Paiz-Candia Bertin, Islas Angel A, Sánchez-Solano Alfredo, Mancilla-Simbro Claudia, Scior Thomas, Millan-PerezPeña Lourdes, Salinas-Stefanon Eduardo M
Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, 14 Sur 6301, CU, San Manuel, Puebla, México.
Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico; Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, 14 Sur 6301, CU, San Manuel, Puebla, México.
Eur J Pharmacol. 2017 Feb 5;796:215-223. doi: 10.1016/j.ejphar.2017.01.002. Epub 2017 Jan 3.
Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Na1.4 currents (IC =60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Na1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources.
甲氟喹是一种多靶点抗疟药，可抑制阳离子电流。然而，该化合物对钠通道的作用及结合位点尚不清楚。为了探究甲氟喹的作用机制，我们在非洲爪蟾卵母细胞上采用双电极电压钳记录技术、对大鼠钠通道进行定点诱变，并结合使用分子动力学和对接协议的计算机模拟方法。我们发现甲氟喹：i）抑制Na1.4电流（IC =60μM），ii）显著延迟快速失活，但不影响失活后的恢复，iii）使稳态失活曲线明显向超极化电位偏移。β1亚基的存在显著降低了甲氟喹的效力，但该药物在重复去极化时会导致显著的频率依赖性衰减。计算和实验结果表明，甲氟喹通过对接在结构域DIII和DIV之间的疏水腔中与局部麻醉药结合位点重叠，该疏水腔将局部麻醉药结合位点与选择性过滤器相连。这一观点得到了以下事实的支持：甲氟喹在突变型Na1.4通道F1579A上的效力显著降低，而在K1237S通道上的效力显著增加。在计算机模拟中，该化合物对接在F1579上方并与该残基形成稳定的π-π相互作用。我们提供了关于阳离子两亲性化合物如何通过对接在哺乳动物钠通道的局部麻醉药结合位点和选择性过滤器之间发挥抑制作用的构效关系见解。我们提出的实验和计算研究的协同循环可能有助于阐明其他药物的结合位点，从而节省体外和计算机模拟资源。
