Phosphorylation Biosensors for Drug Discovery

蛋白激酶是化学上易于处理的药物靶点，但只有不到5%的人类激酶组被选择性小分子抑制剂彻底探索，以证明其治疗效用。制药公司有兴趣评估靶向激酶的治疗潜力，以确定解决未满足医疗需求的新机会。Lucidicor, Inc.已经确定并广泛设计了一种不寻常的海洋来源蛋白质，称为PhosFluor，它在磷酸化时改变其荧光。我们以蛋白激酶A为原型，广泛地设计了原生PhosFluor，并将其开发为基于细胞的荧光测定，提供蛋白质磷酸化的实时体内可视化。该平台可以应用于人类kinome的518种蛋白激酶中的任何一种。本应用程序的目的是优化我们的新细胞为基础的平台，以响应蛋白激酶抑制剂。工作假设是，“PhosFluor”可以很容易地被设计成响应其他蛋白激酶发出荧光，因此可以用来监测已经开发出来的调节蛋白激酶的化合物的作用。我们的实验方法将是通过将工程磷酸化位点转化为酪氨酸激酶（Fyn激酶）和细胞周期蛋白依赖性蛋白激酶（cdk5）的底物序列来创建额外的PhosFluor衍生物。提出这项研究的基本原理是，已经为这些激酶开发了许多具有良好表征的化合物，并且这些生物传感器的成功测试将使我们能够确定我们筛选方法的有效性。这项提议的研究意义重大，因为它为靶向激酶和磷酸酶的新药提供了一个新的细胞筛选平台，这可能会导致癌症、炎症、疼痛和神经精神疾病等疾病的创新治疗。最后，这项新技术将转化为广泛的蛋白激酶，极大地扩大了可能被筛选的药物靶点的数量。

DESCRIPTION: Protein kinases are chemically tractable drug targets, yet less than 5% of the human kinome has been thoroughly explored with selective small molecule inhibitors to demonstrate their therapeutic utility. Pharmaceutical companies are interested in evaluation of the therapeutic potential of targeting kinases to define new opportunities to address unmet medical needs. Lucidicor, Inc. has identified and extensively engineered an unusual marine-derived protein, termed PhosFluor", which varies its fluorescence upon phosphorylation. We have extensively engineered native PhosFluor" using Protein Kinase A as a prototype, and are developing this into a cell-based fluorescence assay that provides real-time in vivo visualization of protein phosphorylation. This platform can be applied to virtually any of the 518 protein kinases of the human kinome. The objective of this application is to optimize our novel cell-based platform to respond to protein kinase inhibitors. The working hypothesis is that PhosFluor" can easily be engineered to fluoresce in response to other protein kinases, and can therefore be used monitor the action of compounds that have been developed to modulate protein kinases. Our experimental approach will be to create additional PhosFluor" derivatives by converting engineered phosphorylation sites into substrate sequences for a tyrosine kinase (Fyn kinase) and a cyclin-dependent protein kinase (cdk5). The rationale for the proposed research is that many well-characterized compounds have already been developed for these kinases, and that the successful testing of these biosensors will enable us to determine the effectiveness of our screening method. The proposed research is significant, because it is offers a novel cellular screening platform for new drugs to target kinases and phosphatases, which may result in innovative treatments for disorders such as cancer, inflammation, pain and neuropsychiatric disorders. Finally, this new technology will be convertible to a wide array of protein kinases, greatly expanding the number of drug targets that may potentially be screened.