Cell-Penetrating Peptides: design strategies beyond primary structure and amphipathicity

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies

Algorithm-supported, mass and sequence diversity-oriented random peptide library design.

Random peptide libraries that cover large search spaces are often used for the discovery of new binders, even when the target is unknown. To ensure an accurate population representation, there is a tendency to use large libraries. However, parameters such as the synthesis scale, the number of library members, the sequence deconvolution and peptide structure elucidation, are challenging when increasing the library size. To tackle these challenges, we propose an algorithm-supported approach to peptide library design based on molecular mass and amino acid diversity. The aim is to simplify the tedious permutation identification in complex mixtures, when mass spectrometry is used, by avoiding mass redundancy. For this purpose, we applied multi (two- and three-)-objective genetic algorithms to discriminate between library members based on defined parameters. The optimizations led to diverse random libraries by maximizing the number of amino acid permutations and minimizing the mass and/or sequence overlapping. The algorithm-suggested designs offer to the user a choice of appropriate compromise solutions depending on the experimental needs. This implies that diversity rather than library size is the key element when designing peptide libraries for the discovery of potential novel biologically active peptides

Jumping Hurdles: Peptides Able To Overcome Biological Barriers

The cell membrane, the gastrointestinal tract, and the blood–brain barrier (BBB) are good examples of biological barriers that define and protect cells and organs. They impose different levels of restriction, but they also share common features. For instance, they all display a high lipophilic character. For this reason, hydrophilic compounds, like peptides, proteins, or nucleic acids have long been considered as unable to bypass them. However, the discovery of cell-penetrating peptides (CPPs) opened a vast field of research. Nowadays, CPPs, homing peptides, and blood–brain barrier peptide shuttles (BBB-shuttles) are good examples of peptides able to target and to cross various biological barriers. CPPs are a group of peptides able to interact with the plasma membrane and enter the cell. They display some common characteristics like positively charged residues, mainly arginines, and amphipathicity. In this field, our group has been focused on the development of proline rich CPPs and in the analysis of the importance of secondary amphipathicity in the internalization process. Proline has a privileged structure being the only amino acid with a secondary amine and a cyclic side chain. These features constrain its structure and hamper the formation of H-bonds. Taking advantage of this privileged structure, three different families of proline-rich peptides have been developed, namely, a proline-rich dendrimer, the sweet arrow peptide (SAP), and a group of foldamers based on γ-peptides. The structure and the mechanism of internalization of all of them has been evaluated and analyzed. BBB-shuttles are peptides able to cross the BBB and to carry with them compounds that cannot reach the brain parenchyma unaided. These peptides take advantage of the natural transport mechanisms present at the BBB, which are divided in active and passive transport mechanisms. On the one hand, we have developed BBB-shuttles that cross the BBB by a passive transport mechanism, like diketoperazines (DKPs), (N-MePhe)n, or (PhPro)n. On the other hand, we have investigated BBB-shuttles that utilize active transport mechanisms such as SGV, THRre, or MiniAp-4. For the development of both groups, we have explored several approaches, such as the use of peptide libraries, both chemical and phage display, or hit-to-lead optimization processes. In this Account, we describe, in chronologic order, our contribution to the development of peptides able to overcome various biological barriers and our efforts to understand the mechanisms that they display. In addition, the potential use of both CPPs and BBB-shuttles to improve the transport of promising therapeutic compounds is described

Bottom-Up Design Approach for OBOC Peptide Libraries

One-bead-one-compound peptide libraries, developed following the top-down experimental approach, have attracted great interest in the identification of potential ligands or active peptides. By exploiting a reverse experimental design approach based on the bottom-up strategy, we aimed to develop simplified, maximally diverse peptide libraries that resulted in the successful characterization of mixture components. We show that libraries of 32 and 48 components can be successfully detected in a single run using chromatography coupled to mass spectrometry (UPLC-MS). The proposed libraries were further theoretically evaluated in terms of their composition and physico-chemical properties. By combining the knowledge obtained on single libraries we can cover larger sequence spaces and provide a controlled exploration of the peptide chemical space both theoretically and experimentally. Designing libraries by using the bottom-up approach opens up the possibility of rationally fine-tuning the library complexity based on the available analytical methods

Blood-brain barrier shuttle peptides: an emerging paradigm for brain delivery

Brain delivery is one of the major challenges in drug development because of the high number of patients suffering from neural diseases and the low efficiency of the treatments available. Although the blood-brain barrier (BBB) prevents most drugs from reaching their targets, molecular vectors - known as BBB shuttles - offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins

Exploration of the One-Bead One-Compound Methodology for the Design of Prolyl Oligopeptidase Substrates

Here we describe the design, synthesis and evaluation of the first solid-phase substrates for prolyl oligopeptidase (POP), a cytosolic serine peptidase associated with schizophrenia, bipolar affective disorder and related neuropsychiatric disorders. This study seeks to contribute to the future design of a one-bead one-compound (OBOC) peptide library of POP substrates, based on an intramolecular energy transfer substrate. Unexpectedly, the enzymatic evaluation of the substrates attached on solid-phase by means of the HMBA linker were cleaved through the ester bond, thereby suggesting an unknown esterase activity of POP, in addition to its known peptidase activity. By performing multiple activity assays, we have confirmed the esterase activity of this enzyme and its capacity to process the substrates on solid-phase. Finally, we tested a new linker, compatible with both the solid-phase peptide-synthesis used and the enzymatic assay, for application in the future design of an OBOC library

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)