Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets
Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets
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DOI：
10.1111/jth.12861
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发表时间：
2015-05-01
影响因子：
10.4
通讯作者：
Nieswandt, B.
中科院分区：
文献类型：
--
作者：
Duetting, S.;Heidenreich, J.;Nieswandt, B.
研究背景血管损伤部位的血小板聚集是正常止血所必需的，但也可能导致病理性血管闭塞。Rho GTPases是调节重要细胞过程的分子开关，并且它们在心血管系统中具有关键功能。Rac 1是血小板细胞骨架重组的重要调节因子，并有助于血小板活化。Rac 1抑制剂被认为在广泛的治疗环境中是有益的，因此已经针对各种疾病进行了体内测试。两种小分子抑制剂NSC 23766和EHT 1864已在不同的细胞类型中进行了表征，分别对Rac 1和Rac表现出高度特异性。目的分析NSC 23766和EHT 1864的特异性。方法通过流式细胞术分析细胞活化和聚集度来评估小鼠野生型和Rac 1缺陷型血小板的血小板功能。结果NSC 23766和EHT 1864在100 m处对血小板功能有明显的Rac 1非依赖性作用。这两种抑制剂诱导Rac 1特异性抑制血小板扩散，但也明显损害激动剂诱导的Rac 1（-/-）血小板活化。此外，糖蛋白Ib介导的信号转导显着抑制NSC 23766在野生型和Rac 1缺陷型血小板。重要的是，这些抑制剂直接影响Rac 1效应子p21激活激酶（PAK）1和PAK 2的激活。结论我们的结果揭示了NSC 23766和EHT 1864在哺乳动物细胞中100 μ m处的关键脱靶效应，提出了关于它们在这些浓度下作为特异性Rac 1/Rac抑制剂在生化研究中的效用以及可能作为治疗剂的问题。
BackgroundPlatelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathologic vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes, and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganization, and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings, and have therefore been tested invivo for a variety of disorders. Two small-molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 and Rac, respectively.ObjectivesTo analyze the specificity of NSC23766 and EHT1864.MethodsPlatelet function was assessed in mouse wild-type and Rac1-deficient platelets by the use of flow cytometric analysis of cellular activation and aggregometry. Platelet spreading was analyzed with differential interference contrast microscopy, and activation of effector molecules was analyzed with biochemical approaches.ResultsNSC23766 and EHT1864 showed strong and distinct Rac1-independent effects at 100m in platelet function tests. Both inhibitors induced Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, glycoproteinIb-mediated signaling was dramatically inhibited by NSC23766 in both wild-type and Rac1-deficient platelets. Importantly, these inhibitors directly affected the activation of the Rac1 effectors p21-activated kinase (PAK)1 and PAK2.ConclusionsOur results reveal critical off-target effects of NSC23766 and EHT1864 at 100m in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents.