Fluorescent Cellular Assay for Kinase Inhibitors

激酶抑制剂的荧光细胞测定

• 批准号: 10114746
• 负责人: Evgueni E Nesterov
• 金额: $ 39.82万
• 依托单位: NORTHERN ILLINOIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10114746
• 关键词: Antineoplastic Agents; Autoimmune Process; Binding; Biological Assay; Cancer-Related Condition; Cell Density; Cells; Cellular Assay; Complex; Cytolysis; Detection; Development; Disease; Drug Costs; Environment; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; FDA approved; Failure; Fluorescence; Foundations; Future; Generations; Goals; Illinois; In Vitro; Inflammatory; Infrastructure; Kinetics; Methodology; Modeling; Molecular; Patients; Permeability; Phosphotransferases; Process; Protein Conformation; Protein Tyrosine Kinase; Proteins; Protocols documentation; Recombinants; Research; Resort; Series; Signal Transduction; Specificity; Tyrosine Kinase Inhibitor; Universities; Western Blotting; Work; assay development; base; cancer therapy; career; cell behavior; cost efficient; design; drug candidate; drug development; efficacy evaluation; experience; high throughput screening; improved; improved outcome; inhibitor/antagonist; kinase inhibitor; nanomolar; non-Native; protein kinase inhibitor; response; screening; sensor; small molecule; small molecule inhibitor; success; therapy outcome; undergraduate student; uptake

PROJECT SUMMARY Over the past 30 years, the field of protein kinase inhibitors development has rapidly expanded. As a result, several dozens of small-molecule kinase inhibitors have been approved by FDA, that led to improved outcomes in cancer treatment for thousands of patients. Moreover, kinase inhibition was recently extended towards non- oncology applications such as autoimmune and inflammatory diseases. The ultimate success of a drug development process relies on the availability of prompt, reliable, and cost- efficient analytical methodologies for potential inhibitor profiling. In the case of kinase inhibition, the screening is often performed against purified recombinant kinase domains in vitro. While this approach has led to successful identification of kinase inhibitors, it suffers from some major limitations that hamper drug development process. A critical limitation is the poor correlation between in vitro and cellular behavior of inhibitor candidates. This limitation is a reason for late-in-the-process failures of some drug candidates, that overall contribute to high cost of the drug development process. This limitation can be overcome via development and use of highly efficient cellular assays for inhibitor profiling. However, currently there are no efficient cellular assays for kinase inhibitor profiling in native environments. In the proposed work, we will design and develop a new cellular assay for profiling inhibitors of a model kinase, EGFR tyrosine kinase. The assay will enable screening inhibitor activity in native conditions, inside the living cells. To develop the cellular assay, we will accomplish three specific aims: (1) rational design, synthesis, and in vitro characterization of the new cell-permeable fluorescent sensors that selectively respond on low-nanomolar concentrations of EGFR; (2) development and characterization of intracellular delivery, localization, and EGFR binding of these new sensors; (3) development of a competitive cellular assay for inhibitors profiling and quantitative assessment of the inhibitor binding. Upon successful completion of this work, the new assay will not only provide a foundation for the high-throughput profiling of EGFR inhibitors, but also will establish general development principles applicable for other protein targets. In addition, this project will enhance research environment and infrastructure at Northern Illinois University. Thus, generations of undergraduate students will get valuable experience in design and synthesis of the new sensors, characterizing biomolecule/sensor interaction, working on developing in vitro and cellular assays. Eventually, participation in this project will prepare and empower the undergraduate students for future careers in biomedical field.

项目摘要 在过去的30年里，蛋白激酶抑制剂的开发领域迅速扩大。因此，在本发明中， FDA已经批准了几十种小分子激酶抑制剂，从而改善了结果 治疗癌症的方法。此外，激酶抑制最近扩展到非- 肿瘤学应用，如自身免疫性和炎症性疾病。 药物开发过程的最终成功依赖于及时、可靠和成本低廉的可用性， 潜在抑制剂分析的有效分析方法。在激酶抑制的情况下，筛选 通常在体外针对纯化的重组激酶结构域进行。虽然这种方法导致了 尽管成功鉴定了激酶抑制剂，但它受到一些阻碍药物开发的主要限制 过程一个关键的限制是抑制剂候选物的体外和细胞行为之间的相关性差。 这种局限性是一些候选药物在后期失败的原因，总体上导致高风险。 药物开发过程的成本。这种局限性可以通过开发和使用高度 用于抑制剂谱分析的高效细胞测定。然而，目前还没有针对激酶的有效细胞测定法。 天然环境中的抑制剂分析。 在拟议的工作中，我们将设计和开发一种新的细胞分析模型激酶的抑制剂， EGFR酪氨酸激酶。该测定将能够在天然条件下，在活体内筛选抑制剂活性。 细胞为了发展细胞分析，我们将完成三个具体目标：（1）合理的设计，合成， 选择性响应低纳摩尔浓度的新型细胞可渗透荧光传感器的体外表征 EGFR浓度;（2）细胞内递送、定位和EGFR表达的发展和表征 这些新传感器的结合;（3）开发用于抑制剂谱分析的竞争性细胞测定， 抑制剂结合的定量评估。 一旦成功完成这项工作，新的检测方法不仅将为高通量检测奠定基础， EGFR抑制剂的分析，但也将建立适用于其他蛋白质的一般开发原则 目标的 此外，该项目还将改善北方伊利诺伊大学的研究环境和基础设施。 因此，一代又一代的本科生将在设计和合成新的 传感器，表征生物分子/传感器相互作用，致力于开发体外和细胞分析。 最终，参与本项目将为本科生未来的职业生涯做好准备并赋予他们权力 在生物医学领域。