Structure-Based Design of Novel EphA2 Agonistic Agents with Nanomolar Affinity in Vitro and in Cell

Abstract

EphA2 overexpression is invariably associated with poor prognosis and development of aggressive metastatic cancers in pancreatic, prostate, lung, ovarian, and breast cancers and melanoma. Recent efforts from our laboratories identified a number of agonistic peptides targeting the ligand-binding domain of the EphA2 receptor. The individual agents, however, were still relatively weak in affinities (micromolar range) that precluded detailed structural studies on the mode of action. Using a systematic optimization of the 12-mer peptide mimetic 123B9, we were able to first derive an agent that displayed a submicromolar affinity for the receptor. This agent enabled cocrystallization with the EphA2 ligand-binding domain providing for the first time the structural basis for their agonistic mechanism of action. In addition, the atomic coordinates of the complex enabled rapid iterations of structure-based optimizations that resulted in a novel agonistic agent, named 135H11, with a nanomolar affinity for the receptor, as demonstrated by in vitro binding assays (isothermal titration calorimetry measurements), and a biochemical displacement assay. As we have recently demonstrated, the cellular activity of these agents is further increased by synthesizing dimeric versions of the compounds. Hence, we report that a dimeric version of 135H11 is extremely effective at low nanomolar concentrations to induce cellular receptor activation, internalization, and inhibition of cell migration in a pancreatic cancer cell line. Given the pivotal role of EphA2 in tumor growth, angiogenesis, drug resistance, and metastasis, these agents, and the associated structural studies, provide significant advancements in the field for the development of novel EphA2-targeting therapeutics or diagnostics.

Engineering nanomolar peptide ligands that differentially modulate EphA2 receptor signaling

“…To determine whether the loss of the positive charge in the side chain of Lys 14 may contribute to the agonistic properties of the biotinylated peptides, we examined a version of ␤A-WLA-YRPK (16) with acetylation of the Lys 14 side chain ( Table 1, ␤A-WLA-YRPacK (21)). We found that the acetylated peptide has only slightly increased agonistic ability compared with ␤A-WLA-YRPK (16) (Fig. 4, E and G), suggesting that the Lys 14 positive charge has only minor detrimental effects on EphA2 activation.…”

“…2A, compare ␤A-WLA-YR (15) with ␤A-WLA-Yam (9)). Because Arg 12 could introduce sensitivity to proteolytic degradation of C-terminal peptide extensions (16), we included a proline at position 13 because arginine followed by a proline is resistant to cleavage by trypsin-like proteases (16). We also included a lysine at position 14 to allow attachment of biotin or other tags (Table 1, ␤A-WLA-YRPK (16)).…”

“…2A, ␤A-WLA-YRPK-bio (17)). Replacement of Arg 12 with Ser, to eliminate possible residual cleavage by trypsin-like proteases (16), yielded a peptide with only slightly decreased potency but with the disadvantage of being poorly soluble in aqueous solutions (Table 1, ␤A-WLA-YSPK-bio (18)). Thus, Arg 12 does not seem to be critical for high potency (Fig.…”

Prostate Cancer Metastases Are Strongly Inhibited by Agonistic Epha2 Ligands in an Orthotopic Mouse Model

“…Because the dimerization of the receptor is a first critical event in the activation process, dimeric versions of the ephrinA1 ligand (ephrinA1-Fc), and of synthetic agonistic compounds (agent 135H12 [ 14 , 15 ] and Supplementary Table S1 ) were used as agents to probe the effect of EphA2 suppression in cellular and in vivo studies. Indeed, the ephrinA1 ligand is less effective as an agonist compared to the Fc dimerized molecule [ 16 ], and similarly, we recently reported that the synthetic agonistic agent 135H11 [ 14 , 15 ] is significantly less effective in activating the receptor in cellular assays than its dimeric version 135H12 [ 14 , 15 ]. The description of these agents, their affinity and selectivity for the EphA2-ligand binding domain, including biophysical characterization and X-ray structure in complex with EphA2, can be found in our recent publications [ 14 , 15 ].…”

Preparative Method for Asymmetric Synthesis of (S)-2-Amino-4,4,4-trifluorobutanoic Acid

“…As presented in Figure 1, the differences projected by several biphenyl rotational values are relatively modest, suggesting steric mimicry between these two groups, though the effective functional mimicry is very much dependent upon the actual biochemical settings [67]. In this context, the bioisosteric relationships between CF3 and i-Pr can be used for 2-amino-4,4,4-trifluorobutanoic acid 1 [68][69][70][71][72][73][74][75][76] substitution for leucine 2 in the de novo design of biologically active peptides and peptidomimetics [77][78][79][80][81][82][83]. As part of our ongoing research program focused on the application of fluorinated tailor-made AAs in pharmaceutical discovery, we needed a convenient access to large quantities of Fmoc-2amino-4,4,4-trifluorobutanoic acid 1 in both enantiomeric forms.…”