Dynamics & energetics of p38a kinase regulation by ligands

描述（由申请人提供）：高亲和力蛋白复合物对大量复杂的调控过程至关重要。它们的形成涉及到各种各样复杂的相互作用。这种复杂性反映在仅用分子结构计算蛋白质之间相互作用的能量学的困难上。事实上，基于结构的药物设计已经被这个障碍严重阻碍了。了解蛋白质与天然配体和药理学配体相互作用的能量学和动力学的基本起源，显然对“合理”药物设计的优化至关重要。核磁共振（NMR）弛豫方法的最新进展使得利用蛋白质构象状态之间的运动测量作为构象熵的代理成为可能。最近利用这种方法的研究强有力地表明，构象熵的变化可以显著地影响小分子配体与蛋白质相互作用的热力学。因此，我们将在丝氨酸/苏氨酸激酶p38¿的背景下研究这一问题和相关问题。P38¿与多种疾病状态密切相关，包括癌症和神经系统疾病，并且是药物开发的活跃靶点。我们提出了实验来研究这种蛋白质在与天然和药理学小分子配体相互作用时快速内部运动的变化。将采用先进的核磁共振弛豫方法来测量主链和侧链运动。各种分析策略将被用来深入了解复杂地层热力学的定量贡献，并发现它们的结构起源。此外，调控蛋白结合的动力学效应也将被检查。这些数据将深入研究这种关键激酶被天然效应蛋白和人造分子激活和失活的物理机制的核心。互补氢交换研究也将进行，目的是揭示p38¿内的合作相互作用。这一观点对于新兴的伪变构药类尤其有用。一种新的基于核磁共振的方法，使用高压扰动和快速三维径向采样，将被用来克服标准的“天然状态”氢交换方法在大蛋白质的背景下的局限性，如p38¿。总的来说，该建议建立在初步结果的重要基础上，包括一个异常深厚和强大的丝氨酸/苏氨酸激酶共振分配库。

DESCRIPTION (provided by applicant): High-affinity protein complexes are critical to a large number of intricate regulatory processes. Their formation involves a complicated manifold of interactions that are diverse and complex. This complexity is reflected in the difficulty of computing the energetics of interactions between proteins using molecular structure alone. Indeed, the structure-based design of pharmaceuticals has been significantly impeded by this barrier. Understanding the fundamental origins of the energetics and dynamics of the interactions of proteins with both natural and pharmacological ligands is clearly critical to the optimization of "rational" drug design. Recent advances in nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. There is now a strong indication from recent studies utilizing this approach that changes in conformational entropy can significantly influence the thermodynamics of the interaction of small molecule ligands with proteins. Therefore, we will examine this and related issues in the context of the ser/thr kinase p38¿. p38¿ is intimately associated with a variety of disease states, including cancer and neurological diseases, and is an active target for pharmaceutical development. Experiments are proposed to examine the changes in fast internal motion in this protein upon interaction with both natural and pharmacological small molecule ligands. Advanced NMR relaxation methods will be employed to measure main chain and side chain motion. A variety of analytical strategies will be used to gain insight into the quantitative contributions to the thermodynamics of complex formation and to discover their structural origins. In addition, the dynamical effects of regulatory protein bindng will also be examined. These data will go to the heart of the physical mechanism for activation and deactivation of this critical kinase by both natural effector proteins and man-made molecules. Complementary hydrogen exchange studies will also be carried out with the goal of exposing cooperative interactions within p38¿. This view will be particularly informative with respect to the emerging class of pseudo-allosteric drugs. A novel NMR-based approach using high-pressure perturbation and rapid three dimensional radial sampling will be employed to overcome limitations in the standard "native state" hydrogen exchange method in the context of large proteins, such as p38¿. Overall, the proposal rests on a significant foundation of preliminary results including an unusually deep and robust library of resonance assignments for a ser/thr kinase.