Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor α

Abstract

The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

“…trx-AF17121 showed inhibition of IL5R␣ binding to IL5 in a dosedependent manner (Fig. 4B), although inhibition was incomplete, as previously found in the competition assay for the AF17121 synthetic peptide (21). As expected, trx-AF17121(AA) did not inhibit the IL5-IL5R␣ interaction at trx-AF17121(AA) concentrations up to 12 M (Fig.…”

“…trx-AF17121 exhibited a dose-dependent inhibition of proliferation, whereas trx-AF17121(AA) showed no significant inhibitory activity. The IC 50 value for trx-AF17121 (560 nM) was similar to the value previously determined for the AF17121 synthetic peptide (21), suggesting that the physical size of thioredoxin makes no steric contribution to the inhibitory effect of AF17121. We found a Ͼ10-fold difference between this biological IC 50 value and the IC 50 value (18 nM) determined from the binding assay described above.…”

“…showed inhibitory effects on IL5 function and proved to be a promising lead for rational drug design as reported previously (20,21). To elucidate the detailed mechanism of these effects, we first carried out an interaction assay using the AF17121 synthetic peptide by SPR-based technology.…”

“…The first set of experiments measured the ability of the fusion peptides to compete with IL5 binding to sIL5R␣ as described previously (21). For this competition assay, 4 nM sIL5R␣ in the absence and presence of different concentrations of trx-AF17121 or trx-AF17121(AA) was passed over the IL5-captured surface (ϳ200 RU).…”

“…A potent disulfide-cyclized 18-mer peptide, AF17121, inhibits the binding of IL5 to IL5R␣ and blocks IL5-dependent eosinophil activation (20). Using mutational analysis of AF17121, we have recently found that the sixth arginine residue plays a crucial role in the antagonist effect and that N/C-terminal acidic residues make a finite but lesser contribution (21). Although AF17121 proved to be a specific antagonist of IL5R␣, showing the importance of its charged clusters for its antagonism, the binding mechanism for IL5R␣, including the binding epitopes on the receptor, has remained incomplete.…”