Modifying the PSD-95 inhibitor NA-1 for brain delivery through proteolytic stabilization and conjugation to the...

Delivering peptide-based therapeutics to the brain through systemic administration is limited by poor blood-brain barrier permeation, rapid proteolytic degradation, and off-target accumulation. The peptide NA-1, targeting PSD-95 interactions, holds promise for stroke treatment but suffers from these limitations. With this study, we explored whether complete D-amino acid substitution of NA-1 and conjugation to the potentially brain-homing peptide BR1 would improve NA-1 plasma stability and brain delivery, while retaining receptor binding and limiting off-target organ accumulation. Four peptide constructs, L- and D-versions of NA-1 and BR1-NA-1, were synthesized and characterized using circular dichroism spectroscopy, surface plasmon resonance, mouse plasma stability assays, cellular uptake studies, and biodistribution analysis in mice. D-amino acid substitution significantly enhanced peptide stability in plasma but compromised receptor binding and to some extent cellular uptake. L-BR1-NA-1 showed improved accumulation in whole brain lysates as compared to L-NA-1, however parenchymal delivery did not differ between the two peptides, indicating vascular retention of the L-BR1-NA-1 form. The present study demonstrated that substitution of L-amino acids with their D-amino acid counterparts improved the proteolytic stability of the constructs. However, this came at the expense of target affinity. BR1 conjugation to L-NA-1 might increase vascular retention, but did not increase parenchymal delivery, indicating that BR1 does not mediate transendothelial passage of the construct.