Molecular Mechanism of Flexible Carbohydrate-Protein Interaction in CD44

描述（由申请人提供）：本研究的目的是通过加深对CD44：透明质酸蛋白：碳水化合物结合相互作用的理解，为合理开发具有侵袭性/转移性癌症和炎症性疾病潜在治疗价值的药物样小分子化合物奠定基础。这种结合相互作用是抗癌和抗炎治疗的有效靶点，一直是核磁共振和x射线晶体学结构生物学研究的主题，这些研究导致了原子分辨率模型。然而，结合位点的构象灵活性，一个大的蛋白质：碳水化合物结合界面，以及变构位点的有序-无序转变都是使试图在原子水平上理解结合细节复杂化的因素。这一结果对于理解CD44：透明质酸分子识别的分子基础和基于结构的CD44：透明质酸结合药物样抑制剂的发现都是一个关键的障碍。为了克服这一障碍，原子细节的分子模拟将被应用于实现三个特定的目标：1。确定结合位点灵活性对结合亲和力的贡献，2。2 .寻找大型绑定接口上对绑定亲和度贡献大于预期的“热点”；确定变构位点有序到无序的转变对结合位点柔韧性和亲和力的贡献。研究设计包括系统地确定弱和动态蛋白质：碳水化合物相互作用的构象景观的能量学，其作用尚未通过实验或计算方法进行广泛探索。使用的方法是全原子显式溶剂分子动力学模拟，包括偏抽样和计算炼金术方法，提供有关CD44：透明质酸复合物构象和组成变化的自由能后果的信息。实现上述目标将导致原子分辨率的理解(a)结合位点的灵活性和亲和力，以及(b)灵活性和亲和力如何受到有序到无序转变的影响，从而进一步了解CD44：透明质酸分子识别及其应用于基于结构的CD44:HA抑制剂的发现。

DESCRIPTION (provided by applicant): The purpose of the proposed research is to strengthen the foundation for the rational development of drug-like small-molecule compounds with potential therapeutic value in invasive/metastatic cancers and inflammatory disorders by advancing the understanding of the CD44:hyaluronan protein:carbohydrate binding interaction. This binding interaction, which is a validated target for anti-cancer and anti-inflammatory therapy, has been the subject of both NMR and X-ray crystallography structural biology studies that have led to atomic-resolution models. However, conformational flexibility of the binding site, a large protein:carbohydrate binding interface, and an order-to-disorder transition at an allosteric site are all factors that complicate attempts to understand binding at an atomic level of detail. The result is a critical barrier to progress both with respect to understanding the molecular basis for CD44:hyaluronan molecular recognition and the structure-based discovery of drug-like inhibitors of CD44:hyaluronan binding. Toward overcoming this barrier, atomically- detailed molecular simulations will be applied to achieve three specific aims: 1. Determining the contribution of binding site flexibility to binding affinity, 2. Looking for "hot spots" on the large binding interface that make greater than expected contributions to binding affinity, and 3. Determining the contribution of the allosteric site order-to-disorder transition on binding site flexibility and affinity. The research design involves systematically determining the energetics of the conformational landscape underlying weak and dynamic protein:carbohydrate interactions, the role of which has not been explored extensively via either experimental or computational approaches. The methodology to be used is all-atom explicit-solvent molecular dynamics simulations, including biased sampling and computational alchemy approaches that provide information regarding the free-energy consequences of conformational and compositional changes in the CD44:hyaluronan complex. Achieving the stated aims will lead to atomic- resolution understandings of (a) binding site flexibility and affinity, and (b) how flexibility and affinity are impacted by the order-to-disorder transition, thereby furthering the fundamental understanding of CD44:hyaluronan molecular recognition and its application to structure-based discovery of CD44:HA inhibitors.) PUBLIC HEALTH RELEVANCE: The binding interaction of the CD44 protein and the hyaluronan carbohydrate plays roles in certain invasive/metastatic cancers and inflammatory disorders. Targeting this interaction may therefore be a new way to help treat these diseases. Toward this goal of new therapeutics, and also to enhance the limited understanding of molecular flexibility in protein:carbohydrate complexes, the present work seeks to strengthen the fundamental molecular-level understanding of this binding interaction through the application of cutting- edge computer simulation methods.