ITC analysis of ephrinA5-EphA3 binding.

<p>(A) Raw data showing the heat pulses resulting from a titration of ephrin-A5 (10 μM) in the calorimetric cell with an initial 5 μl injection of 100 μM EphA3 followed by 19 subsequent 15 μl injections. (B) Integrated heat pulses normalized per mole of injectant as a function of the molar ratio.</p

Ephrin-A5 interactions with EphA3.

<p>BB, backbone; SC, side chain.</p><p>Modes of interaction are marked as follows: van der Waals interactions in plain text, hydrogen bonds in bold, and salt bridges in bold and underlined.</p><p>Ephrin-A5 interactions with EphA3.</p

Ephrin tilt angles and interface areas in Eph/ephrin complexes.

<p>*Out, not part of the interface area; in, part of the interface area.</p><p>**Amine-methylated protein. Low resolution structures are marked with bold italic.</p><p>Ephrin tilt angles and interface areas in Eph/ephrin complexes.</p

Surface properties of the EphA3 LBD.

<p>(A) Sequence conservation of the EphA3 LBD. Residues of EphA3 forming part of the interface with ephrin-A5 are colored by sequence conservation and other residues are colored wheat. The core of the EphA3 ephrin-binding pocket is lined by conserved residues, highlighted as blue spheres. EphA3 is shown in surface representation and the ephrin-A5 GH loop in cartoon representation. (B) Surface representation of the EphA3 LBD colored by diffusion accessibility. The two regions with poor diffusion accessibility (dark blue) are the ephrin-binding pocket (top left) and a channel near the previously described tetramerization surface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127081#pone.0127081.ref030" target="_blank">30</a>] (bottom right). While the EphA3 LBD alone is not sufficient to form a heterotetramer, the structure nevertheless reveals a framework for residues D130, H131, G132 and V133, which have been proposed to be part of the tetramerization surface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127081#pone.0127081.ref030" target="_blank">30</a>]. This framework includes residues R83, N85, W86 and Y180 located in a channel with poor diffusion accessibility.</p

ITC data for EphA3-ephrin-A5 binding^*.

<p>* Data are averages from 2 measurements ± SD.</p><p>ITC data for EphA3-ephrin-A5 binding^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127081#t001fn001" target="_blank">*</a>}.</p

Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the EphA2 Receptor

Designing potent and selective peptides and small molecules that target Eph receptor tyrosine kinases remains a challenge, and new strategies are needed for developing novel and potent ligands for these receptors. In this study, we performed a structure–activity relationship study of a previously identified 12 amino acid-long peptide, SWL, by alanine scanning to identify residues important for receptor binding. To further enhance and optimize the receptor binding affinity of the SWL peptide, we applied the concept of bivalent ligand design to synthesize several SWL-derived dimeric peptides as novel ligands capable of binding simultaneously to two EphA2 receptor molecules. The dimeric peptides possess higher receptor binding affinity than the original monomeric SWL peptide, consistent with bivalent binding. The most potent dimeric peptide, a SWL dimer with a six-carbon linker, has about 13-fold increased potency as compared to SWL. Furthermore, similar to SWL, the dimeric peptide is an agonist and can promote EphA2 tyrosine phosphorylation (activation) in cultured cells

Data collection and refinement statistics.

<p>Numbers in parentheses represent data in the highest resolution shell (2.34–2.26 Å).</p><p>^a R_merge(<i>I</i>) = Σ_hkl((Σ<i>i</i>|<i>I</i>_{<i>hkl</i>,<i>i</i>}—[<i>I</i>_<i>hkl</i>]|)/Σ_<i>i</i><i>I</i>_{<i>hkl</i>,<i>i</i>}).</p><p>^b R_cryst = Σ_<i>hkl</i>|<i>F</i>_obs|—|<i>F</i>_calc|/Σ_<i>hkl</i>|<i>F</i>_obs|. <i>R</i>_free was computed identically, except that 5% of the reflections were omitted as a test set.</p><p>Data collection and refinement statistics.</p

Features of the EphA3/ephrin-A5 interface.

<p>(A) The L111 cap. The ephrin-binding pocket of EphA3 (in wheat cartoon representation, with select residues represented by sticks) is extended at the top by residue L111^R, which accommodates a shift in conformation of residue F121^L. In the bottom part of the pocket, a sharp twist around residues S56^R and G57^R brings side chains from the C^R and D^R strands into contact with ephrin-A5. (B) The interaction between ephrin-A5 and EphA3 extends outside the ephrin-binding pocket. Atoms in contact with their binding partner are modeled as spheres, colored dark blue for N atoms or red for O atoms. Side chains of residues in the ephrin-GH loop (shown in Fig 4A) are hidden for clarity in Fig 4B.</p

Development and Structural Analysis of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor

The EphA4 receptor is highly expressed in the nervous system, and recent findings suggest that its signaling activity hinders neural repair and exacerbates certain neurodegenerative processes. EphA4 has also been implicated in cancer progression. Thus, EphA4 inhibitors represent potential therapeutic leads and useful research tools to elucidate the role of EphA4 in physiology and disease. Here, we report the structure of a cyclic peptide antagonist, APY, in complex with the EphA4 ligand-binding domain (LBD), which represents the first structure of a cyclic peptide bound to a receptor tyrosine kinase. The structure shows that the dodecameric APY efficiently occupies the ephrin ligand-binding pocket of EphA4 and promotes a “closed” conformation of the surrounding loops. Structure-guided relaxation of the strained APY β-turn and amidation of the C terminus to allow an additional intrapeptide hydrogen bond yielded APY-βAla8.am, an improved APY derivative that binds to EphA4 with nanomolar affinity. APY-βAla8.am potently inhibits ephrin-induced EphA4 activation in cells and EphA4-dependent neuronal growth cone collapse, while retaining high selectivity for EphA4. The two crystal structures of APY and APY-βAla8.am bound to EphA4, in conjunction with secondary phage display screens, highlighted peptide residues that are essential for EphA4 binding as well as residues that can be modified. Thus, the APY scaffold represents an exciting prototype, particularly since cyclic peptides have potentially favorable metabolic stability and are emerging as an important class of molecules for disruption of protein–protein interactions

Modifications of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor To Achieve High Plasma Stability

EphA4 is a receptor tyrosine kinase with a critical role in repulsive axon guidance and synaptic function. However, aberrant EphA4 activity can inhibit neural repair after injury and exacerbate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s. We previously identified the cyclic peptide <b>APY-d2</b> (APYCVYRβASWSC-nh₂, containing a disulfide bond) as a potent and selective EphA4 antagonist. However, <b>APY-d2</b> lacks sufficient plasma stability to be useful for EphA4 inhibition <i>in vivo</i> through peripheral administration. Using structure–activity relationship studies, we show that protecting the peptide N-terminus from proteolytic degradation dramatically increases the persistence of the active peptide in plasma and that a positively charged peptide N-terminus is essential for high EphA4 binding affinity. Among several improved <b>APY-d2</b> derivatives, the cyclic peptides <b>APY-d3</b> (<u>βA</u>PYCVYRβASWSC-nh₂) and <b>APY-d4</b> (<u>βA</u>PYCVYRβA<u>E</u>W<u>E</u>C-nh₂) combine high stability in plasma and cerebrospinal fluid with slightly enhanced potency. These properties make them valuable research tools and leads toward development of therapeutics for neurological diseases