MULTIDENTATE HIGH AFFINITY LIGANDS FOR AB5 TOXINS

本提案的目标是利用多聚体蛋白的结构对称性，这是一个很少被探索的特性，最终达到具有超高亲和力和特异性的结构互补的多齿状蛋白配体。长期目标是阐明一个基本的生物学领域：多齿状蛋白质配体的分子识别特性和使用这些配体来控制蛋白质功能。具体来说，本提案包括设计、合成和评估针对一对理想模型系统的多齿配体：大肠杆菌（LT）的热不稳定肠毒素和霍乱弧菌（CT）分泌的密切相关的霍乱毒素，它们都是AB5异六聚体。LT和CT作用的生物学机制包括B五聚体在靶细胞上识别受体的关键步骤。LT和CT的B亚基的五重对称性为开发整体结构与毒素受体结合位点排列互补的五齿配体提供了良好的机会。这种配体将使用模块化方法逐步创建，每个模块都为进一步优化提供机会。基于分子识别原理，我们提出的工作将结合组合化学和基于结构的设计的力量，以达到超高亲和力的五齿酸配体。得到的配体的亲和力将用各种分析工具进行研究。配体-蛋白质相互作用的详细热力学将使用一系列单齿到五齿配体进行研究。所提出的研究对分子识别领域具有广泛的意义，因为它处于研究多齿配体与多聚体蛋白相互作用的前沿。此外，从我们的工作中获得的高亲和力配体具有潜在的健康益处，因为它们可能导致用于检测，治疗和预防AB5毒素相关的肠毒素疾病的药物的开发。

The goal of this proposal is to take advantage of the structural symmetry of multimeric proteins, a rarely explored property, to arrive eventually at structurally complementary multidentate protein ligands with ultra-high affinity and specificity. The long term objective is to illuminate an area of fundamental biological interest: the molecular recognition properties of multidentate protein ligands and the use of such ligands to control protein functions. Specifically, this proposal encompasses the design, synthesis and evaluation of multidentate ligands targeting a pair of ideal model systems: the heat-labile enterotoxin from E. coli (LT) and the closely related cholera toxin secreted by V. cholerae (CT), which are both AB5 heterohexamers. The biological mechanism of the actions of LT and CT includes a critical step of receptor recognition on the target cell by the B pentamer. The five-fold symmetry of the B subunits of LT and CT offers good opportunities to develop pentadentate ligands with overall structures complementary to the arrangement of toxin receptor binding sites. Such ligands will be created stepwise using a modular approach with each module providing opportunities for further optimization Building on the principles of molecular recognition, our proposed work will combine the powers of combinatorial chemistry and structure-based design to arrive at ultrahigh affinity pentadentate ligands. The affinity of the ligands obtained will be investigated with a variety of analytical tools. Detailed thermodynamics of ligand-protein interaction will be studied using a series of mono- to penta-dentate ligands. The proposed research has broad implications for the field of molecular recognition in general since it is at the frontiers of investigations focusing on multidentate ligands interacting with multimeric proteins. In addition, high affinity ligands derived from our work have potential health benefits, as they may lead to the development of agents useful for the detection, treatment, and prevention of AB5 toxin-related enterotoxigenic diseases.