d‐Amino acid substitutions and dimerization increase the biological activity and stability of an IL‐15 antagonist peptide

• Published in: Journal of peptide science Vol. 27; no. 3; pp. e3293 - n/a
• Main Authors: Rodríguez‐Álvarez, Yunier, Cabrales‐Rico, Ania, Diago‐Abreu, David, Correa‐Arguelles, Elianys, Reyes‐Acosta, Osvaldo, Puente‐Pérez, Pedro, Pichardo‐Díaz, Dagmara, Urquiza‐Noa, Dioslaida, Hernández‐Santana, Amalia, Garay‐Pérez, Hilda E.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.03.2021
• Subjects: Amino acids; Antibodies; Biological activity; Cell proliferation; Chymotrypsin; CTLL‐2 cells; dimer; Dimerization; Dimers; d‐amino acid; Immunogenicity; Inflammatory diseases; Interleukins; interleukin‐15; Isomers; Pepsin; peptide; Peptides; Stability; Substitutes; Trypsin; interleukin-15; peptide; biological activity; CTLL-2 cells; dimer; d-amino acid; stability
• ISSN: 1075-2617; 1099-1387
• DOI: 10.1002/psc.3293

Interleukin (IL)‐15 plays an important role in several inflammatory diseases. We have previously identified an IL‐15 antagonist called P8 peptide, which binds specifically to IL‐15 receptor alpha subunit. However, the P8 peptide rapidly degraded by proteases, limiting its therapeutic application. Thus, we replaced each P8 peptide l‐amino acid by its corresponding d‐isomers. First, we determined the biological activity of the resulting peptides in a proliferation assay by using CTLL‐2 cells. The substitution of l‐Ala by d‐Ala ([A4a]P8 peptide) increased the inhibitory effect of the P8 peptide in CTLL‐2 cells in five‐fold. In addition to that, the [A4a]P8 peptide dimer showed the most inhibitory effect. To protect the [A4a]P8 peptide and its dimer against exopeptidase activity, we acetylated the N‐terminal of these peptides. At least a three‐fold reduction in antagonist activity of acetylated peptides was exhibited. However, the substitution of the N‐terminal l‐Lys residue of [A4a]P8 peptide and its dimer by d‐Lys ([K1k;A4a]P8 peptide) did not affect the antagonist effect of the aforementioned peptides. The [K1k;A4a]P8 peptide dimer was stable to the degradation of trypsin, chymotrypsin, and pepsin up until 48 min. Also, the safety and immunogenicity studies in healthy BALB/c mice demonstrated that the administration of this peptide did not affect the clinical parameters of the animals nor generated antipeptide antibodies. Our findings reveal that two distinct d‐amino acid substitutions and dimerization increase the biological activity and stability of P8 peptide. The resulting peptide constitutes a novel IL‐15 antagonist with potential applicability in inflammatory diseases. The substitution of l‐Ala at position 4 and N‐terminal l‐Lys residue by its corresponding d‐isomers increased the inhibitory effect of the P8 peptide in CTLL‐2 cells. The dimer of the resulting peptide [K1k;A4a]P8 showed the most inhibitory effect and was stable to the degradation of trypsin, chymotrypsin, and pepsin. in vivo studies demonstrated that the administration of the [K1k;A4a]P8 peptide dimer did not affect the clinical parameters of healthy BALB/c mice nor generated antipeptide antibodies.