Studies of Molecular Recognition in Biological Systems

描述(由申请人提供)：将小分子的结构改变与其蛋白质结合亲和力的变化相关联是化学生物学中的一个基本问题。与预测蛋白质-配体相互作用中的能量学相关的困难由于严重缺乏关于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热和结合熵的补偿变化的详细实验数据而加剧。这个问题的复杂性之一是缺乏关于配基结构的变化如何影响蛋白质-配基复合体中的蛋白质动力学以及这种动力学中的差异变化是否对结合能量学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、多学科的方法，将合成有机化学、微量热学、蛋白质结晶学、核磁共振光谱和计算化学整合到系统研究中，以明确地研究配体结构的特定变化如何影响明确定义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，我们将设计和合成来源于pTyr-Val-Asn的假肽，它们的刚性和/或预组织性、疏水性、阳离子稳定能力和氢键接受能力都不同。用等温滴定量热法测定了这些假肽与Grb2SH2结构域结合的热力学参数，并通过溶剂分配实验测定了代表化合物的溶剂转移自由能。通过X射线结晶学和核磁共振研究配体结构变化对蛋白质在络合物中的结构和动力学的影响。分子动力学模拟将使用实验数据来改进模型和方法，这样我们就可以计算相对结合能，并探测具有不同结构和亲和力的结合配体时蛋白质动力学的变化。将对结果进行分析，并确定配体结构的特定变化与热力学结合参数和蛋白质柔性的变化之间的相关性，以便确定配体结构的变化是否与结合热和结合熵的变化相关，以及Grb2 SH2结构域的柔性变化是否对配体结合的能量学有显著贡献。从这些研究中获得的见解将被用来设计对Grb2 SH2结构域具有更高结合亲和力的第二代伪肽，因为这些伪肽将作为信号生物学的工具和潜在的治疗线索。与公共健康相关：这里提出的高度集成的实验是独一无二的，旨在确定配体结构的具体变化如何影响蛋白质-配体复合体中的焓、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了解，并有助于开发实验和计算方法，促进基于结构的小分子对蛋白质靶标具有高亲和力的设计。这些工具对于药物化学家来说是不可或缺的，因为它们优化了配体结合亲和力，并将新的先导化合物转化为治疗疾病的选择性和有效的治疗剂。

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.