Targeting the IKK-binding Domain of NEMO for Inhibitors Discovery

描述（由申请人提供）：NF-kB必需调节剂（NEMO）是一种支架蛋白，是IkB激酶（IKK）复合物的重要组成部分。IKK复合体被多种细胞刺激激活，包括细胞因子、细菌和病毒产物以及应激，是核因子kB （NF-kB）信号通路的中心节点，它调节细胞增殖和凋亡、免疫反应和炎症以及应激反应。由NEMO和两种激酶（IKK- α和IKK- β）形成的IKK复合物是药物开发的主要靶点，因为NF-kB激活在自身免疫性疾病（包括类风湿关节炎、牛皮癣和肌肉相关疾病）的发病机制中起作用。IKK抑制的一种方法是针对IKK- α和IKK- β激酶与NEMO之间的相互作用。该策略被证明是有效的，利用肽，对应IKKs的nemo结合域（NBD），作为IKK复合物抑制剂在体外和体内模型炎症性关节炎，肌肉萎缩症和其他。了解目标蛋白（NEMO的ikk结合结构域）的高分辨率结构，将极大地促进NEMO- ikk相互作用的多肽和小分子量抑制剂的开发。在其无配体状态下，NEMO既没有x射线结构，也没有核磁共振结构。在本提案中，我们的目标是设计和表征包含NEMO的ikk结合域的新型蛋白质结构，这将有助于结构确定（目标1），并通过核磁共振或x射线晶体学解决非配位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理、生化和细胞分析来评估ikk结合新结构的能力，以及包括圆二色性和核磁共振光谱在内的生物物理研究，以确定NEMO变体的折叠和稳定性。NEMO的N端结构域（包含IKK结合的区域）将允许识别基于结构设计的可药物口袋，这是开发IKK复合物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.