Studies of Molecular Recognition in Biological Systems

描述（由申请人提供）：将小分子的结构修饰与其蛋白质结合亲和力的变化联系起来是化学生物学中的一个基本问题。由于严重缺乏与配体结构的特定变化如何影响蛋白质-配体相互作用中结合焓和熵的补偿变化有关的详细实验数据，预测蛋白质-配体相互作用中能量学的困难加剧了。导致问题复杂的原因是缺乏关于配体结构的变化如何影响蛋白质-配体复合物中的蛋白质动力学以及这种动力学的差异变化是否对结合能产生重大影响的信息。为了更好地理解生物系统中的分子识别，我们采用了一种独特的多学科方法，将合成有机化学、微热法、蛋白质晶体学、核磁共振波谱学和计算化学集成到系统研究中，明确地研究配体结构的特定变化如何影响一个定义良好的生物系统中蛋白质-配体相互作用的能量学、结构和动力学。简而言之，我们将设计和合成从pTyr-Val-Asn衍生的假肽，这些假肽在刚性和/或预组织、疏水性、阳离子@稳定能力和氢键接受能力方面各不相同。这些伪多肽与Grb2 SH2结构域结合的热力学参数将通过等温滴定量热法确定，代表性化合物的溶剂转移自由能将通过溶剂分配实验确定。不同配体结构对复合物中蛋白质的结构和动力学的影响将通过x射线晶体学和核磁共振进行研究。利用实验数据进行分子动力学模拟，完善模型和方法，计算相对结合能，探测结合配体具有不同结构和亲和力时蛋白质动力学的变化。对结果进行分析，确定配体结构的具体变化与结合热力学参数和蛋白质柔韧性的变化之间的相关性，从而确定配体结构的变化是否与结合焓和熵的变化相关，以及Grb2 SH2结构域柔韧性的变化是否对配体结合的能量学有重要贡献。从这些研究中获得的见解将用于设计对Grb2 SH2结构域具有更高结合亲和力的第二代假肽，因为这些假肽将作为信号生物学的工具和潜在的治疗线索。公共卫生相关性：本文提出的高度整合的实验是独特设计的，旨在确定配体结构的特定变化如何影响蛋白质配体复合物的焓、熵和动力学。这些研究的结果将增强我们对生物系统中分子识别的认识，并有助于开发实验和计算方法，这些方法将促进对蛋白质靶点具有高亲和力的小分子的基于结构的设计。这些工具对于药物化学家来说是必不可少的，因为它们可以优化配体结合亲和力，并将新的先导物转化为选择性和有效的治疗药物来治疗疾病。

DESCRIPTION (provided by applicant): Correlating structural modifications of small molecules with changes in their protein binding affinities is a fundamental problem in chemical biology. The difficulty associated with predicting energetics in protein-ligand interactions is exacerbated by an acute lack of detailed experimental data pertaining to how specific variations in ligand structure affect compensating changes in binding enthalpies and entropies in protein-ligand interactions. Contributing to the complexity of the problem is a paucity of information regarding how variations in ligand structure affect protein dynamics in protein-ligand complexes and whether differential changes in such dynamics have a significant effect upon binding energetics. Toward developing a better understanding of molecular recognition in biological systems, we have adopted a unique, multidisciplinary approach in which synthetic organic chemistry, microcalorimetry, protein crystallography, NMR spectroscopy, and computational chemistry are integrated in systematic studies to investigate explicitly how specific variations in ligand structure affect energetics, structure and dynamics in protein-ligand interactions in a well-defined biological system. Briefly, we will design and synthesize pseudopeptides that are derived from pTyr-Val-Asn and vary in their rigidity and/or preorganization, hydrophobicity, cation-@ stabilizing ability, and hydrogen bond accepting ability. The thermodynamic parameters for binding of these pseudopeptides to the Grb2 SH2 domain will be determined using isothermal titration calorimetry, and free energies of solvent transfer of representative compounds will be determined by solvent partition experiments. The consequences of varying ligand structure upon structure and dynamics of the protein in the complex will be studied by X-ray crystallography and NMR. Molecular dynamics simulations will be conducted using experimental data to refine the models and methods, so we can calculate relative binding energetics and probe changes in protein dynamics that occur upon binding ligands having different structures and affinities. The results will be analyzed, and correlations between specific changes in ligand structure with variations in thermodynamic binding parameters and protein flexibility will be identified so it can be ascertained whether changes in ligand structure can be correlated with changes in binding enthalpies and entropies and whether changes in flexibility of the Grb2 SH2 domain contribute significantly to the energetics of ligand binding. Insights obtained from these studies will be used to design second generation pseudopeptides having higher binding affinities for the Grb2 SH2 domain as these will be useful as tools for signal biology and as potential therapeutic leads. PUBLIC HEALTH RELEVANCE: The highly integrated experiments proposed herein are uniquely designed to determine how specific changes in ligand structure affect enthalpies, entropies and dynamics in protein-ligand complexes. The results of these studies will enhance our knowledge of molecular recognition in biological systems and contribute to developing experimental and computational methods that will facilitate the structure-based design of small molecules having high affinities for protein targets. Such tools are indispensable to medicinal chemists as they optimize ligand binding affinities and transform novel leads into selective and potent therapeutic agents to treat diseases.