Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK-Paxillin Interaction.
Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK-Paxillin Interaction.
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DOI：
10.1177/2472555219874313
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发表时间：
2020-01
期刊：
影响因子：
3.1
通讯作者：
Cance, William
中科院分区：
文献类型：
--
作者：
Marlowe, Timothy;Alvarado, Carlos;Rivera, Andrew;Lenzo, Felicia;Nott, Rohini;Bondugji, Dena;Montoya, Justin;Hurley, Alana;Kaplan, Matt;Capaldi, Andrew;Cance, William
粘着斑激酶（FAK）是一种有前途的癌症药物靶点，这是由于其在多种实体瘤中的大量过表达以及在控制增殖、侵袭、凋亡和转移的信号整合中的关键作用。以前的FAK药物发现和高通量筛选仅集中在鉴定靶向FAK激酶结构域的抑制剂。由于FAK既是激酶又是支架蛋白，因此开发检测FAK蛋白-蛋白相互作用抑制剂的新型筛选测定仍然是一个关键需求。在这份报告中，我们描述了一个高通量的荧光偏振（FP）筛选分析，测量FAK和桩蛋白，一个焦点粘附相关蛋白之间的相互作用的发展。我们基于桩蛋白LD 2基序设计了TAMRA标记的桩蛋白肽，其以显著的动态范围、特异性、可变性、稳定性和适于高通量筛选的Z '因子结合粘着斑靶向（FAT）结构域。此外，我们使用该FP测定法对1，593种化合物进行了中试筛选，显示了其用于高通量药物筛选的可行性。最后，我们鉴定了3种化合物，其显示出对脂肪-桩蛋白结合的剂量依赖性竞争。该试验代表了FAK支架抑制剂的第一个高通量筛选试验，可以加速这种有前途的药物靶点的药物发现工作。
Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening has exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein-protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion-associated protein. We designed a TAMRA-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1,593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified 3 compounds that show dose-dependent competition of FAT-paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.
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影响因子：
8.8
作者：
Nishimura, M;Machida, K;Imaizumi, M;Abe, T;Umeda, T;Takeshima, E;Watanabe, T;Ohnishi, Y;Takagi, K;Hamaguchi, M
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作者：
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通讯作者：
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