A claudin5-binding peptide enhances the permeability of the blood-brain-barrier

Abstract

ABSTRACTThe blood-brain barrier (BBB) is essential to maintain brain homeostasis and healthy conditions but it also prevents drugs from reaching brain cells. In the BBB, tight junctions (TJs) are multi-protein complexes located at the interface between adjacent brain endothelial cells that regulate paracellular diffusion and claudin-5 (CLDN5) is the major component of the TJ portfolio, playing a pivotal role in restricting the paracellular traffic. In view of obtaining fine control over the transport across the BBB, the use of competing peptides able to bind CLDN5 to induce transient and regulated permeabilization of the paracellular passage is emerging as a potentially translatable strategy for clinical applications. In this work, we designed and tested short peptides with improved solubility and biocompatibility using a combined approach that involved structural modeling techniques andin vitrovalidation, generating a robust workflow for the design, screening, and optimization of peptides for the modulation of the BBB paracellular permeability. We designed a selection of 11- to 16-mer compounds derived from the first CLDN5 extracellular domain and from the CLDN5-binding domain ofClostridium perfringensenterotoxin and determined their efficiency in enhancing BBB permeability. The computational analysis classified all tested peptides based on solubility and affinity to CLDN5, and provided atom-level details of the binding process. From our screening, we identified a novel CLDN5-derived peptide, here calledf1-C5C2, which demonstrated good solubility in biological media, efficient binding to CLDN5 subunits, and capability to increase permeability at low concentrations. The peptidomimeticin silico/in vitrostrategy described here can achieve a transient and reversible permeabilization of the BBB with potential applications in the pharmacological treatment of brain diseases.HIGHLIGHTSWater-soluble peptidomimetics are used to competitively bind claudin-5 tight junction proteins and increase the permeability of the blood-brain barrier;Trans-endothelial electrical resistance and dissociation constant measurements demonstrate the binding affinity of the peptidef1-C5C2for claudin-5;Unbinding free energy calculations correlated with experimental results and provided information on the protein-peptide binding interface.Incubation with the peptidef1-C5C2allows paracellular transport of 4K, but not 70K, dextran.GRAPHICAL ABSTRACT