Locating the Route of Entry and Binding Sites of Benzocaine and Phenytoin in a Bacterial Voltage Gated Sodium Channel

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

钠通道阻滞剂（sodium channel blockers）可通过调控细胞电兴奋性，用于治疗癫痫发作与心律失常，亦可实现短期镇痛。开发能够选择性靶向体内9种已表达的电压门控钠通道（voltage-gated sodium channel）中任意一种的新一代药物，亟需更深入地阐明当前钠通道阻滞剂的作用机制。本研究借助近期解析的细菌来源电压门控钠通道NavAb的晶体结构，开展分子动力学模拟（molecular dynamics simulations），以明确钠通道阻滞药苯佐卡因（benzocaine）与苯妥英（phenytoin）与该蛋白的结合位点，并解析此类药物进入静息态通道的具体路径。结果显示，两种药物在通道孔道内均存在两个以非特异性疏水相互作用维系的潜在结合位点：一个紧邻激活门（activation gate）上方，另一个位于脂质填充的侧向窗孔入口处。三种独立分析方法均得到一致的结合位点，且均表明结合于激活门的稳定性略高于窗孔位点。研究还发现，两种药物均可穿过窗孔进入脂质双分子层，仅伴随较低的能量壁垒，这表明该路径即为学界长期推测的中性药物疏水进入通道。本项模拟研究凸显了多个氨基酸残基在引导药物进入并通过窗孔、以及构建药物结合位点过程中的关键作用。