Engineering Gain-of-Function Analogues of the Spider Venom Peptide HNTX-I, A Potent Blocker of the hNa1.7 Sodium Channel.

Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. The voltage-gated sodium channel (VGSC) human Na1.7 (hNa1.7) is a compelling target that plays a key role in human pain signaling. The 33-residue peptide µ-TRTX-Hhn2b (HNTX-I), a member of Na-targeting spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian VGSCs, including the hNa1.7 channel. We engineered analogues of HNTX-I based on sequence conservation in NaSpTx family 1. Substitution of Asn for Ser at position 23 or Asp for His at position 26 conferred potent activity against hNa1.7. Moreover, multiple site mutations combined together afforded improvements in potency. Ultimately, we generated an analogue E1G⁻N23S⁻D26H⁻L32W with >300-fold improved potency compared with wild-type HNTX-1 on hNa1.7 (IC 0.036 ± 0.007 µM). Structural simulation suggested that the charged surface and the hydrophobic surface of the modified peptide are responsible for binding affinity to the hNa1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNa1.7 channel.