Abstract
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 Na(V)1.7 (hNa(V)1.7) 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(V)-targeting spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian VGSCs, including the hNa(V)1.7 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 hNa(V)1.7. 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 hNa(V)1.7 (IC(50) 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 hNa(V)1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNa(V)1.7 channel.