Targeting EphA2‐Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues

Abstract

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam-Sam interactions. Because EphA2-Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam-Sam interactions involving EphA2.

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study

“…SPR : SPR experiments were performed as previously reported with a Biacore 3000 instrument and associated reagents (GE Healthcare). A CM5 sensor chip was used and EphA2‐SAM was immobilized on it at a concentration equal to 300 μg mL −1 in 10 m m acetate buffer, pH 4.5, by injecting sample (35 μL) with a flow rate of 5 μL min −1 and reaching an immobilization level of 1047 RU.…”

“…CD : CD spectra were recorded on a Jasco J‐810 spectropolarimeter (JASCO Corp, Milan, Italy), as previously reported –. Spectra were obtained by averaging three scans, subtracting blanks, and converting the signal to mean residue ellipticity [deg cm 2 dmol −1 res −1 ].…”

The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors

“…The interaction between the (KRI) 3 peptide and Ship2-Sam was further investigated by MST (Microscale Thermophoresis) experiments (Supplemental Fig. S6 ) 23 . MST studies clearly show a protein/peptide interaction but, they do not allow an accurate K D measurement due to precipitate formation at the highest (KRI) 3 peptide concentrations (Supplemental Fig.…”

“…CD spectra were recorded in a 0.1 cm path-length quartz cuvette on a Jasco J-810 spectropolarimeter (JASCO Corp, Milan, Italy) as already described 20 , 23 . Spectra derived from the average of three scans, the subtraction of blanks and the conversion of the signal to mean residue ellipticity (deg × cm 2 × dmol −1 × res −1 ).…”

Therapeutic potential of targeting the Eph/ephrin signaling complex

“…Even the very C-terminal SAM domain could offer an attractive drug target, since it operates as a docking surface for several cytoplasmic down-stream signaling proteins (Wang et al, 2016). Targeting SAM for drug discovery (Mercurio and Leone, 2016; Mercurio et al, 2016) should gain more interest in the future taking into account the fact that Eph’s have an unusual property to be able to signal even in the absence of cytoplasmic tyrosine phosphorylation (Truitt and Freywald, 2011). Importantly, high-resolution structures of all the domains, either as isolated molecules or in complex with ligands and other binding partners, including peptides, and small molecular inhibitors/modifiers, have been determined and are available (Barton et al, 2004; Noberini et al, 2012b).…”