this work provide valuable guidance that should facilitate future discovery of new complex self-replicating molecules. They may also assist in the development of new self-synthesizing materials, where self-assembly drives the synthesis of the very molecules that self-assemble. To illustrate the potential of this concept, the present system enables access to self-assembling materials made from self-synthesizing macrocycles with tunable ring size ranging from trimers to octamers

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein–ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption