HTS for FADD kinase inhibitors using molecular imaging

使用分子成像对 FADD 激酶抑制剂进行 HTS

• 批准号: 7502826
• 负责人: Alnawaz Rehemtulla
• 金额: $ 6.84万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-03 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7502826
• 关键词: Adaptor Signaling Protein; Adult; Amino Acids; Antibodies; Apoptotic; Biochemical; Biological Assay; Biological Factors; Biological Markers; Bioluminescence; Brain; C-terminal; CI-1033; Cancer Patient; Cancer cell line; Cell Cycle Progression; Cells; Cessation of life; Chemicals; Clinical; Collection; Complex; Condition; Cyclin D1; Data; Death Domain; Development; Diagnosis; Disease; Distress; Dose; Embryonic Development; Ensure; Environment; Enzymes; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Erlotinib; Event; Facility Construction Funding Category; Family; Fluorescent Dyes; G2/M Transition; Gene Mutation; Genetically Engineered Mouse; Genomics; Germ cell tumor; Goals; Head and Neck Cancer; Head and neck structure; Human; Image; Imaging Device; Immunohistochemistry; In Vitro; Inhibitory Concentration 50; Knockout Mice; Lead; Letters; Libraries; Localized; Luciferases; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediator of activation protein; Methodology; Methods; Michigan; Microarray Analysis; Molecular Profiling; Molecular and Cellular Biology; Monitor; Noise; Nuclear; Numbers; Operative Surgical Procedures; Outcome; Pan Genus; Peptides; Permeability; Persons; Phenotype; Phosphorylation; Phosphotransferases; Play; Prevalence; Probability; Protein Kinase; Protein Overexpression; Proteins; Publishing; Quantitative Evaluations; Radiation therapy; Rate; Regulation; Relative (related person); Reporter; Reporting; Resort; Role; Science; Screening procedure; Sensitivity and Specificity; Series; Serine; Signal Transduction; Signal Transduction Pathway; Solubility; Specificity; Stream; Survival Rate; T-Cell Proliferation; Technology; Time; Tissue Microarray; Toxic effect; Two-Dimensional Gel Electrophoresis; United States Food and Drug Administration; Universities; Western Blotting; base; cancer cell; cancer therapy; chemotherapeutic agent; clinically significant; cyclin B1; cytotoxic; data mining; desire; enzyme activity; high throughput screening; inhibitor/antagonist; inorganic phosphate; kinase inhibitor; male; molecular imaging; mortality; mutant; neoplastic cell; novel; novel therapeutics; prognostic; research study; small molecule libraries; success; therapy resistant; tumor; two-dimensional

DESCRIPTION (provided by applicant): Using differential expression profiling, quantitative two-dimensional (2-D) gel electrophoresis and data mining we recently identified a new prognostic biomarker, Fas-associated death domain (FADD), which is overexpressed in a number of human malignancies such as lung, head and neck, brain and adult male germ cell tumors. Studies in lung cancer revealed that overexpression of FADD significantly associated with poor clinical outcome. Immunohistochemistry-based tissue microarray analysis confirmed the association between FADD over-expression and the poor outcome, and also revealed the presence of nuclear localized phosphorylated FADD (p-FADD). Tumors with increased p-FADD expression also showed elevated NF-?B activation. Taken together, published results from our lab and others suggest a causal relationship between the phosphorylation of FADD and NF-?B activation, a hallmark of an aggressive therapy resistant cancer phenotype. Thereby, we hypothesize that inhibiting FADD phosphorylation in tumor cells may sensitize cancer cells to chemotherapeutic agents. To aid in experimentation of this hypothesis we have resorted to molecular imaging tools and developed a pan FADD kinase reporter (FKR) which non-invasively senses FADD-kinase activity in real time. In Specific Aim 1, we will characterize the sensitivity and specificity of FKR. In Specific Aim 2A we will perform a high throughput screen to identify molecules from a diverse set of compound libraries that target FADD phosphorylation. Utilizing secondary screens with cells expressing either mutant FKR or luciferase, the toxic and less sensitive lead molecules will be eliminated. In Specific Aim 2B we will evaluate the relative efficacy of the candidate molecules by quantifying IC50 of the top leads. In Specific Aim 2C the specificity of candidate molecules in inhibiting FADD kinases will be investigated using western blotting and protein kinase arrays. The utility of these compounds and their derivatives in the treatment of cancers will be investigated in subsequent years.

描述（由申请人提供）：通过差异表达分析、定量二维 (2-D) 凝胶电泳和数据挖掘，我们最近鉴定了一种新的预后生物标志物 Fas 相关死亡结构域 (FADD)，它在多种人类恶性肿瘤中过度表达，例如肺、头颈、脑和成年男性生殖细胞肿瘤。肺癌研究表明 FADD 过度表达与不良临床结果显着相关。基于免疫组织化学的组织微阵列分析证实了 FADD 过度表达与不良结果之间的关联，并且还揭示了核定位磷酸化 FADD (p-FADD) 的存在。 p-FADD 表达增加的肿瘤也表现出 NF-κB 激活升高。总而言之，我们实验室和其他实验室发表的结果表明 FADD 磷酸化和 NF-κB 激活之间存在因果关系，NF-κB 激活是侵袭性治疗耐药癌症表型的标志。因此，我们假设抑制肿瘤细胞中的 FADD 磷酸化可能会使癌细胞对化疗药物敏感。为了帮助验证这一假设，我们采用了分子成像工具，并开发了一种泛 FADD 激酶报告基因 (FKR)，它可以非侵入性地实时感知 FADD 激酶活性。在具体目标 1 中，我们将描述 FKR 的敏感性和特异性。在特定目标 2A 中，我们将进行高通量筛选，从一组不同的化合物库中识别针对 FADD 磷酸化的分子。利用表达突变 FKR 或荧光素酶的细胞进行二次筛选，有毒且不太敏感的先导分子将被消除。在特定目标 2B 中，我们将通过量化顶级先导化合物的 IC50 来评估候选分子的相对功效。在 Specific Aim 2C 中，将使用蛋白质印迹和蛋白激酶阵列研究候选分子抑制 FADD 激酶的特异性。这些化合物及其衍生物在癌症治疗中的效用将在随后的几年中进行研究。