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 kinase 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过程中，它经常被过度激活。因此，调节JAK3过表达的合适的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，开发一种敏感、精确和坚固的筛选工具来选择调节JAK3过度激活的药物是非常重要的。目前可用的筛选ELISA工具仅使用JAK3的活性位点。一般采用定量夹心酶免疫分析技术。简单地说，将JAK3特异性抗体预先包被在微孔板上。标准品和样品被转移到孔中，任何存在的JAK3都被固定的抗体结合。在去除任何未结合的物质后，将针对JAK3的生物素偶联抗体添加到孔中。在洗涤以去除任何未结合的亲和素酶试剂后，将底物溶液添加到孔中，并且在初始步骤中与JAK3结合的量成比例显色。另一种技术利用了与经典的酶活性“锁与钥匙模型”不同的“诱导拟合”概念，因为人们认为酶活性位点的3D结构通过与其底物/抑制剂的相互作用而不断重塑。总的来说，目前所有可用的筛选工具都有几个共同的缺陷，包括灵敏度低、需要外部衬底和需要放射性检测。作为我们在免疫介导疾病研究中持续努力的一部分，我们一直致力于改进筛选技术以寻找更好的JAK3抑制剂；我们在此介绍一种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)显著降低背景噪声，(2)更高的灵敏度，(3)不需要外部底物，(4)不需要使用放射性方法，以及(5)能够测量自（酪氨酸）磷酸化。该项目的最终目标是制造和销售一种显著改进的ELISA试剂盒，用于筛选JAK3抑制剂。这项I期SBIR研究的目标是展示开发这种新型试剂盒作为优质商业产品的可行性。本SBIR I期研究的具体目的是：1)进一步证明在筛选试剂盒中使用全长JAK3抗体的概念。2)检测试剂盒中多种化合物（阳性对照和潜在化合物）。我们发表了阳性对照，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，并从购买的文库中购买一系列化合物来测试新的ELISA试剂盒。3)验证我们的筛选方法是准确的、精确的、可重复的和坚固的。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。将现有技术与我们正在开发的新技术进行比较和对比。