Institute for Molecular Bioscience, The University of Queensland, Qld 4072, Australia.
Institute for Molecular Bioscience, The University of Queensland, Qld 4072, Australia; School of Chemistry and Molecular Biosciences, The University of Queensland, Qld 4072, Australia.
Biochim Biophys Acta Biomembr. 2017 May;1859(5):835-844. doi: 10.1016/j.bbamem.2017.01.020. Epub 2017 Jan 20.
The human voltage-gated sodium channel sub-type 1.7 (hNa1.7) is emerging as an attractive target for the development of potent and sub-type selective novel analgesics with increased potency and fewer side effects than existing therapeutics. HwTx-IV, a spider derived peptide toxin, inhibits hNa1.7 with high potency and is therefore of great interest as an analgesic lead. In the current study we examined whether engineering a HwTx-IV analogue with increased ability to bind to lipid membranes would improve its inhibitory potency at hNa1.7. This hypothesis was explored by comparing HwTx-IV and two analogues [E1PyrE]HwTx-IV (mHwTx-IV) and [E1G,E4G,F6W,Y30W]HwTx-IV (gHwTx-IV) on their membrane-binding affinity and hNa1.7 inhibitory potency using a range of biophysical techniques including computational analysis, NMR spectroscopy, surface plasmon resonance, and fluorescence spectroscopy. HwTx-IV and mHwTx-IV exhibited weak affinity for lipid membranes, whereas gHwTx-IV showed improved affinity for the model membranes studied. In addition, activity assays using SH-SY5Y neuroblastoma cells expressing hNa1.7 showed that gHwTx-IV has increased activity at hNa1.7 compared to HwTx-IV. Based on these results we hypothesize that an increase in the affinity of HwTx-IV for lipid membranes is accompanied by improved inhibitory potency at hNa1.7 and that increasing the affinity of gating modifier toxins to lipid bilayers is a strategy that may be useful for improving their potency at hNa1.7.
人类电压门控钠离子通道亚型 1.7（hNa1.7）作为一种有吸引力的靶点，正在开发具有更高效力和更少副作用的新型、亚型选择性镇痛药物。HwTx-IV 是一种源自蜘蛛的肽毒素，对 hNa1.7 具有高抑制活性，因此作为一种潜在的镇痛先导化合物具有很大的研究价值。在本研究中，我们研究了通过工程设计增加与脂质膜结合能力的 HwTx-IV 类似物是否会提高其对 hNa1.7 的抑制效力。通过比较 HwTx-IV 及其两种类似物 [E1PyrE]HwTx-IV（mHwTx-IV）和 [E1G，E4G，F6W，Y30W]HwTx-IV（gHwTx-IV）在与脂质膜结合亲和力和 hNa1.7 抑制效力方面的差异，我们探索了这一假说。我们使用一系列生物物理技术，包括计算分析、NMR 光谱学、表面等离子体共振和荧光光谱学，对该假说进行了研究。HwTx-IV 和 mHwTx-IV 与脂质膜的亲和力较弱，而 gHwTx-IV 与所研究的模型膜的亲和力增强。此外，使用表达 hNa1.7 的 SH-SY5Y 神经母细胞瘤细胞进行的活性测定表明，gHwTx-IV 在 hNa1.7 上的活性高于 HwTx-IV。基于这些结果，我们假设 HwTx-IV 与脂质膜的亲和力增加伴随着其对 hNa1.7 的抑制效力增强，并且增加门控修饰毒素与脂质双层的亲和力是一种可能有助于提高其对 hNa1.7 效力的策略。
