Identification of a NEP1–35 recognizing peptide that neutralizes CNS myelin inhibition using phage display library

Nogo-A has been identified as an inhibitory molecule to neurite outgrowth after injury in adult mammalian central nervous system (CNS). The C-terminal fragment of Nogo-A, Nogo-66, inhibits axonal regrowth through NgR1 signaling. Residues 1–32 of Nogo-66 cover two regions that contribute most affinity of Nogo-66 to NgR1. It is unclear whether blocking the two regions with specific small ligands could neutralize the inhibition of Nogo-66. Therefore in this study we explored two phage display peptide libraries to screen small peptides that might bind Nogo-66. NEP1–35 containing 1–33 residues of Nogo-66 was taken as the target for panning. We found that phage-borne peptides with stronger affinity to NEP1–35 contained a relatively conserved motif, RRXXXXXXXRRX. Afterwards one identified peptide, NH2-RRQTLSHQMRRP-COOH was synthesized and tested in neurite outgrowth assay, in which this small molecule showed moderate ability to neutralize CNS myelin inhibition in vitro. Our results demonstrated that short peptides could act as adaptors to Nogo-66 and neutralize CNS myelin inhibition in vitro. Additionally, the results also suggested that phage display could help to discover novel small molecules with high affinity to CNS regrowth inhibitors, which might be able to promote CNS regeneration with fewer side effects since they could block only the corresponding regions of inhibitors.

► NEP1–35 covering active regions of Nogo-66 is taken as the target for screening.
► Small peptides with high affinity to NEP1–35 are selected from phage display peptide libraries.
► Selected peptides have preference for proline and arginine and some peptides contain relatively conserved primary structure.
► One selected peptide with strong affinity to NEP1–35 is identified to neutralize CNS myelin inhibition in vitro.