Cell-Based Assays for Identifying Anti-RANK Signaling Agents

用于识别抗 RANK 信号转导剂的细胞测定法

• 批准号: 7426192
• 负责人: XU FENG
• 金额: $ 19.36万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7426192
• 关键词: Adaptor Signaling Protein; Adverse effects; Binding; Biological Assay; Biological Process; Biology; Bone Diseases; Cells; Complex; Cytoplasmic Tail; Disease; Ligands; Macrophage Colony-Stimulating Factor; Macrophage Colony-Stimulating Factor Receptor; Mediating; Metastatic Neoplasm to the Bone; Molecular Bank; Molecular Probes; Osteoclasts; Osteoporosis; Pathogenesis; Pharmaceutical Preparations; Play; Process; Proteins; Recruitment Activity; Rheumatoid Arthritis; Robotics; Role; Screening procedure; Signal Pathway; Signal Transduction; Signaling Protein; System; TNF Receptor-Associated Factors; TNF receptor-associated factor 6; TNFSF11 gene; TRAF6 gene; Therapeutic; Tumor Necrosis Factor Receptor; base; bone loss; high throughput screening; immune function; member; monocyte; novel; receptor; tumor

DESCRIPTION (provided by applicant): Elevated osteoclast (OC) formation and/or activity are implicated in the pathogenesis of various bone disorders. Current antiresorptive (anti-OC formation and/or function) drugs either lack satisfactory efficacy or cause serious side effects. OC formation requires two key factors: the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-?B ligand (RANKL). RANKL regulates OC formation and function by activating its receptor RANK (the receptor activator of NF-?B) on OC precursors and mature OCs. RANK, a member of the tumor necrosis factor receptor (TNFR) family, transduces intracellular signals by recruiting adaptor proteins including TNFR associated factors (TRAFs) through specific motifs in the cytoplasmic domain. The unraveling of the RANKL/RANK system has not only greatly advanced our understanding of OC biology but also provided an opportunity to develop more effective antiresorptive drugs. The current effort has primarily been focused on developing therapeutics blocking RANK/RANKL interactions, which are likely to have side effects due to the involvement of the system in multiple biological processes. Recently we identified three functional TRAF-binding motifs in the RANK cytoplasmic domain (Motif 1:PFQEP369-373; Motif 2: PVQEET559-564 & Motif 3: PVQEQG604-609). These motifs regulate OC formation and function by utilizing different TRAF proteins to form distinct signaling complexes. However, while it has been established that Motif 1 recruits TRAF6 to form a multiple protein signaling complex to activate numerous signaling pathways, the precise signaling mechanism by which Motif 2 and Motif 3 activate downstream signals have not been fully understood. Moreover, these motifs regulate OC formation and function to different extent. While Motif 2 and Motif 3 are very potent in mediating OC formation and function, Motif 1 plays a minimal role in these processes, instead being the primary actor in RANKL-mediated immune function. Thus, Motifs 2 and 3 can serve as potent and specific antiresorptive targets. In this application, we propose to develop and configure novel cell-based assay systems for high throughput screen (HTS) to identify compounds blocking signaling from Motif 2 and 3. To this end, we seek to achieve the following two specific aims: Aim 1: Develop highly efficient and robotic cell-based assays for identifying compounds blocking the signaling pathways activated by Motif 2 and Motif 3; Aim 2: Configure the cell-based assays for HTS; Aim 3: Submit the cell based assays to the Molecular Libraries Screening Center Network (MLSCN) to identify compounds inhibiting Motif 2- and Motif-3 mediated signaling. Significantly, the compounds can not only serve as useful molecular probes for elucidating the signaling mechanism of Motif 2-/Motif 3 but may also be further developed as therapeutics for osteoporosis, bone loss in rheumatoid arthritis (RA) and tumor bone metastasis.

描述（由申请人提供）：破骨细胞（OC）形成和/或活性升高与各种骨疾病的发病机制有关。目前的抗再吸收（抗OC形成和/或功能）药物要么缺乏令人满意的疗效，要么引起严重的副作用。 OC 的形成需要两个关键因素：单核细胞/巨噬细胞集落刺激因子 (M-CSF) 和 NF-κB 配体受体激活剂 (RANKL)。 RANKL 通过激活 OC 前体和成熟 OC 上的受体 RANK（NF-κB 受体激活剂）来调节 OC 的形成和功能。 RANK 是肿瘤坏死因子受体 (TNFR) 家族的成员，通过细胞质结构域中的特定基序招募包括 TNFR 相关因子 (TRAF) 在内的接头蛋白来转导细胞内信号。 RANKL/RANK系统的阐明不仅极大地增进了我们对OC生物学的理解，而且为开发更有效的抗骨吸收药物提供了机会。 目前的工作主要集中在开发阻断 RANK/RANKL 相互作用的疗法，由于该系统参与多个生物过程，这可能会产生副作用。 最近，我们在 RANK 胞质结构域中鉴定了三个功能性 TRAF 结合基序（基序 1：PFQEP369-373；基序 2：PVQEET559-564 和基序 3：PVQEQG604-609）。这些基序通过利用不同的 TRAF 蛋白形成不同的信号复合物来调节 OC 的形成和功能。然而，虽然已经确定Motif 1招募TRAF6形成多蛋白信号传导复合物以激活多种信号传导途径，但Motif 2和Motif 3激活下游信号的精确信号传导机制尚未完全了解。此外，这些基序在不同程度上调节OC的形成和功能。虽然 Motif 2 和 Motif 3 在介导 OC 形成和功能方面非常有效，但 Motif 1 在这些过程中发挥的作用很小，而是 RANKL 介导的免疫功能的主要参与者。因此，基序 2 和 3 可以作为有效且特异性的抗再吸收靶点。在此应用中，我们建议开发和配置新型基于细胞的检测系统，用于高通量筛选 (HTS)，以识别阻断基序 2 和 3 信号传导的化合物。 为此，我们寻求实现以下两个具体目标： 目标 1 ：开发高效且基于机器人的细胞测定法，用于识别阻断 Motif 2 和 Motif 3 激活的信号通路的化合物；目标 2：配置 HTS 的基于细胞的测定；目标 3：将基于细胞的检测提交给分子库筛选中心网络 (MLSCN)，以鉴定抑制 Motif 2- 和 Motif-3 介导的信号传导的化合物。值得注意的是，这些化合物不仅可以作为有用的分子探针来阐明Motif 2-/Motif 3的信号传导机制，而且还可以进一步开发为骨质疏松症、类风湿性关节炎（RA）骨质流失和肿瘤骨转移的治疗药物。