GWAS to drugs: small molecule screen for inhibitors of CD40 signaling pathway

GWAS 到药物：CD40 信号通路抑制剂的小分子筛选

• 批准号: 8577387
• 负责人: Jose R. Perez
• 金额: $ 34.18万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8577387
• 关键词: Alleles; Amino Acids; Autoimmune Diseases; B-Lymphocytes; Binding; Biological; Biological Assay; Biology; Blood Platelets; CD40 Ligand; Cell Culture Techniques; Cell Line; Cell Surface Receptors; Cell physiology; Cell surface; Cells; Chronic; Collaborations; Complex; Data; Development; Disease; Ensure; Event; Experimental Designs; Genes; Genetic; Genomics; Goals; Human; Immune response; Inflammation; Inflammatory; Institutes; Lead; Ligand Binding; Luciferases; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Molecular; Monoclonal Antibodies; Mus; NF-kappa B; Pathogenesis; Pathway interactions; Patients; Performance; Pharmaceutical Preparations; Phenotype; Phosphorylation; Pilot Projects; Play; Poison; Preclinical Drug Evaluation; Protein Binding; Proteins; RNA Interference; Reagent; Reporter; Rheumatoid Arthritis; Risk; Role; Signal Pathway; Signal Transduction; Stable Isotope Labeling; TNF gene; TNFRSF5 gene; TNFSF5 gene; Techniques; Technology; Testing; Therapeutic; Toxic effect; Western Blotting; base; clinical efficacy; disorder risk; genetic association; genome wide association study; high throughput screening; inhibitor/antagonist; man; new therapeutic target; novel; novel therapeutics; p65; public health relevance; small molecule; therapeutic effectiveness; trait

DESCRIPTION (provided by applicant): Genome-wide association studies (GWAS) of disease risk serve as a promising strategy to identify novel therapeutic targets. However, going from SNP association to a high-throughput screen (HTS) to identify novel drugs requires many steps and has not yet been successfully accomplished. Our team has exciting preliminary data demonstrating that HTS assays can be developed from GWAS of complex traits. We will perform one of the first HTS based on GWAS findings - a confirmed association of a common allele at the CD40 locus and risk of a common autoimmune disease, rheumatoid arthritis (RA). We will identify novel intracellular inhibitors of CD40-mediated NF-kB signaling in B cells, which can ultimately be used to treat RA. Although others have searched for inhibitors of NF-kB signaling, and others have searched for molecules that disrupt CD40-CD40L binding, no screen has been conducted in B cells to identify for CD40-specific inhibitors of NF-kB signaling - ours will be the first. In our revised application, we present new preliminary data that demonstrate our ability to perform counter screens that will identify small molecules that are specific to CD40-mediated signaling inside of human B cells. We have also revised Aim 3 to overcome the "target ID problem" inherent in pathway-based screens. Specific Aim 1: Collaborate with BIPDeC to implement a validated HTS (luciferase assay in our BL2- NFkB-luc cells stimulated with tCD40L) to identify inhibitors of the CD40 signaling pathway. We will use a cell-based, NF-kB luciferase assay to screen ~450,000 MLPCN probe compounds. Specific Aim 2: Employ established reagents to conduct secondary counter screens to validate that our hits are specific to the CD40 signaling pathway. To ensure that hit compounds are specific to intracellular CD40-mediated signaling, we will perform counter screens at the Broad Institute to (a) remove compounds that inhibit TNF? and LPS signaling, (b) remove compounds that interfere with the luciferase assay, and (c) remove compounds that are toxic to BL2 cells. We will test hit compounds in primary B-cells. Specific Aim 3: Apply SILAC (stable isotope labeling by amino acids in cell culture)-based target identification technology to identify the protein target from our drug screen. The approach is based on protein mass-spectrometry. We will apply SILAC to identify proteins that bind to small molecules identified in Aim 2. We will validate SILAC hits using a variety of molecular techniques, including Western blots, enzymatic assays and RNAi in cell lines and primary cells. IMPACT: We have preliminary data to demonstrate our ability to conduct all aspects of the study - including new data on counter screens and SILAC. Upon completion of our study, we will have identified small molecule inhibitors that are specific to CD40-mediated NF-kB signaling in human B cells.

描述（由申请人提供）：疾病风险的全基因组关联研究（GWAS）是确定新型治疗靶标的有前途的策略。但是，从SNP关联到高通量屏幕（HTS）以识别新型药物需要许多步骤，并且尚未成功完成。我们的团队有令人兴奋的初步数据，表明可以从复杂特征的GWA开发HTS分析。我们将根据GWAS发现进行最早的HTS之一 - CD40基因座的普通等位基因的确认关联，并患有常见自身免疫性疾病的风险类风湿关节炎（RA）。我们将确定B细胞中CD40介导的NF-KB信号传导的新型细胞内抑制剂，最终可用于治疗RA。尽管其他人已经搜索了NF-KB信号传导的抑制剂，而其他人则搜索了破坏CD40-CD40L结合的分子，但在B细胞中尚未进行筛选以识别NF-KB信号的CD40特异性抑制剂 - 我们的S-KB信号 - 我们的S将是第一个。在我们修订的应用程序中，我们提供了新的初步数据，证明了我们的 能够执行可以识别针对人类B细胞内部CD40介导的信号传导的小分子的计数器筛选的能力。我们还修改了AIM 3，以克服基于途径的屏幕固有的“目标ID问题”。具体目标1：与BIPDEC合作实施经过验证的HTS（在我们的BL2-NFKB-LUC细胞中用TCD40L刺激的荧光素酶测定），以鉴定CD40信号通路的抑制剂。我们将使用基于细胞的NF-KB荧光素酶测定法来筛选约45万MLPCN探针化合物。特定目标2：采用已建立的试剂进行二级计数器筛查，以验证我们的命中是否特定于CD40信号通路。为了确保命中化合物特异性针对细胞内CD40介导的信号传导，我们将在Broad Institute执行柜台屏幕以（a）去除抑制TNF的化合物？和LPS信号传导，（b）去除干扰荧光素酶测定的化合物，（c）去除对BL2细胞有毒的化合物。我们将测试主要B细胞中的命中化合物。具体目标3：应用SILAC（氨基酸在细胞培养中稳定的同位素标记）基于细胞培养的靶标识别技术，以从我们的药物筛选中识别蛋白质靶标。该方法基于蛋白质质谱法。我们将使用SILAC识别与AIM 2中鉴定的小分子结合的蛋白质。我们将使用各种分子技术验证SILAC命中，包括蛋白质印迹，酶促测定和细胞系和原代细胞中的RNAi。影响：我们有初步数据来证明我们进行研究的各个方面的能力 - 包括计数器屏幕和SILAC上的新数据。完成研究后，我们将确定针对人B细胞中CD40介导的NF-KB信号传导的小分子抑制剂。