NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors
NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors
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DOI：
10.1124/jpet.113.207266
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发表时间：
2013-10-01
影响因子：
3.5
通讯作者：
Wieland, Thomas
中科院分区：
文献类型：
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作者：
Levay, Magdolna;Krobert, Kurt Allen;Wieland, Thomas
干扰rac1活性的小分子被认为是潜在的药物，并且已经在动物模型中进行了研究。一种被广泛使用但没有RhoA活性减弱报道的抑制剂是NSC23766[(N-6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4，6-quinolinediamine三盐酸盐]。我们发现NSC23766抑制M-2mAChR(M-2mAChR)诱导的新生大鼠心肌细胞rac1的激活。令人惊讶的是，NSC27366同时抑制了卡巴胆碱诱导的RhoA激活和M-2 mAChR诱导的变力反应，这些反应需要激活Rho依赖的激酶。因此，我们的目标是确定NSC23766干扰差异介导的M-2 mAChR诱导的反应的机制。有趣的是，NSC23766导致正性肌力反应的卡巴胆碱浓度反应曲线右移，而不改变卡巴胆碱的疗效。为了分析NSC23766的特异性，我们比较了卡巴胆碱和类似的G(I)β-γ介导的腺苷诱导的人心房肌细胞G(I)蛋白调节钾通道(GIRK)通道的激活。应用NSC23766可阻断氨基甲胆碱诱发的K+电流，但不影响腺苷诱导的GIRK电流。同样，NSC23766不能减弱腺苷A(1)受体在新生大鼠心脏中诱导的正性变力反应。为了研究其对不同mAChR类型的特异性，我们研究了卡巴胆碱对表达M-1、M-2或M-3mAChR的人胚胎肾293(HEK-293)细胞内钙离子浓度的影响。NSC23766使所有mAChRs的卡巴胆碱浓度反应曲线右移。因此，NSC23766不仅是rac1激活的抑制剂，而且在相同的浓度范围内是mAChRs的竞争性拮抗剂。M-2和M-3mAChR晶体结构的分子对接分析证实了这一解释。
Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N-6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M-2 muscarinic acetylcholine receptor (M-2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M-2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M-2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly G(i)beta gamma-mediated, adenosine-induced activation of G(i) protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K+ current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A(1) receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca2+ concentrations in human embryonic kidney 293 (HEK-293) cells expressing M-1, M-2, or M-3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M-2 and M-3 mAChR crystal structures confirmed this interpretation.