Zamolo Laura, Busini Valentina, Moiani Davide, Moscatelli Davide, Cavallotti Carlo
Dept. Chimica, Materiali e Ingegneria Chimica G. Natta, Politecnico di Milano, Via Mancinelli 7, 20131 Milano, Italy.
Biotechnol Prog. 2008 May-Jun;24(3):527-39. doi: 10.1021/bp070469z. Epub 2008 May 2.
Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose). The study was carried out performing molecular dynamics simulations of the support-spacer-ligand-IgG complex in explicit water. Binding energies between IgG and two supported ligands, a disubstituted derivative of trichlorotriazine and a tetrameric peptide, were determined with the linear interaction energy and MM-GBSA approaches. A detailed study of the possible binding sites of the considered ligands was performed exploiting docking protocols and MD simulations. It was found that both ligands bind IgG in the same site as protein A, which is the hinge region between the CH2 and CH3 domains of IgG. However this site is not easily accessible and requires a high mobility of the ligands. The energetic analysis revealed that van der Waals and electrostatic energies of interaction of the triazine ligand with the support are significant and comparable to those with the protein, so that they limit its capability to reach the protein binding site. A similar result was found also for the tetrameric peptide, which is however able to circumvent the problem; for steric reasons only two of its arms can interact at the same time with the agarose support, thus leaving the remaining two available to bind the protein. These results indicate that the interaction between ligand and support material is an important parameter, which should be considered in the computational and experimental design of ligands for affinity chromatography.
在过去几十年中，基于单克隆抗体的诊断和治疗方法变得越来越重要，但它们的大规模应用需要优化纯化过程。为了实现这一目标，研究集中在亲和色谱技术和新型合成配体的开发上。在这项工作中，我们通过研究亲和配体与单克隆抗体（以人IgG的Fc结构域为模型）和模型支持材料（琼脂糖）之间的相互作用，进行了一项计算研究，旨在为亲和配体的合理设计获得一些指导原则。该研究是在显式水中对支持物-间隔臂-配体-IgG复合物进行分子动力学模拟来进行的。使用线性相互作用能和MM-GBSA方法确定了IgG与两种固定化配体（三氯三嗪的二取代衍生物和四聚体肽）之间的结合能。利用对接协议和分子动力学模拟对所考虑配体的可能结合位点进行了详细研究。发现两种配体都在与蛋白A相同的位点结合IgG，该位点是IgG的CH2和CH3结构域之间的铰链区。然而，该位点不容易接近，并且需要配体具有高流动性。能量分析表明，三嗪配体与支持物相互作用的范德华力和静电能很显著，与和蛋白质相互作用的能量相当，因此限制了其到达蛋白质结合位点的能力。对于四聚体肽也发现了类似的结果，不过它能够规避这个问题；由于空间位阻原因，其只有两个臂可以同时与琼脂糖支持物相互作用，从而使其余两个臂可用于结合蛋白质。这些结果表明，配体与支持材料之间的相互作用是一个重要参数，在亲和色谱配体的计算和实验设计中应予以考虑。
