A disalicylic acid-furanyl derivative inhibits ephrin binding to a subset of Eph receptors

Abstract

Eph receptor tyrosine kinases and ephrin ligands control many physiological and pathological processes, and molecules interfering with their interaction are useful probes to elucidate their complex biological functions. Moreover, targeting Eph receptors might enable new strategies to inhibit cancer progression and pathological angiogenesis as well as promote nerve regeneration. Because our previous work suggested the importance of the salicylic acid group in antagonistic small molecules targeting Eph receptors, we screened a series of salicylic acid derivatives to identify novel Eph receptor antagonists. This identified a disalicylic acid-furanyl derivative that inhibits ephrin-A5 binding to EphA4 with an IC(50) of 3 μm in ELISAs. This compound, which appears to bind to the ephrin-binding pocket of EphA4, also targets several other Eph receptors. Furthermore, it inhibits EphA2 and EphA4 tyrosine phosphorylation in cells stimulated with ephrin while not affecting phosphorylation of EphB2, which is not a target receptor. In endothelial cells, the disalicylic acid-furanyl derivative inhibits EphA2 phosphorylation in response to TNFα and capillary-like tube formation on Matrigel, two effects that depend on EphA2 interaction with endogenous ephrin-A1. These findings suggest that salicylic acid derivatives could be used as starting points to design new small molecule antagonists of Eph receptors.

Figure 1

The 76D10 salicylic acid-furanyl derivative inhibits ephrin binding to a subset of Eph receptors. (A) IC₅₀ values for inhibition of EphA4 AP binding to immobilized ephrin Fc fusion proteins by 76D10. (B) IC₅₀ values for inhibition of ephrin-A5 AP binding to immobilized EphA receptor Fc fusion proteins and ephrin-B2 AP binding to immobilized EphB receptor Fc fusion proteins by 76D10. It should be noted that we typically obtain somewhat lower IC₅₀ values in this version of the ELISA assay with immobilized Eph receptors compared to the arrangement with immobilized ephrins. Error bars in A and B represent the standard errors for IC₅₀ values calculated from 3–5 experiments. (C) Curves showing binding of ephrin-A5 AP to immobilized EphA4 Fc in the presence of the indicated concentrations of 76D10. The curves were fitted according to the Michaelis-Menten equation. Error bars represent the standard errors from triplicate measurements.

Figure 2

Compound 76D10 causes chemical shifts in residues of the EphA4 ligand-binding pocket. (A) Superposition of ¹H-¹⁵N NMR HSQC spectra of the EphA4 ligand-binding domain (at a concentration of 60 μM) in the absence (red) and in the presence (green) of 300 μM 76D10. The residues whose peaks disappeared after the addition of 76D10 are labeled. (B) Residue-specific chemical shift perturbation (CSP) values for the EphA4 ligand-binding domain in the presence of 76D10. Peaks that disappear from the EphA4 spectrum in panel A are shown as purple bars in the histogram and assigned an arbitrary 0.16 Δppm value. A threshold dash line is drawn at 0.06 Δppm to indicate residues with significant (Δδ > 30 Hz) perturbation, which are shown as red bars in the histogram. The amino acids that are part of the EphA4 ligand-binding pocket shown in C are labeled. (C) Ribbon representation of residues in the EphA4 ligand-binding domain that show significant perturbation in the presence of 76D10. The residues that disappeared from the EphA4 spectrum in A are colored in purple and labeled and the residues with Δppm between 0.06 and 0.16 are shown in red. The structure of the EphA4 ligand-binding domain is from the PDB structure 3CKH, with the missing residues in the loop regions calculated using SWISS-MODEL.

Figure 3

Compound 76D10 inhibits EphA4 and EphA2 activation and cell retraction after ephrin stimulation. (A) Cells pretreated with the indicated concentrations of 76D10 for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compound. COS cells were stimulated with 0.2 μg/mL ephrin-A1 or 0.8 μg/mL ephrin-B2 Fc and used to immunoprecipitate EphA2 and EphB2, while HT22 neuronal cells were stimulated with 0.2 μg/mL ephrin-A1 and used to immunoprecipitate EphA4. Eph immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for the Eph receptor immunoprecipitated. (B) PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.2 μg/mL ephrin-A1 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. The histogram shows the average level of phosphorylated EphA2 normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. Error bars represent standard errors from 4–10 measurements. The levels of EphA2 phosphorylation in cells treated with ephrin-A1 Fc and compound were compared to those in cells treated only with ephrin-A1 Fc by one-way ANOVA and Dunnett’s post test. ***P<0.001 by one-way ANOVA. (C–D)PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.5 μg/ml ephrin-A 5Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. (C) The histogram shows the average area of the cells normalized to the value obtained for the Fc-treated cells. Error bars represent standard errors from three wells. The average cell areas in cells treated with ephrin-A1 Fc and compound were compared to that in cells treated only with ephrin-A1 Fc by one-way ANOVA and Bonferroni’s post test, showing that 76D10 significantly (***P<0.001) inhibits ephrin-A1-dependent cell retraction at concentrations between 100 and 25 μM. The effect of ephrin-A1 was reverted completely by 100 μM 76D10 and partially by 50 and 25 μM (comparison between Fc and ephrin-A1 Fc treated samples at each compound concentration yielded P values of >0.05 for 100μM, <0.05 for 50 μM and <0.001 for 25μM 76D10. (D) Representative images of cells stained with rhodamine-phalloidin to label actin filaments (red) and DAPI to label nuclei (blue). Scale bar = 50 μm.

Figure 4

Compound 76D10 inhibits ephrin- and TNFα-induced tyrosine phosphorylation and capillary-like tube formation in HUVE cells. (A) Cells plated on Matrigel were treated with the indicated concentrations of 76D10 or DMSO and imaged 18 hours later. The number of polygons present in each picture and the average tube length were quantified. The histograms show averages from 4 independent experiments and the error bars represent the standard errors. **P<0.01 and ***P<0.001 by one-way ANOVA and Dunnett’s post test. (B) HUVE cells were left unstimulated or stimulated with 20 nM TNFα for 2 hours in the presence of the indicated concentrations of 76D10. EphA2 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphA2. (C) MTT assay to determine the number of viable HUVE cells after growth in the presence of the indicated concentrations of 76D10 for 1 or 3 days. Only DMSO was used in the “0 μM” sample, as a control. The histogram shows average absorbance at 570 nm in the presence of 76D10 normalized to the absorbance in the absence of the compound. Error bars represent standard error from 3 measurements in each of two experiments. *P<0.05 by one-way ANOVA and Dunnett’s post test for the comparison to cells not treated with compound (0 μM).

Scheme 1

Synthesis of 76D10

Scheme 2

Synthesis of 76A5 and 76B8