Structure-Based Design of Eph Receptor Antagonists

描述(申请人提供)：EPH受体是最大的蛋白酪氨酸激酶家族。近年来研究表明，Eph受体与其蛋白配基ephins之间的蛋白质-蛋白质相互作用以及随后的双向信号转导在病理性血管生成以及肿瘤的进展和转移中起着重要作用。最近解决了EphB2/ePhrin-B2复合体的X射线晶体结构，使得基于结构的小肽设计和模拟多肽作为蛋白质-蛋白质相互作用的抑制剂成为可能。对受体-配体复合体的X射线晶体结构的检查发现，在受体-配体界面上有一个“热点”，即由六个氨基酸组成的GHL环，深入到Eph受体表面的通道中。对简单二硫键环肽的初步研究表明，含有环残基的短构象限制性环肽可以与EphB2受体以亚微摩尔亲和力结合。在这一建议中，设计了新的环肽类似物和新的肽仿制药来检验以下两个假设：i)更准确地模拟与Eph受体结合的G-HL环的构象可能提供更好的亲和力，ii)基于两个亚类eparin的不同保守序列的分子可能导致发现亚类特异性的小分子拮抗剂(也可能是激动剂)。我们实验室已经合成了两个构象受限的环肽类似物，并正在由我们的合作者进行评估。本项目的具体目标是：1)确定已合成的构象受限环肽与EphB2和EphB1受体的受体结合亲和力，相对于EphA2和EphA3受体，并将构象受限环肽中的残基修饰为EphA中常见的残基，以期获得EphA特异性小分子拮抗剂；2)以D-葡萄糖为骨架合成并评价模拟肽，其中包含被认为在与EphB受体结合中起关键作用的基团；以及3)合成并评价包含D-葡萄糖支架和环肽的杂交结构，以进一步限制构象并增加对EphB受体的亲和力。成功的拮抗剂可作为药物探针，用于阐明Ephn和Eph在各种生理和病理过程中的功能，并可作为潜在的抗血管生成和抗癌药物用于癌症治疗。

DESCRIPTION (provided by applicant): Eph receptors are the largest family of protein tyrosine kinases. The protein-protein interaction between Eph receptors and their protein ligand ephrins and the subsequent bi-directional signaling were recently shown to play an important role in pathological angiogenesis as well as in cancer progression and metastasis. The recent solution of the X-ray crystal structure of EphB2/ephrin-B2 complex made it possible to perform structure-based design of small peptides and peptidomimetics as inhibitors of the protein-protein interaction. Examination of the X-ray crystal structure of the receptor-ligand complex identified a "hot spot" in the receptor-ligand interface, a GHL loop consisting of six amino acids protruding deep into a channel on the Eph receptor surface. Preliminary studies with simple disulfide-linked cyclic peptides indicate that short conformationally restricted cyclic peptides containing the loop residues could bind to the EphB2 receptor with submicromolar affinity. New cyclic peptide analogues and new peptidomimetics were designed in this proposal to test the following two hypotheses: i) better conformational restriction mimicking more accurately the conformation of the G-HL loop as bound to the Eph receptor might offer even better affinity, and ii) molecules based on the distinct conserved sequences of two subclass ephrins might lead to the discovery of subclass-specific small molecule antagonists (possibly also agonists). Two of the conformationally restricted cyclic peptide analogues were already synthesized in our laboratory and are being evaluated by our collaborators. The specific aims of this project are: 1) to determine the receptor binding affinity towards EphB2 and EphB1 receptors of the conformationally restricted cyclic peptides already synthesized, relative to EphA2 and EphA3 receptors, and to modify residues in the conformationally restricted cyclic peptides to those commonly found in ephrin-As in the hope of achieving EphA-specific small molecule antagonists; 2) to synthesize and evaluate peptidomimetics using D-glucose as a scaffold incorporating groups believed to be critical in binding to EphB receptors; and 3) to synthesize and evaluate a hybrid structure containing the D-glucose scaffold and a cyclic tripeptide in efforts to further restrict the conformation and increase the affinity towards EphB receptors. Successful antagonists can be used as pharmacological probes for the elucidation of ephdn and Eph functions in various physiological and pathological processes and as potential antiangiogenic and anticancer agents for the treatment of cancer.