Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide

Abstract

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.

Figure 1. Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays.

(A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). Kinetic parameters (k_a, k_d) were obtained by a global fit using three concentrations in triplicate. K_D determined by the standard equation, K_D = k_d/k_a. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). Data are mean and error bars of +/− standard deviation of duplicated experiments.

Figure 2. Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation.

For clarity, different molecules are colored differently. Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively. Picture prepared using program CCP4MG. (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. (B) Close-in view of the IL-17A/HAP structure. IL-17A β-strands are labelled. Each of the two bound HAP interacts with both monomers of the IL-17A dimer. (C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation. Three distinct areas IL-17A/IL-17RA interface are labeled.

Figure 3. Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP.

(A) HAP binds at region I of IL-17A. IL-17A dimer is in surface presentation (β-strands 0 shown as ribbons for clarity). Polar interactions are shown in dashes. HAP residues as well as key IL-17A residues are labeled. For clarity, a few HAP residues are also shown in stick model with carbon atoms colored green, oxygen in red and nitrogen in blue. (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure.