Fluorescent Cellular Assay for Kinase Inhibitors

项目总结 在过去的30年里，蛋白激酶抑制剂的开发领域迅速扩大。结果, 数十种小分子激酶抑制剂已被FDA批准，从而改善了结果 为数以千计的患者治疗癌症。此外，激酶抑制最近被扩展到非 肿瘤学应用，如自身免疫性疾病和炎症性疾病。 药物开发过程的最终成功依赖于及时、可靠和成本- 潜在抑制剂分析的有效分析方法。在激酶抑制的情况下，筛查 通常在体外针对纯化的重组激酶域进行。虽然这种方法导致了 虽然成功鉴定了激酶抑制剂，但它存在一些阻碍药物开发的主要限制。 进程。一个关键的限制是候选抑制剂的体外行为和细胞行为之间的相关性很差。 这一限制是一些候选药物后期失败的原因，总体上导致了高 药物开发过程的成本。这一限制可以通过开发和使用High 抑制剂谱的高效细胞分析方法。然而，目前还没有有效的细胞检测方法。 本机环境中的抑制剂分析。 在这项拟议的工作中，我们将设计和开发一种新的细胞检测方法，用于分析模型激酶的抑制剂， EGFR酪氨酸激酶。该检测将能够在自然条件下，在活体内筛选抑制物的活性 细胞。为了发展细胞测试，我们将实现三个具体目标：(1)合理的设计、合成和 新型细胞透过性荧光传感器对低纳米分子选择性响应的体外表征 EGFR的浓度；(2)细胞内递送、定位和EGFR的发展和特征 这些新传感器的结合；(3)开发一种竞争性的细胞检测方法，用于抑制剂的分析和 对抑制物结合的定量评估。 在这项工作成功完成后，新的检测不仅将为高通量提供基础 EGFR抑制剂的概况，但也将建立适用于其他蛋白质的一般开发原则 目标。 此外，该项目还将改善北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新的设计和合成方面获得宝贵的经验 传感器，表征生物分子/传感器的相互作用，致力于开发体外和细胞分析。 最终，参与这个项目将为本科生未来的职业生涯做好准备并增强他们的能力 在生物医学领域。

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.