Synthesis and evaluation of the GluA2 endocytic blocker bicyclic peptide CMT-C3Y

To enhance the cyclic peptide compound’s membrane permeability, structural stability, and neuroprotective activity, based on the amino acid sequence of peptides of Tat-GluA2-3Y, by designing and synthesizing a serial of cyclic peptides through strategies including polypeptide cyclization, replacement of the cell-penetrating peptide, substitution with D-amino acids, and incorporation of mini polyethylene glycol fragments.

The target peptides were synthesized based on standard Fmoc solid-phase method, followed by analysis and purification via reverse phase high-performance liguid chromatography (RP-HPLC). The cytoprotective activity of the peptides was evaluated by using the HT-22 cell model. The transmembrane transport efficiency of the peptides was determined based on the Caco-2 monolayer intestinal epithelial cell model. Plasmatic plasma and metabolic stability of the peptides were measured by in vitro co-incubation experiments with rat plasma and human liver microsomes. Finally, the in vivo neuroprotective activity of the peptides was validated by using a mouse middle cerebral artery occlusion model.

Seven cyclic peptides were successfully designed and synthesized by using the standard Fmoc solid-phase method, with purities exceeding 90% as confirmed by RP-HPLC. Cytoprotective activity assay demonstrated that both Tat-GluA2-3Y and CMT-C3Y exhibited activity at concentrations above 125 nmol/L, with CMT-C3Y showing superior activity as compared to Tat-GluA2-3Y. The results of the transmembrane assay demonstrated that, compared to Tat-GluA2-3Y, CMT-C3Y exhibited significant transmembrane capabilities at all tested concentrations (P<0.001). CMT-C3Y was classified as a highly permeable compound, whereas Tat-GluA2-3Y was categorized as a moderately permeable compound. Plasma stability studies indicated that over 50% of Tat-GluA2-3Y was metabolized after 4 h of co-incubation with rat plasma. After 8 h of coincubation with CMT-C3Y, the remaining amount was 88.1%, and no obvious degradation phenomenon occurred. In human liver microsomal stability tests, the half-life of Tat-GluA2-3Y was 26.1 min, as compared to 103.8 min for CMT-C3Y, highlighting the enhanced stability of CMT-C3Y. Tat-GluA2-3Y and CMT-C3Y were classified as a fast-metabolizing drug and a moderate-metabolizing drug, respectively. Animal experiments further demonstrated that at a dose of 8 mg/kg the neuroprotective activity of CMT-C3Y was significantly superior to that of Tat-GluA2-3Y (P<0.001).

The designed bicyclic peptide CMT-C3Y demonstrates significantly higher cell-penetrating efficiency and superior plasma stability as compared to Tat-GluA2-3Y, along with enhanced neuroprotective activity at both cellular and animal levels.