Targeting the IKK-binding Domain of NEMO for Inhibitors Discovery

靶向 NEMO 的 IKK 结合域以发现抑制剂

• 批准号: 8822424
• 负责人: MARIA Margherita PELLEGRINI
• 金额: $ 8.1万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-20 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822424
• 关键词: Affinity; Apoptosis; Arthritis; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Calorimetry; Cell Proliferation; Cells; Cellular Assay; Characteristics; Circular Dichroism; Competence; Complement; Complex; Crystallization; Data; Development; Disease; Drug Design; Drug Targeting; Engineering; Enzyme-Linked Immunosorbent Assay; Fluorescence Polarization; Foundations; Funding; Future; Goals; Hand; Health; Heterogeneity; Hot Spot; Immune response; In Vitro; Inflammation; Inflammatory; Knowledge; Label; Lead; Length; Ligand Binding; Ligands; Molecular Weight; Muscle; Muscular Dystrophies; Musculoskeletal Diseases; N-terminal; NF-kappa B; NMR Spectroscopy; Nuclear; Pathogenesis; Pathway interactions; Peptides; Pharmaceutical Preparations; Phosphotransferases; Play; Positioning Attribute; Protein Engineering; Proteins; Psoriasis; Reagent; Regulation; Research; Research Project Grants; Resolution; Rheumatoid Arthritis; Roentgen Rays; Role; Scaffolding Protein; Series; Signal Pathway; Signal Transduction; Staging; Stimulus; Stress; Structure; Surface Plasmon Resonance; Techniques; Tertiary Protein Structure; Testing; Time; Variant; Viral; Work; X-Ray Crystallography; base; biological adaptation to stress; biophysical techniques; cytokine; design; design and construction; drug development; drug discovery; experience; improved; in vivo Model; inhibitor/antagonist; insight; interdisciplinary approach; novel; preference; protein phosphatase inhibitor-2; protein protein interaction; research study; screening; skin disorder; small molecule; success; three dimensional structure; tool

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.

描述（由申请人提供）：NF-kB 必需调节剂（NEMO）是一种支架蛋白，也是 IkB 激酶（IKK）复合物的必需成分。 IKK复合物被多种细胞刺激（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）信号通路的中心节点，调节细胞增殖和凋亡、免疫反应和炎症以及应激反应。 IKK 复合物由 NEMO 和两种激酶（IKK-α 和 IKK-β）形成，由于 NF-kB 激活在自身免疫性疾病（包括类风湿性关节炎、牛皮癣和肌肉相关疾病）发病机制中的作用，因此成为药物开发的主要靶点。 IKK 抑制的一种方法针对 IKK-α 和 IKK-β 激酶与 NEMO 之间的相互作用。在炎性关节炎、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 IKK 的 NEMO 结合域 (NBD) 相对应的肽作为 IKK 复合物抑制剂。 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 结合域）的高分辨率结构的知识。对于未配体状态的 NEMO，X 射线和 NMR 结构均不可用。在本提案中，我们的目标是设计和表征包含 NEMO IKK 结合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 射线晶体学解析未配体 NEMO 的三维结构（目标#2）。我们的多学科方法将蛋白质设计原理与生化和细胞测定相结合，以评估 IKK 结合新结构的能力，以及包括圆二色性和 NMR 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 N 端结构域的结构涵盖了 IKK 结合的区域，将允许识别可药物口袋以进行基于结构的设计，这是开发 IKK 复合物形成调节剂的重要一步。