Abstract：
The epidermal growth factor receptor EGFR allows targeted delivery of macromolecular drugs to tumors. Its ligand, epidermal growth factor, binds EGFR with high affinity but acts mitogenic. Non-mitogenic peptides are utilized as targeting ligands, like the dodecapeptide GE11, although its low binding affinity warrants improvement. We applied a two-step computational approach with database search and molecular docking to design GE11 variants with improved binding. Synthesized peptides underwent binding studies on immobilized EGFR using surface plasmon resonance. Conjugates of peptides coupled via heterobifunctional PEG linker to linear polyethylenimine (LPEI) were used for transfection studies on EGFR-overexpressing cells using reporter gene encoding plasmid DNA. Docking studies unraveled similarities between GE11 and the EGFR dimerization arm. By skipping non-overlapping amino acids, a less hydrophobic segment (YTPQNVI) was identified to be directly involved in EGFR binding. By replacing valine by tyrosine, a full-length version with proposed enhanced binding (GE11m3) was developed. While hydrophobic or hydrophilic segments and variations thereof exhibited low binding, GE11m3 exhibited 3-fold increase in binding compared to GE11, validating in silico predictions. In transfection studies, polyplexes with GE11m3 induced a significantly higher reporter gene expression when compared to GE11 polyplexes both on murine and human cancer cells overexpressing EGFR.
摘要：
表皮生长因子受体EGFR允许将大分子药物靶向递送至肿瘤。它的配体，表皮生长因子，以高亲和力结合EGFR，但有丝分裂作用。非促有丝分裂肽被用作靶向配体，与十二肽GE11一样，尽管其低结合亲和力需要改进。我们应用了具有数据库搜索和分子对接的两步计算方法来设计具有改进的结合的GE11变体。使用表面等离子体共振对合成的肽进行了固定化EGFR的结合研究。通过异双功能PEG接头与线性聚乙烯亚胺(LPEI)偶联的肽的缀合物用于使用编码质粒DNA的报告基因对EGFR过表达细胞的转染研究。对接研究揭示了GE11和EGFR二聚化臂之间的相似性。通过跳过不重叠的氨基酸，疏水性较低的片段(YTPQNVI)被鉴定为直接参与EGFR结合。用酪氨酸代替缬氨酸,开发了具有建议的增强绑定（GE11m3）的全长版本。虽然疏水或亲水链段及其变体表现出低结合，与GE11相比，GE11m3的结合增加了3倍，验证了计算机预测。在转染研究中,与GE11复合物相比，具有GE11m3的复合物在过表达EGFR的鼠和人癌细胞上诱导了显着更高的报告基因表达。
