Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells.

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar Ki) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Discovery of α-Substituted Imidazole-4-acetic Acid Analogues as a Novel Class of ρ1 γ-Aminobutyric Acid Type†A Receptor Antagonists with Effect on Retinal Vascular Tone.

The ρ-containing γ-aminobutyric acid type A receptors (GABAA Rs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABAA Rs are of interest. In this study, we demonstrate that the partial GABAA R agonist imidazole-4-acetic acid (IAA) is able to penetrate the blood-brain barrier in vivo; we prepared a series of α- and N-alkylated, as well as bicyclic analogues of IAA to explore the structure-activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l-histidine by an efficient minimal-step synthesis, and their pharmacological properties were characterized at native rat GABAA Rs in a [3 H]muscimol binding assay and at recombinant human α1 ß2 γ2S and ρ1  GABAA Rs using the FLIPR™ membrane potential assay. The (+)-α-methyl- and α-cyclopropyl-substituted IAA analogues ((+)-6 a and 6 c, respectively) were identified as fairly potent antagonists of the ρ1  GABAA R that also displayed significant selectivity for this receptor over the α1 ß2 γ2S GABAA R. Both 6 a and 6 c were shown to inhibit GABA-induced relaxation of retinal arterioles from porcine eyes.

Molecular, cellular, and muscle strip mechanics of the mdx mouse diaphragm.

Duchenne muscular dystrophy (DMD) is a lethal disorder caused by defects in the dystrophin gene, which leads to respiratory or cardiac muscle failure. Lack of dystrophin predisposes the muscle cell sarcolemmal membrane to mechanical damage. However, the role of myosin in this muscle weakness has been poorly addressed. In the current study, in addition to measuring the velocity of actin filament propulsion (υmax) of mdx myosin molecules purified from 3- and 12-mo-old control (C57Bl/10) and mdx (C57Bl/10mdx) mouse diaphragms, we also measured myosin force production. Furthermore, we measured cellular and muscle strip force production at three mo of age. Stress (force/cross-sectional area) was smaller for mdx than control at the muscle strip level but was not different at the single fiber level. υmax of mdx myosin was not different from control at either 3 or 12 mo nor was their relative myosin force. The type I and IIb myosin heavy chain composition was not different between control and mdx diaphragms at 3 or 12 mo. These results suggest that the myosin function, as well as the single fiber mechanics, do not underlie the weakness of the mdx diaphragm. This weakness was only observed at the level of the intact muscle bundle and could not be narrowed down to a specific mechanical impairment of its individual fibers or myosin molecules.