Ruchala Piotr, Varadi Gyorgyi, Ishino Tetsuya, Scibek Jeffery, Bhattacharya Madhushree, Urbina Cecilia, Ryk Donald Van, Uings Iain, Chaiken Irwin
Department of Medicine, University of Pennsylvania, 522 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA.
Biopolymers. 2004 Apr 5;73(5):556-68. doi: 10.1002/bip.20001.
The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.
通过重组随机肽库筛选及后续合成变异得到了抑制白细胞介素5（IL-5）功能和IL-5受体α链（IL-5Rα）结合的环肽AF17121（Ac-VDECWRIIASHTWFCAEE）。为了更好地理解其拮抗活性的结构基础，通过固相肽合成制备了AF17121及其一系列母肽类似物。序列变异集中在带电荷的残基Asp(2)、Glu(3)、Arg(6)、Glu(17)和Glu(18)上。其中两个残基，即Glu(3)和Arg(6)，形成了一个EXXR基序，发现该基序在源自肽库的IL-5拮抗剂中很常见。EXXR基序中的E和R的间距类似于先前确定的受体α结合区域（人IL-5的C螺旋和D螺旋之间的β链）中的带电荷残基。通过测量与高活性单链IL-5的竞争情况，利用光学生物传感器相互作用动力学和细胞增殖试验来评估纯化合成肽的拮抗活性。将酸性残基（Asp(2)、Glu(3)、Glu(17)和Glu(18)）逐个替换为Ala的类似物保留了相当的竞争活性，这些残基的多个替换最多导致效力部分丧失。相反，R6A类似物的竞争活性大幅降低。结果表明，精氨酸残基对AF17121与IL-5Rα的结合至关重要，而酸性残基虽可能对稳定复合物特别是在Asp(2)-Glu(3)区域有作用，但并非必不可少。通过圆二色光谱（CD）分析，AF17121大多呈现无序结构，有少量β折叠结构的证据，精氨酸的替换对观察到的肽二级结构没有影响。AF17121活性中Arg(6)的主导地位与先前关于IL-5的反平行β折叠中由（88）EERRR（92）组成的一条链以及相邻β链中的Arg(32)所构成的正电荷平衡主导地位的研究结果一致。这些结果表明，AF17121及相关的源自肽库的肽通过模拟IL-5中的CD转角受体α识别表位发挥作用，为小分子拮抗剂的设计开辟了道路。
