Lopez Ludivine, Montnach Jérôme, Oliveira-Mendes Barbara, Khakh Kuldip, Thomas Baptiste, Lin Sophia, Caumes Cécile, Wesolowski Steven, Nicolas Sébastien, Servent Denis, Cohen Charles, Béroud Rémy, Benoit Evelyne, De Waard Michel
L'institut du Thorax, INSERM, CNRS, UNIV NANTES, Nantes, France.
Xenon Pharmaceuticals, Burnaby, BC, Canada.
Front Cell Dev Biol. 2021 Dec 20;9:798588. doi: 10.3389/fcell.2021.798588. eCollection 2021.
Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider , has been reported to be a potent antinociceptive compound due to its action on the genetically-validated Na1.7 pain target. Using this peptide for antinociceptive applications suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the Na1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) Na1.7/Na1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV EK was significantly improved for hNa1.6, leading to a decreased hNa1.7/hNa1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV EG/EG and HwTx-IV EK/RQ). In contrast, increased selectivity ratios (>46) were obtained if the EK mutation was combined to an additional double substitution ( A/YW) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNa1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNa1.6 and/or hNa1.7 subtypes. In addition, selective analogues for hNa1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.
虎纹毒素-IV（HwTx-IV）是在中国鸟蛛毒液中发现的一种肽，据报道，由于其作用于经过基因验证的疼痛靶点Na1.7，它是一种有效的抗伤害感受化合物。将这种肽用于抗伤害感受应用存在一个主要缺点，即它对神经肌肉系统有负面影响。尽管最近才进行研究，但这种影响似乎是由于该肽与位于突触前水平的Na1.6通道亚型之间的相互作用所致。这项工作的目的是研究如何对HwTx-IV进行修饰，以改变其最初23的人（h）Na1.7/Na1.6选择性比率。化学合成了19种HwTx-IV类似物，并测试了它们对在细胞系中稳定表达的这两种通道亚型所通过的钠电流的阻断作用。由于使用了自动膜片钳系统，生成了这些类似物的剂量反应曲线。根据早期构效关系（SAR）研究提供的信息，确定了几个关键氨基酸位置。在所测试的类似物中，HwTx-IV EK对hNa1.6的效力显著提高，导致hNa1.7/hNa1.6选择性比率降低（接近1）。对于在第4位有取代并对氨基酸1或26进行修饰的HwTx-IV类似物（HwTx-IV EG/EG和HwTx-IV EK/RQ），观察到了类似的选择性比率降低，但两种亚型的效力均有所提高。相比之下，如果将EK突变与额外的双取代（A/YW）相结合，或者简单地将肽的C末端酰胺化进一步替换为羧化基序，则会得到提高的选择性比率（>46），在后一种情况下，hNa1.6的效力会显著降低。这些结果表明，可以显著调节这两种通道亚型的选择性比率，以提高给定类似物对hNa1.6和/或hNa1.7亚型的效力。此外，还制备了具有更好安全性的hNa1.7选择性类似物，以限制神经肌肉损伤。
