Targeting the IKK-binding Domain of NEMO for Inhibitors Discovery

 描述(由申请人提供)：核因子-kB必需调节剂(NEMO)是一种支架蛋白，也是IKB激酶(IKK)复合体的重要组成部分。受多种细胞刺激，包括细胞因子、细菌和病毒产物以及应激，IKK复合体的激活是核因子kB信号通路的中心节点，它调节细胞的增殖和凋亡、免疫反应和炎症反应以及应激反应。由NEMO和两个激酶(IKK-α和IKK-β)形成的IKK复合体是药物开发的主要靶点，因为核因子-kB激活在自身免疫性疾病的发病机制中发挥作用，包括类风湿性关节炎、牛皮癣和肌肉相关疾病。一种抑制IKK的方法是针对IKK-α和IKK-β激酶与NEMO之间的相互作用。在炎症性关节炎、肌营养不良等的体外和体内模型中，该策略被证明是有效的，利用与IKKS(NBD)的Nemo结合结构域相对应的多肽作为IKK复杂抑制剂。了解靶蛋白的高分辨结构(NEMO的IKK结合域)将对NEMO-IKK相互作用的多肽和小分子量抑制剂的开发大有裨益。对于处于未连接状态的NEMO，无论是X射线结构还是核磁共振结构都不可用。在这项提案中，我们的目标是设计和表征包含NEMO的IKK结合域的新的蛋白质结构，这将有助于结构确定(目标1)，并通过核磁共振或X射线结晶学解决未连接的NEMO的三维结构(目标2)。我们的多学科方法将结合蛋白质设计原则与生化和细胞分析来评估新构建物与ikk结合的能力，并进行生物物理研究，包括圆二色谱和核磁共振光谱，以确定Nemo变体的折叠和稳定性。NEMO的N-末端结构域的结构包括IKK结合区域，这将使识别可药物口袋用于基于结构的设计，这是开发IKK复合体形成调节因子的重要一步。

 DESCRIPTION (provided by applicant): The NF-kB essential modulator (NEMO) is a scaffolding protein and an essential component of the of the IkB kinase (IKK) complex. Activation of the IKK complex by a number of cellular stimuli, including cytokines, bacterial and viral products and stress, is the central node of the nuclear factor kB (NF-kB) signaling pathway, which regulates cell proliferation and apoptosis, immune response and inflammation, and stress response. The IKK complex, formed by NEMO and two kinases (IKK-alpha and IKK-beta), is a primary target for drug development due to the role of NF-kB activation in the pathogenesis of autoimmune disorders, including rheumatoid arthritis, psoriasis and muscle related disorders. One approach to IKK inhibition targets the interaction between the IKK-alpha and IKK-beta kinases and NEMO. The strategy was shown to be effective utilizing a peptide, corresponding to the NEMO-binding domain of the IKKs (NBD), as an IKK complex inhibitor in in vitro and in vivo models of inflammatory arthritis, muscular dystrophy and others. The development of peptides and small molecular weight inhibitors of the NEMO-IKK interaction would greatly benefit by the knowledge of the high resolution structure of the target protein (the IKK-binding domain of NEMO). Neither X-ray nor NMR structures are available for NEMO in its unliganded state. In this proposal we aim to design and characterize novel protein constructs encompassing the IKK-binding domain of NEMO which will facilitate structural determination (aim #1) and to solve the three-dimensional structure of unliganded NEMO by NMR or X-ray crystallography (aim #2). Our multidisciplinary approach will combine protein design principles with biochemical and cellular assays to assess competence of the new constructs for IKK-binding, and biophysical studies including circular dichroism and NMR spectroscopy, to determine folding and stability of the NEMO variants. The structure of the N- terminal domain of NEMO, which encompasses the region where the IKKs bind, would allow the identification of druggable pockets for structure based design, an important step in the development of regulators of the formation of the IKK complex.