Engineered Cyclotide\nBlocks Neuronal Excitotoxicity

Cyclotides\nare naturally occurring cyclic peptides with three disulfide\nbonds, offering remarkable stability. In neurological disorders, the\nformation of a complex between postsynaptic density protein 95 and\nNMDA receptors (NMDARs) can lead to neuronal cell death. In this study,\nwe modified the MCoTI-II cyclotide backbone with polyarginines for\nenhanced intracellular delivery and grafted a 9-amino acid PSD-95-NMDAR\ninhibitor sequence, NR2B9c, into loop 6. We found that incorporating\npolyarginines into the cyclotide backbone significantly improved uptake\ninto neuronal cells. Primary neurons treated with the NR2B9c cyclotide\n(c5R-NR2B9c) prevented cell death in response to high concentrations\nof <i>N</i>-methyl-d-aspartate (NMDA), demonstrating\nprotection from excitotoxicity. Administration of c5R-NR2B9c in a\nchemically induced seizure model in mice resulted in increased survival\nand reduced seizure severity. Overall, we show that modifying cyclotides\nwith a polyarginine backbone can enhance the delivery of therapeutic\npeptides into neuronal cells, which can be utilized to administer\ntherapeutic peptides for the protection of neuronal cells from excitotoxicity.