Development of a Novel ELISA Kit for Screening Potential JAK3 Inhibitors

开发用于筛选潜在 JAK3 抑制剂的新型 ELISA 试剂盒

• 批准号: 8641503
• 负责人: Jiajiu Shaw
• 金额: $ 33.87万
• 依托单位: 21ST CENTURY THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-05 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8641503
• 关键词: Active Sites; Antibodies; Autoimmune Diseases; Avidin; B-Lymphocytes; Binding; Biological Assay; Biological Sciences; Biotin; Color; Contractor; Cytokine Receptors; Detection; Development; Devices; Disease; Electrophoretic Mobility Shift Assay; Enzyme Immunoassay; Enzyme-Linked Immunosorbent Assay; Enzymes; Equipment; Family; Fc Receptor; Fluorescence; Future; Goals; Health; Human Resources; Immune; Immune response; Inflammatory; JAK2 gene; Janus kinase 1; Janus kinase 3; Label; Length; Libraries; Marketing; Measurement; Measures; Mediating; Methods; Modeling; Natural Killer Cells; Noise; Paper; Peptides; Phase; Phenotype; Phosphorylation; Phosphotransferases; Process; Production; Protein Tyrosine Kinase; Proteins; Publishing; Radio; Radioactive; Reaction; Reagent; Receptor Signaling; Research; Running; Sales; Sampling; Series; Severe Combined Immunodeficiency; Small Business Innovation Research Grant; Solutions; Streptavidin; Study Section; T-Lymphocyte; Techniques; Technology; Testing; Texas; Time; Tyrosine Phosphorylation; Validation; Work; antibody conjugate; base; cost; drug candidate; drug discovery; enzyme activity; flexibility; improved; inhibitor/antagonist; inorganic phosphate; instrument; large scale production; microchip; new technology; novel; overexpression; phase 1 study; phase 2 study; programs; public health relevance; scale up; screening; three dimensional structure; tool; user-friendly

DESCRIPTION (provided by applicant): Janus kinase 3 (JAK3) is an intracellular kinase involved in cytokine and antibody receptor signaling; it is frequently over-activated during the course of inflammatory autoimmune diseases. Therefore, suitable JAK3 inhibitors that modulate the overexpression of JAK3 may be potential agents for treating autoimmune diseases. Logically, it is of significant importance to develop a sensitive, precise, and rugged screening tool to select agents that modulate the over-activation of JAK3. Currently available screening ELISA tools use only the active site of JAK3. Generally, the assay employs the quantitative sandwich enzyme immunoassay technique. Briefly, antibody specific for JAK3 is pre-coated onto a microplate. Standards and samples are transferred into the wells and any JAK3 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for JAK3 is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of JAK3 bound in the initial step. Another technology utilizes an "induced fit" concept different from the classical 'lock and key model' of enzyme activity, in thatit was believed that the 3D structures of the enzyme active sites are continuously reshaped by interactions with their substrates/inhibitors. Overall, all currently available screening tools hav several common deficiencies including low sensitivity, requiring an external substrate, and requiring a radioactive detection. As part of our continued efforts in the research in immune- mediated disorders, we have been working on improving the screening technology for finding better JAK3 inhibitors; we herein present the novel screening kit under development. Briefly, the proposed JAK3 screening tool comprises the use of full-length JAK3, different from currently available in that only the active site of JAK3 is used. The advantages of our novel screening kit include (1) significantly lower background noise, (2) higher sensitivity, (3) no need for the external substrate, (4) no need to use the radioactive method, and (5) capable of measuring auto-(tyrosine)phosphorylation. The ultimate goal of this project is to manufacture and sell a significantly improved ELISA kit to new drug discovery companies for screening JAK3 inhibitors. The goal of this Phase I SBIR study is to show the feasibility of developing this novel kit as a superior commercial product. The specific aims for this SBIR Phase I study are: 1) To further prove the concept of using the full-length JAK3 antibody in the screening kit. 2) To test the kit o a number of compounds (positive control and potential compounds). We have published positive control as shown in Preliminary Studies section. In this SBIR Phase I study, we will synthesize at least 12 potential JAK3 inhibitors and purchase a series of compounds from purchased library to test the novel ELISA kit. 3) To validate that our screening method is accurate, precise, reproducible, and rugged. We will choose two suitable JAK3 inhibitors for the validation of our screening kit. 4) To compare and contrast between already existing technology and our new technology under development.

描述（由申请人提供）：Janus 激酶 3 (JAK3) 是一种参与细胞因子和抗体受体信号传导的细胞内激酶；在炎症性自身免疫性疾病的过程中，它经常被过度激活。因此，调节JAK3过度表达的合适JAK3抑制剂可能是治疗自身免疫性疾病的潜在药物。从逻辑上讲，开发一种灵敏、精确且坚固的筛选工具来选择调节 JAK3 过度激活的药物具有重要意义。目前可用的筛选 ELISA 工具仅使用 JAK3 的活性位点。通常，该测定采用定量夹心酶免疫测定技术。简而言之，JAK3 特异性抗体被预包被到微孔板上。标准品和样品被转移到孔中，任何存在的 JAK3 都会被固定化抗体结合。去除所有未结合的物质后，将 JAK3 特异性的生物素偶联抗体添加到孔中。清洗以除去任何未结合的亲和素-酶试剂后，将底物溶液添加到孔中，颜色的显色与初始步骤中结合的 JAK3 的量成比例。另一种技术利用与酶活性的经典“锁和钥匙模型”不同的“诱导契合”概念，因为人们相信酶活性位点的3D结构通过与其底物/抑制剂的相互作用而不断重塑。总体而言，目前所有可用的筛查工具都有几个共同的缺陷，包括灵敏度低、需要外部基质以及需要放射性检测。作为我们在免疫介导疾病研究方面持续努力的一部分，我们一直致力于改进筛选技术以寻找更好的 JAK3 抑制剂；我们在此介绍正在开发的新型筛选试剂盒。简而言之，所提出的JAK3筛选工具包括使用全长JAK3，与目前可用的仅使用JAK3的活性位点不同。我们的新型筛选试剂盒的优点包括（1）背景噪音显着降低，（2）灵敏度更高，（3）无需外部基质，（4）无需使用放射性方法，（5）能够测量自（酪氨酸）磷酸化。该项目的最终目标是制造并向新药发现公司销售显着改进的 ELISA 试剂盒，用于筛选 JAK3 抑制剂。第一阶段 SBIR 研究的目标是展示将这种新型试剂盒开发为优质商业产品的可行性。本次SBIR I期研究的具体目标是：1）进一步证明在筛选试剂盒中使用全长JAK3抗体的概念。 2) 测试试剂盒中的多种化合物（阳性对照和潜在化合物）。我们已经发布了阳性对照，如初步研究部分所示。在这项SBIR I期研究中，我们将合成至少12种潜在的JAK3抑制剂，并从购买的库中购买一系列化合物来测试新型ELISA试剂盒。 3) 验证我们的筛选方法准确、精确、可重复且坚固。我们将选择两种合适的 JAK3 抑制剂来验证我们的筛选试剂盒。 4）对现有技术和我们正在开发的新技术进行比较和对比。