Engineered Cyclotide Blocks Neuronal Excitotoxicity

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