Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target NaV1.7
Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target NaV1.7
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DOI：
10.1124/mol.115.100784
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发表时间：
2015-12-01
影响因子：
3.6
通讯作者：
Mobli, Mehdi
中科院分区：
文献类型：
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作者：
Klint, Julie K.;Chin, Yanni K. -Y.;Mobli, Mehdi
已知许多蜘蛛毒液肽可调节电压门控钠（Na-V）亚型1.7 （Na(V)1.7）通道的活性，这已成为一种有希望的镇痛靶点。特别是，一类蜘蛛毒液肽（NaSpTx1）已被发现能有效抑制NaV1.7（纳摩尔IC50），并在动物中显示出镇痛作用。然而，该家族的一个成员[mu- trtx -Hhn2b (Hhn2b)]在高达100 μ m的浓度下不抑制哺乳动物背根神经节中表达的NaV通道。该肽由于其半胱氨酸间距和在功能重要残基上的序列保守而被归类为NaSpTx1成员。在这里，我们对Hhn2b进行了详细的结构和功能分析，从而确定了两个非药效团残基，它们通过非重叠机制抑制人类Na(V)1.7 （hNa(V)1.7）。这些发现使我们产生了Hhn2b的双突变体，显示出纳摩尔抑制hNa(V)1.7。传统的结构/功能分析不能提供足够的分辨率来确定观察到的功能增益的机制。然而，通过使用先进的多维核磁共振实验解决野生型和突变肽的高分辨率结构，我们能够发现一个以前未知的相互作用网络，稳定这类毒液肽的药效团区域。我们进一步监测了肽的脂质结合特性，并发现其中一个关键氨基酸取代也选择性地调节了肽与阴离子脂质的结合。这些结果将进一步有助于以肽为基础的镇痛药治疗慢性疼痛的发展。
Many spider-venom peptides are known to modulate the activity of the voltage-gated sodium (Na-V) subtype 1.7 (Na(V)1.7) channel, which has emerged as a promising analgesic target. In particular, a class of spider-venom peptides (NaSpTx1) has been found to potently inhibit NaV1.7 (nanomolar IC50), and has been shown to produce analgesic effects in animals. However, one member of this family [mu-TRTX-Hhn2b (Hhn2b)] does not inhibit mammalian NaV channels expressed in dorsal root ganglia at concentrations up to 100 mu M. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b, leading us to identify two nonpharmacophore residues that contribute to human Na(V)1.7 (hNa(V)1.7) inhibition by nonoverlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNa(V)1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild-type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.