Original articles
LIANG Ruiguo, YAN Zhixiang, ZHU Hongyu, DONG Mingxin
Objective 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. Methods 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. Results 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）. Conclusion 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.
