Ingolf E Blasig
Leibniz-Institut für Molekulare Pharmakologie, Germany
Title: Protein-protein and peptide-protein interactions to elucidate protein and cell structures as well as to modulate pharmacological barriers
Biography
Biography: Ingolf E Blasig
Abstract
Pharmacological barriers are formed via extracellular loops (ECLs) of tight junction (TJ) proteins, such as claudins (Cldns). Thus, Cldn petidomimetics were designed as drug enhancer of the blood-brain barrier (BBB). Cldns, transmembrane proteins, limit paracellular permeation of pharmaceuticals. The tightening is achieved by protein-protein interaction between Cldn-ECLs from opposing cells. Consequently, modification of Cldn integrity by the respective peptidomimetics is proposed to enhance drug delivery through the BBB. The Cldn1 and Cldn5 derived peptides C1C2 and C5C2 were tested for structural, binding and barrier modulating properties. C1C2 revealed a beta-sheet flanked by an alpha-helix, a structure modeled in the Cldn1-ECL1 also. C1C2 affected the TJ strand morphology and transiently increased the permeability through a cell culture model of the BBB. Redistribution of various Cldns from TJs to cytosol suggested interactions with other Cldns subtypes also. FRET measurements verified heterophilic interactions between different Cldns isoforms. Analysis in TJ-free HEK-293 cells transfected with Cldn1, -2, -3, -4 or -5 identified Cldn1 and 5 as direct targets. Binding measurements (microscale thermophoresis) with full-length Cldns and recombinant ECLs confirmed these findings with kd-values in nanomolar range. Association studies of peptides and recombinant ECLs on live-cells further confirmed the target selectivity. Freeze-fracture electron microscopy exhibited alterations in the TJ-architecture of Cldn5 by drastic P to E face transition and altered shape of the Cldn1 TJ-network with enhanced number of parallel strands. C5C2 increased the permeability of a brain endothelial cell barrier. Transmission electron microscopy showed opening of interendothelial TJs. Binding of C5C2 to Cldn5 showed also nanomolar affinity. C5C2 administration in mice resulted in concentration and time dependent BBB opening (marker uptake into brain, magnet resonance imaging). In summary, the Cldn peptidomimetics C1C2 and C5C2 transiently enhanced the paracellular permeability of Cldn1 and Cldn5 expressing cell barriers by affecting TJ localization and structure. The findings recommend Cldn peptidomimetics as templates to elucidate molecular and cellular TJ structures and as candidates to improve drug delivery to the brain.