Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran.
FEBS J. 2014 Oct;281(19):4479-94. doi: 10.1111/febs.12956. Epub 2014 Sep 1.
Angiogenesis is a hallmark of various pathological conditions and is controlled by a variety of angiogenic factors. Blockade of vascular endothelial growth factor (VEGF) as the most pivotal stimulator of angiogenesis offers a promising therapeutic approach for some diseases, typically cancer. In the present study, a heterodimeric antagonistic VEGF was precisely designed based on structural information of recently-crystallized VEGF/VEGF receptor-2 (VEGFR-2/fetal liver kinase 1/kinase domain region) complex. Directed blocking of kinase domain region occurs via substitution of a VEGF receptor binding site by two peptide segments in one pole, whereas the binding domain of the other pole of VEGF was intact. Candidate peptides for substitution were selected considering to some sequence and structural criteria. A reliable model of modified VEGF was built, refined using molecular dynamics simulation and docked with VEGFR-2. Docking analysis revealed that binding affinity of mutant VEGF was notably diminished, corroborating our design. Heterodimeric VEGF was expressed, refolded and highly purified by two-step affinity chromatography. Dimerization of this antagonist was confirmed using some analytical techniques. Spectroscopic studies assured us to obtain the heterodimeric form of VEGF. Some angiogenic in vitro assays such endothelial cell proliferation and tube formation indicated that this antagonist is not only strongly capable of inhibiting angiogenesis (half maximal inhibitory concentration of 33 and 24 ng · mL(-1) , respectively), but also showed the highest inhibitory effect compared to all other heterodimeric VEGF variants. The high anti-angiogenic potency of this VEGF antagonist may allow its future use as an anti-tumor agent.
血管生成是各种病理状况的标志，由各种血管生成因子控制。阻断血管内皮生长因子（VEGF）作为血管生成的最关键刺激因子，为一些疾病（通常是癌症）提供了一种有前途的治疗方法。在本研究中，根据最近结晶的 VEGF/VEGF 受体-2（VEGFR-2/胎肝激酶 1/激酶结构域）复合物的结构信息，精确设计了一种异二聚体拮抗 VEGF。通过在一个极中的两个肽段取代 VEGF 受体结合位点，定向阻断激酶结构域区域，而 VEGF 的另一极的结合域保持完整。考虑到一些序列和结构标准，选择了候选肽段进行取代。建立了可靠的修饰 VEGF 模型，使用分子动力学模拟进行了细化，并与 VEGFR-2 对接。对接分析表明，突变 VEGF 的结合亲和力明显降低，证实了我们的设计。通过两步亲和层析表达、复性和高度纯化异二聚体 VEGF。使用一些分析技术证实了该拮抗剂的二聚化。光谱研究使我们确信获得了 VEGF 的异二聚体形式。一些血管生成体外测定，如内皮细胞增殖和管形成实验，表明该拮抗剂不仅具有强烈的抑制血管生成能力（半最大抑制浓度分别为 33 和 24ng·mL-1），而且与所有其他异二聚体 VEGF 变体相比，显示出最高的抑制效果。这种 VEGF 拮抗剂的高抗血管生成效力可能使其将来可作为抗肿瘤剂使用。
