Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analog (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively charged residues that contribute to the activity of the peptide can improve gHwTx-IV’s potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx- IV,
[R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTxIV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTxIV and gHwTx-IV. The lipid affinity of each of the three novel analogs was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable to that of native HwTx- IV. In summary, the present study reports the development of an HwTx-IV analog with improved in vitro selectivity for the pain target NaV1.7 and with an in Vivo efficacy similar to that of native HwTx-IV.