A fluorescent polarization-based assay for the identification of disruptors of the RCAN1-calcineurin A protein complex
A fluorescent polarization-based assay for the identification of disruptors of the RCAN1-calcineurin A protein complex
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DOI：
10.1016/j.ab.2009.10.045
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发表时间：
2010-03-01
影响因子：
2.9
通讯作者：
Perez-Riba, Merce
中科院分区：
文献类型：
--
作者：
Carme Mulero, M.;Orzaez, Mar;Perez-Riba, Merce
钙调神经磷酸酶是一种 Ca2+/钙调蛋白依赖性丝氨酸/苏氨酸蛋白磷酸酶，参与许多生物过程和发育程序，包括免疫反应。钙调神经磷酸酶研究最多的底物之一是负责 T 细胞激活的转录因子 NFAT（激活 T 细胞的核因子）。不同的抗钙调神经磷酸酶药物，如环孢素 A 和 FK506，是移植治疗中最常用的免疫抑制剂。不幸的是，它们的作用机制完全阻断钙调神经磷酸酶活性，同时还需要持续给药，具有严重的副作用。近年来，钙调磷酸酶调节剂家族 (RCAN) 作为钙调磷酸酶信号通路的调节剂已被描述和广泛研究。 RCAN1 区域跨越氨基酸 198 至 218，负责抑制体内钙调神经磷酸酶-NFAT 信号通路。跨越该序列的 RCAN1 衍生肽会干扰钙调磷酸酶-NFAT 相互作用，而不影响一般钙调磷酸酶活性。在这里，我们报告了一种基于 RCAN1(198-218)-CnA 相互作用破坏的优化体外高通量荧光偏振测定的开发，用于识别具有免疫抑制潜力的分子。这种方法使我们确定双嘧达莫是这种相互作用的破坏者。此外，还鉴定出三种具有潜在免疫抑制作用的小分子。 (C) 2009 Elsevier Inc. 保留所有权利。
Calcineurin is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppresants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and Studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin-NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identified. (C) 2009 Elsevier Inc. All rights reserved.