Design, synthesis, and mechanism of action of novel μ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Nav1.7

Abstract

μ-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Na_v isoforms, including Na_v1.7, and has potent analgesic effects, but it contains three pairs of disulfide bonds, making structural modification difficult and synthesis complex. To circumvent these difficulties, we designed and synthesized three KIIIA analogues with one disulfide bond deleted. The most active analogue, KIIIA-1, was further analyzed, and its binding pattern to hNa_v1.7 was determined by molecular dynamics simulations. Guided by the molecular dynamics computational model, we designed and tested 32 second-generation and 6 third-generation analogues of KIIIA-1 on hNa_v1.7 expressed in HEK293 cells. Several analogues showed significantly improved inhibitory activity on hNa_v1.7, and the most potent peptide, 37, was approximately 4-fold more potent than the KIIIA Isomer I and 8-fold more potent than the wildtype (WT) KIIIA in inhibiting hNa_v1.7 current. Intraperitoneally injected 37 exhibited potent in vivo analgesic activity in a formalin-induced inflammatory pain model, with activity reaching ∼350-fold of the positive control drug morphine. Overall, peptide 37 has a simplified disulfide-bond framework and exhibits potent in vivo analgesic effects and has promising potential for development as a pain therapy in the future.

Keywords: KIIIA; analgesia; peptide synthesis; structure optimization; voltage-gated sodium ion channel; μ-conotoxin.

Figure 1

Structure of human (h)Na_v1.7 (Protein Data Bank code:6J8G).A, the hNa_v1.7 structure includes an α (green) and one or more β (cyan) auxiliary subunits. B, top view of hNa_v1.7. The sugar molecules were shown in stick. All structures were analyzed in PyMol.

Figure 2

Effects of disulfide bond deletion on the activity and structure of KIIIA Isomer I.A, inhibitory activity of μ-conotoxin KIIIA Isomer I analogues on hNa_v1.7 at 1 μM (n = 6). B, evolution of the root-mean-square-deviation (RMSD) for the backbone of three KIIIA Isomer I analogues, KIIIA-1 (black), KIIIA-2 (red), and KIIIA-3 (green) in 100-ns molecular dynamic simulations. C, circular dichroism absorption curves of KIIIA Isomer I analogues. All data were presented as mean ± SD (∗∗∗p ≤ 0.001; statistical significance compared with the KIIIA Isomer I was determined using one-way ANOVA followed by Holm–Sidak posttest).

Figure 3

Interactions of the μ-conotoxin KIIIA Isomer I analogue KIIIA-1 with the hNa_v1.7.A, overall showing the interactions between KIIIA-1 and hNa_v1.7. B–F, locally magnified showing the interactions between residues at positions 1, 3, 5, 6, 10, and 15 of KIIIA-1 and residues at hNa_v1.7, respectively. hNa_v1.7 was shown in green, KIIIA-1 was shown in wheat, and hydrogen bonds were shown as yellow dashed lines.

Figure 4

Inhibitory activity of KIIIA-1 analogues against hNa_v1.7 at 1 μM (n > 3, for precise n number seeTable S2).A, example traces of analogues 1, 11, 14, 27, 29, and 31, with inhibition activities against hNa_v1.7 increased by 10% compared with KIIIA-1. hNa_v1.7 current stimuli waveform was shown on the top right. B, percentage of inhibition of all analogues against hNa_v1.7. All data are presented as mean ± SD (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; statistical significance compared with KIIIA-1 was determined using one-way ANOVA followed by Holm–Sidak posttest).

Figure 5

The inhibitory activities of KIIIA-1 analogues on hNa_v1.7.A, example traces for third-generation double mutation analogues against hNa_v1.7. B, inhibitory activity of KIIIA-1 analogues on hNa_v1.7 at 1 μM (33, 34, 35, 36, n = 6; 37, n = 5; 38, n = 4). Dose-dependent curves of 35 (C, Hill slope = 2.032, n = 4) and 37 (D, Hill slope = 1.119, n = 4) on hNa_v1.7 currents were fitted by Prism 7 software. Statistical significance compared with 33 was determined using one-way ANOVA followed by Holm–Sidak posttest (∗p ≤ 0.05, ∗∗p ≤ 0.01). All data are presented as mean ± SD.

Figure 6

NMR structure study of KIIIA Isomer I analogue 37.A, comparison of Hα secondary chemical shifts between KIIIA Isomer I (Biological Magnetic Resonance Data Bank ID: 20048) and 37. The sequence along the x-axis represents that of wildtype peptide. B, NMR solution structure of 37 as an overlay of the 20 lowest-energy states, with disulfides shown in yellow. C, superposition of 37 (cyan) and KIIIA Isomer I (slate), Protein Data Bank ID: 2LXG.

Figure 7

The analgesic activity of KIIIA Isomer I analogues 35 and 37 was evaluated by formalin-induced pain model in mice.A, KIIIA Isomer I analogues and morphine were diluted in saline and i.p. injected in mice 20 min prior to the injection of formalin into a paw. All data are presented as mean ± SD (∗∗∗p ≤ 0.001; negative control and 35, n = 7; 37 and morphine n = 6; statistical significance compared with negative control in each phase was determined using one-way ANOVA followed by Holm-Sidak posttest). B, brief graph shows how noxious stimuli conducted from peripheral terminal to spinal cord through axon via Na_v1.7 mediated action potential propagation.