Inhibitor Screens for Five MAPK Kinase Kinases Important in Human Disease

说明书(申请人提供)：丝裂原活化蛋白激酶(MAPK)通常在所有细胞类型中表达，但调节不同细胞类型不同的非常特定的生物反应。MAPK实际上是由MAPK、MAPK(MKK)和MAPK(MKKK)组成的三个信号模块的成员。MKKKs磷酸化并激活MKKs，MKKs进而磷酸化并激活MAPKs。正是MKKK及其不同的蛋白质-蛋白质相互作用和共价修饰的多样性使得特定的MAPK通路能够整合到细胞对不同刺激的反应中，这些刺激包括细胞因子、生长因子、抗原、毒素、药物、应激和细胞外基质和细胞间相互作用的变化。MAPK激酶(MKKK)是一类在很大程度上未被开发的小分子抑制作用的靶点，以调节信号网络、基因表达和潜在的人类疾病。因此，这项建议的具体目的是：1.建立适用于高通量筛选(HTS)的可靠和可重复性的时间分辨荧光能量转移(TR-FRET)分析方法，以确定五种MAPK激酶的特异性抑制剂。这些激酶包括MEKK1、MEKK2、MEKK3、MEKK4和ASK1。这五种MAPK激酶与慢性炎症、病理性肥大、血管生成和氧化应激诱导的细胞死亡相关的组织重塑密切相关。这些MAPK激酶的抑制剂还没有被描述出来，导致对小分子探索其细胞和生理功能的需求尚未得到满足。2.从HTS中分离出或从一个小的定向激酶抑制剂库中鉴定为特定MKKK的抑制剂的小分子，即MEKK1、MEKK2、MEKK3、MEKK4或ASK1，将与剩余的14个MAPK激酶以及具有代表性的MAPK和MAPK进行筛选，以进行初步的选择性分析。3.将进行基于功能细胞的筛选，以确定五种MAPK激酶(MEKK1、MEKK2、MEKK3、MEKK4或ASK1)中每一种的小分子抑制剂。这些MAPK激酶的基因敲除和RNAi提供了一种独特的表型和对特定刺激的改变反应，为基于机制的选择性分析提供了小分子与基因消融的激酶的容易比较。

DESCRIPTION (provided by applicant): Mitogen-activated protein kinases (MAPKs) are generally expressed in all cell types yet regulate very specific biological responses that differ from cell type to cell type. MAPKs are actually members of a three kinase signaling module composed of the MAPK, MAPK kinase (MKK) and MAPK kinase kinase (MKKK). MKKKs phosphorylate and activate MKKs, which in turn phosphorylate and activate MAPKs. It is the diversity of MKKKs and their different protein-protein interactions and covalent modifications that allows the integration of specific MAPK pathways in the cellular response to diverse stimuli including cytokines, growth factors, antigens, toxins, pharmacological drugs, stress insults, and changes in extracellular matrix and cell- cell interactions. MAPK kinase kinases (MKKKs) are a largely untapped target class for small molecule inhibition to modulate signaling networks, gene expression and potentially human disease. Therefore, the specific aims of this proposal are: 1. Develop robust and reproducible time resolved fluorescence energy transfer (TR-FRET) assays suitable for high-throughput screens (HTS) to identify specific inhibitors for five MAPK kinase kinases. These kinases include MEKK1, MEKK2, MEKK3, MEKK4 and ASK1. These five MAPK kinase kinases are strongly implicated in contributing to tissue remodeling associated with chronic inflammation, pathological hypertrophy, angiogenesis and oxidative stress-induced cell death. No inhibitors for these MAPK kinase kinases have been described resulting in an unmet need for small molecules to explore their cellular and physiological function. 2. Small molecules isolated from HTS or identified from a small directed kinase inhibitor library as inhibitors for a specific MKKK, that is, MEKK1, MEKK2, MEKK3, MEKK4 or ASK1, will be screened against the remaining 14 MAPK kinase kinases, and representative MARK kinases and MAPKs for initial selectivity profiling. 3. Functional cell based screens will be performed for small molecule inhibitors identified for each of the five MAPK kinase kinases (MEKK1, MEKK2, MEKK3, MEKK4 or ASK1). Gene knockouts and RNAi of these MAPK kinase kinases gives a unique phenotype and altered response to specific stimuli providing ready comparison of small molecules to genetic ablation of the kinase for mechanism based selectivity profiling.