Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

Abstract

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence-function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX 1 HNGRHX 2 C, cCX 1 HNGRAX 2 C, and cCX 1 ANGRHX 2 C) are related with the position of His residues and cationic amino acids.

Agonist/Antagonist Activity of Oxytocin Variants Obtained from Free Cyclic Peptide Libraries Generated via Amino Acid Substitution

“…The cyclic peptides used in this study were synthesized on a cellulose membrane using a spot synthesis method. The reaction conditions for disulfide bond formation were established as described in our previous papers. , When we preliminarily synthesized a free cyclic OXT peptide and analyzed the released peptide via mass spectrometry, the peak of the intermolecular dimer was detected (Figure S2). To prevent intermolecular oxidation, the photolinker concentration in the peptide array synthesis was decreased.…”

“…A cyclic peptide library was recently established and applied for the assay of NGR cyclic cell adhesion peptides. 16 Previously, we also established a free peptide library of linear peptides using a photocleavable linker. 17 Oxytocin (OXT) is a natural cyclic nonapeptide with the sequence CYIQNCPLG, which generates a disulfide bond between the two Cys residues.…”

“…In our group, bioactive peptide screening using a peptide array was performed. − It was confirmed that peptide activity could be improved via AA residue substitution. A cyclic peptide library was recently established and applied for the assay of NGR cyclic cell adhesion peptides . Previously, we also established a free peptide library of linear peptides using a photocleavable linker …”