J Pharm Pharmacogn Res 2(Suppl. 1): S41, 2014
Special supplement with the abstract book of LATINFARMA 2013
CO 067: ENHANCEMENT OF THE INHIBITORY EFFECT OF AN IL-15 ANTAGONIST PEPTIDE BY ALANINE SCANNING
Rodríguez Y1, Reyes O1, Gerónimo H1, Chico A2, Garay H1, Ojeda M1, Arrieta C1, Estévez M2, Guillén G1 and Santos A1.
Introduction: IL-15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including Rheumatoid Arthritis (RA), where it had been proposed as a therapeutic target. We recently reported an IL15 antagonist peptide corresponding to sequence 36–45 of IL-15 (KVTAMKCFLL) named P8, which specifically binds to IL-15Rα and inhibits IL-15 biological activity with a half maximal inhibitory concentration (IC50) of 130 μM in CTLL-2 proliferation assay.
Material and methods: In order to improve binding of peptide P8 to the receptor IL-15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide’s antagonist effect. To evaluate the effects of the peptides, CTLL-2 cells were incubated with serial dilutions of peptides in presence of 300 pg of IL15 during 72 hours. Proliferation was measured by MTT mitochondrial staining. Cells from patients with RA were incubated with 50 µg/mL of peptide or 60 ng/mL of IL-15, or a combination of both. After 48 h incubation, supernatants were collected and TNF-α concentration was determined by ELISA.
Results: Here, we found that Phe and Cys are important for peptide binding to IL-15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non-charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 μM. CTLL-2 proliferation assays showed that the dimeric form of [K6T] P8 had a higher inhibitory activity (IC50 8 μM) than the monomeric form. We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from RA patients.
Conclusion The peptide [K6T]P8 described in this work is a new type of IL-15 antagonist and constitutes a potential therapeutic agent for RA.