Targeting the IKK-binding Domain of NEMO for Inhibitors Discovery

 描述（由申请方提供）：NF-κ B必需调节剂（NEMO）是一种支架蛋白，是IkB激酶（IKK）复合物的必需组分。IKK复合物被许多细胞刺激物（包括细胞因子、细菌和病毒产物以及应激）激活是核因子kB（NF-kB）信号传导途径的中心节点，其调节细胞增殖和凋亡、免疫应答和炎症以及应激应答。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标，这是由于NF-κ B活化在自身免疫性疾病（包括类风湿性关节炎、银屑病和肌肉相关疾病）的发病机制中的作用。IKK抑制的一种方法靶向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，对应于NEMO结合结构域的IKK（NBD），作为IKK复合物抑制剂在体外和体内模型的炎性关节炎，肌营养不良症和其他。 NEMO-IKK相互作用的肽和小分子量抑制剂的开发将极大地受益于靶蛋白（NEMO的IKK结合结构域）的高分辨率结构的知识。NEMO在其未配体状态下的X射线和NMR结构都不可用。在该提案中，我们的目标是设计和表征包含NEMO的IKK结合结构域的新型蛋白质构建体，这将有助于结构测定（目标#1），并通过NMR或X射线晶体学解决未配体的NEMO的三维结构（目标#2）。我们的多学科方法将结合联合收割机的蛋白质设计原则与生化和细胞测定，以评估新的构建体的IKK结合能力，和生物物理研究，包括圆二色性和NMR光谱，以确定NEMO变体的折叠和稳定性。NEMO的N-末端结构域（其包含IKK结合的区域）的结构将允许鉴定用于基于结构的设计的可药物化口袋，这是开发IKK复合物形成的调节剂的重要步骤。

 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.