Alanine scan for the YSA peptide. (A) IC₅₀ values for the indicated modified forms of the YSA peptide were calculated from curves of inhibition of ephrin-A5 AP binding to immobilized EphA2 Fc. The table shows average IC₅₀ values, calculated from the indicated number of experiments (n). Ala-1 through Ala-12 are the peptides, where alanine replaces the indicated residue. Ala-3 is the original YSA peptide. For peptides Ala-1 and Ala-5 the IC₅₀ values were too high to measure accurately (>100 μM); for the Ala-4 peptide no inhibition was detectable at 100 μM, which is the highest peptide concentration tested (≫100 μM). (B) The histogram shows the IC₅₀ values ± SE. The sequence of the YSA peptide is shown, with the residues identified as critical for binding to EphA2 in bold.

The YSA-9mer peptide retains substantially high potency and the YSA-5mer peptide retains high selectively for EphA2. (A, B) Inhibition of ephrin-A5 AP binding to immobilized EphA2 Fc by YSA-9mer and YSA-12mer peptides. (C) Binding of EphA2 Fc to immobilized biotinylated YSA-5mer peptide and YSA-12mer peptides. Similar amounts of the two peptides were immobilized on streptavidin-coated ELISA wells. (D) Binding of the indicated Eph receptor Fc fusion proteins, and ephrin-A5 Fc as a negative control, to the immobilized YSA-5mer peptide.

Biological effects of EphA2-targeting peptides. (A) PC3 cells were treated for 20 min with 0.1 μg/ml ephrin-A1 Fc, 50 μM of the indicated peptides, or left untreated as a control (in duplicate samples). EphA2 immunoprecipitates were probed with anti-phosphotyrosine (PTyr) antibodies and reprobed with anti-EphA2 antibodies. (B) Serum-starved PC3 cells were either left untreated (control), treated with 10% FBS (in duplicate samples), or treated with FBS together with 1μg/ml ephrin-A1 Fc or 50 μM of the indicated peptides. Cell lysates were probed with antibodies specific for Akt phosphorylated at T308 or phosphoErk1/Erk2 MAP kinases (pMAPK) and reprobed for total Akt and MAP kinases. (C) Cells were treated for 20 min with 1 μg/ml ephrin-A1 Fc, for 30 min with 100 μM SWL-12mer peptide or 100 μM YSA-12mer peptide, or left untreated as a control. The cells were then fixed in formaldehyde and labeled with phalloidin to stain actin filaments (red) and with DAPI to label nuclei (blue).

(A) Alignment of the sequences of the YSA and SWL 12mer peptides. The aromatic amino acids are in red; other residues conserved in the two peptides are in blue. (B) The two tyrosines in the YSA peptide are critical for binding to EphA2. The curves show the binding of EphA2 Fc to immobilized biotinylated YSA peptide, or peptide in which Y1 or Y4 is mutated to alanine. Similar amounts of the three peptides were immobilized on streptavidin-coated ELISA wells.

Docking of the YSA peptide in the EphA2 ephrin-binding channel. (A) Top 9 docking poses of the YSA peptide after refinement by RDOCK. All top 9 poses adopt a similar conformation in the ephrin-binding channel of EphA2. The pose with lowest RDOCK energy is presented as a stick structure (C colored in green, O colored in red, N colored in blue, S colored in yellow) and the others as line structures (C colored in purple, O colored in red, N colored in blue, S colored in yellow). The 4 amino acids found to be most critical for EphA2 binding based on the alanine scan (Y1, Y4, P5, and D6) are labeled in green. (B) View of the portion of the YSA peptide (stick representation, colored as in A) that is buried in the EphA2 channel (presented as a solid red ribbon with selected residues that are predicted to interact with the peptide shown in blue and labeled in black). Hydrogen bonds are represented by green dashed lines and salt bridges by black lines (distances in angstroms and indicated by orange numbers). (C) Comparison of the best docked conformation of the YSA peptide (stick representation, colored as in A) with the conformation of the G-H loop of ephrin-A1 in complex with EphA2 as determined by X-ray crystallography (35) (C colored in grey, N colored in blue, and O colored in red).

Stability of EphA2 targeting peptides. The indicated peptides were incubated in PC3 conditioned medium for 5 days at 37 °C (+ medium), or not incubated for comparison (Ctrl), and used for inhibition curves of ephrin-A5 AP binding to immobilized EphA2 Fc. The IC₅₀ values are indicated. The increased IC₅₀ values due to incubation of the peptides in medium were statistically significant (P<0.01 for SWL-13mer and SWLA-10mer; P<0.001 for YSA-12mer and SWL-12mer by Student's t test). However, the effects are small and thus may have little practical consequences.