Yang Jie, Gustavsson Anna-Lena, Haraldsson Martin, Karlsson Göran, Norberg Thomas, Baltzer Lars
Department of Chemistry-BMC, Uppsala University, Uppsala, Sweden.
Org Biomol Chem. 2017 May 31;15(21):4644-4654. doi: 10.1039/c7ob00684e.
A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the K of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca ions is low, or where Ca binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the μM to nM range for which no small molecule ligands are known.
一种高亲和力多肽偶联物4-C25L22-DQ已被开发用于分子识别人类C反应蛋白（CRP），CRP是一种著名的炎症生物标志物。CRP是诊断应用中最常被定量检测的目标之一，也是药物开发的靶点。除抗体外，大多数分子构建体利用了磷酸胆碱对CRP的已知亲和力，而磷酸胆碱的结合能力依赖于钙离子。与磷酸胆碱无关的4-C25L22-DQ在无钙离子的情况下结合，解离常数为760 nM，比磷酸胆碱（其K值为5 μM）低一个数量级。基于从一个构建模块集合中选择的一组化合物中的三个命中物的结构相似性设计了小分子2-氧代-1,2-二氢喹啉-8-羧酸（DQ），并通过核磁共振光谱评估其对CRP的亲和力。在竞争实验中显示4-C25L22-DQ结合CRP的能力比DQ本身强三个数量级，在下拉实验中显示4-C25L22-DQ能从人血清中提取CRP。一种强大的、不依赖磷酸胆碱的识别元件的开发，在体内和体外生物分析应用中，在钙离子浓度低或需要使用如EDTA或肝素等钙结合剂来防止血液凝固的条件下，提供了前所未有的机会。从小分子化合物库中鉴定出一种小分子及其与一小部分多肽的偶联，这些多肽以前都不知道能结合CRP，这说明了一种方便且通用的途径，可以得到解离常数在μM到nM范围内的、对蛋白质具有选择性高亲和力的结合剂，而对于这些蛋白质，目前还不知道有小分子配体。
