Dynamics & energetics of p38a kinase regulation by ligands

描述（由申请人提供）：高亲和力蛋白质复合物对大量复杂的调控过程至关重要。它们的形成涉及多种多样和复杂的相互作用。这种复杂性反映在仅使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受到了这一障碍的严重阻碍。理解蛋白质与天然和药理学配体相互作用的能量学和动力学的基本起源对于优化“理性”药物设计显然是至关重要的。核磁共振（NMR）弛豫方法的最新进展，使使用的措施之间的运动的构象状态的蛋白质作为一个代理的构象熵。现在有一个强有力的迹象，从最近的研究利用这种方法，构象熵的变化可以显着影响小分子配体与蛋白质的相互作用的热力学。因此，我们将在ser/thr激酶p38的背景下研究这一点和相关问题。第38页与包括癌症和神经疾病在内的多种疾病状态密切相关，并且是药物开发的活性靶标。实验提出了检查在这种蛋白质的快速内部运动的变化后，与天然和药理学的小分子配体的相互作用。先进的核磁共振弛豫方法将被用来测量主链和侧链的运动。各种分析策略将用于深入了解复杂形成的热力学的定量贡献，并发现其结构起源。此外，还将研究调节蛋白结合的动力学效应。这些数据将进入天然效应蛋白和人造分子激活和失活这一关键激酶的物理机制的核心。还将进行补充氢交换研究，目的是揭示p38内的合作相互作用。这一观点将是特别翔实的关于新兴类的假变构药物。一种新的基于NMR的方法，使用高压扰动和快速三维径向采样将被用来克服在大蛋白质，如p38的背景下，在标准的“天然状态”氢交换方法的局限性。总的来说，该建议依赖于一个显着的基础上的初步结果，包括一个异常深入和强大的库的共振分配的丝氨酸/苏氨酸激酶。

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.