Table 1.
Affinity matured peptide characterization.
Fig 1.
On-& off- rate map of the the two peptide series.
585–1 & 18–1 peptide series are shown in in blue and red, respectively. Parent peptides are shown as stars and the affinity matured offspring as circles. Affinity is indicated by dashed lines.
Fig 2.
Differential HDX of IL-17A in complex with 4 peptides vs the apo state, illustrated on a model of IL-17A built from 4HSA.
The color (generated using the pymol spectrum command) of each alpha carbon represents the magnitude of the change in calculated H-D exchange rate constant upon binding of the corresponding ligand, with purple < = -2.0, blue, green, white = 0.0, yellow, orange, red > = +2.0 log units. The size of each sphere indicates confidence that the apparent signal is truly non-zero, based upon the mean and standard deviation of the acceptable models from the Bayesian analysis, with 0.5 Å radius indicating zero confidence and 1.5 Å indicating a confidence of 1.0 [21]. The blue and green toward the right of the protein upon binding of 585-1-series peptides shows protection to exchange suggestive of binding in that region, while the blue and green closer to the center of the structure in the 18-series peptides suggests binding in that zone. Differential HDX of IL-17A in complex with 585–1 (A), 585–870 (B), 18–1 (C), and 18–902 (D).
Fig 3.
Crystal structure of IL-17A in complex with peptide 18–902.
A) The IL-17A dimer binds two peptide molecules, with each peptide molecule occupying one of the two symmetrically equivalent dimer interfaces. IL-17A is depicted in cartoon representation, with one monomer colored in green and the second monomer colored in orange. The peptide molecules are colored magenta, with one of the molecules depicted in stick representation and the other is shown in cartoon representation to reveal the secondary structure. B) Close-up view of the interactions between IL-17A and 18–902. The peptide interacts with IL-17A through both Van Der Waals and hydrogen bonds. The interaction is mediated primarily by peptide main chain atoms interacting with both main chain and side chain atoms of IL-17A. C) Comparison of the IL-17A:18–902 structure with the structure of the IL-17RA:IL-17A complex. Both structures are shown in cartoon representations. In the IL-17:IL-17RA complex, IL-17RA is colored blue and IL-17A dimer is colored yellow. 18–902 adopts a two-stranded anti-parallel β-sheet linked at the ends by a disulfide, that binds in the same pocket as a pair of anti-parallel β-strands (highlighted in bright blue) of IL-17RA.
Fig 4.
Peptide neutralization of human IL-17A in a cell-based assay.
Peptides were incubated at the indicated concentrations with IL-17A prior to addition to HT-29 cells. After 48 hr. incubation, the culture supernatant was collected and inhibition of CXCL1 production was determined by ELISA. Results are shown as the mean of duplicate treatments ± SD and are representative of two independent experiments.
Fig 5.
Specificity of peptide neutralization on IL-17 family members.
ELISA plates were coated O/N with (A) IL-17RA-Fc, (B) IL-17RE-Fc, (C) IL-17RB-Fc, or (D) IL-17RC-Fc. Indicated concentrations of peptides were pre-incubated with biotinylated (A) IL-17A, (B) IL-17C, (C) IL-17E, or (D) IL-17F for 1 h. Peptide:cytokine mixtures were then added to the respective ELISA plate for 30 min prior to developing. Results are shown as the mean of duplicate treatments ± SD and are representative of two independent experiments.
Fig 6.
Comparison of the crystal structure of IL-17A in complex with peptide 18–902 to the Pfizer macrocyclic ligand and peptide.
A) Comparison of the structure of 18–902 with Pfizer macrocyclic ligand [32]. In the peptide complex, the IL17A monomers are colored green and orange and the peptide molecules are colored magenta. Pfizer macrocyclic is depicted in cyan in stick representation. B) Comparison of the structure of the linear peptide in complex with IL-17A [31], with the structure of the IL17RA:IL17A complex using the same color scheme as Fig 3C. The linear peptide is depicted in magenta cartoon representation.