Štambuk Nikola, Manojlović Zoran, Turčić Petra, Martinić Roko, Konjevoda Paško, Weitner Tin, Wardega Piotr, Gabričević Mario
Center for Nuclear Magnetic Resonance, Ruđer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia.
Croatian Institute for Toxicology and Antidoping, Borongajska 83 g, 10000 Zagreb, Croatia.
Int J Mol Sci. 2014 May 26;15(6):9209-23. doi: 10.3390/ijms15069209.
Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.
反义肽技术是一种用于衍生新的生物活性分子和进行肽-受体调节的重要工具。它基于这样一个事实，即由互补（反义）核苷酸序列指定的肽通常以更高的特异性和效力相互结合。我们以人促红细胞生成素为例测试了这一概念的有效性，人促红细胞生成素是一种特征明确且在药理学上相关的造血生长因子。这项工作的目的是提出并测试一种简单有效的三步程序，用于设计靶向人促红细胞生成素受体结合位点的反义肽。首先，我们选择该分子的羧基末端受体结合区域（表位）作为反义肽建模的模板；其次，我们通过将表位序列在3'→5'方向上进行mRNA转录并利用BLAST对潜在的互补位结构进行计算筛选来设计反义肽；第三，我们通过荧光光谱法和微量热泳法评估正义-反义（表位-互补位）肽的结合和亲和力。两种方法分别显示出相似的解离常数（Kd）值，分别为850和816 μM。这些方法的优点是：筛选速度快，所需样品量少，并且在类似于生理条件的物理化学参数范围内进行测量。靶向特定促红细胞生成素区域的反义肽可用于开发新的免疫化学方法。具有最佳亲和力的选定反义肽是开发新型诊断物质、生物制药和疫苗的潜在先导化合物。
