Interactions of disulfide-deficient selenocysteine analogs of μ-conotoxin BuIIIB with the α-subunit of the voltage-gated sodium channel subtype 1.3


  • Brad R. Green,

    1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia
    2. Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA
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  • Min-Min Zhang,

    1. Department of Biology, University of Utah, Salt Lake City, UT, USA
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  • Sandeep Chhabra,

    1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia
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  • Samuel D. Robinson,

    1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia
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  • Michael J. Wilson,

    1. Department of Biology, University of Utah, Salt Lake City, UT, USA
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  • Addison Redding,

    1. Department of Biology, University of Utah, Salt Lake City, UT, USA
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  • Baldomero M. Olivera,

    1. Department of Biology, University of Utah, Salt Lake City, UT, USA
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  • Doju Yoshikami,

    1. Department of Biology, University of Utah, Salt Lake City, UT, USA
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  • Grzegorz Bulaj,

    Corresponding author
    1. Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA
    2. Department of Biology, University of Utah, Salt Lake City, UT, USA
    • Correspondence

      R. S. Norton, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Vic. 3052, Australia

      Fax: +61 3 9903 9582

      Tel: +61 3 9903 9167


      G. Bulaj, Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA

      Fax: +1 801 581 7087

      Tel: +1 801 581 4371


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  • Raymond S. Norton

    Corresponding author
    1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia
    • Correspondence

      R. S. Norton, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Vic. 3052, Australia

      Fax: +61 3 9903 9582

      Tel: +61 3 9903 9167


      G. Bulaj, Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA

      Fax: +1 801 581 7087

      Tel: +1 801 581 4371


    Search for more papers by this author


Inhibitors of the α-subunit of the voltage-gated sodium channel subtype 1.3 (NaV1.3) are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described μ-conotoxin BuIIIB (μ-BuIIIB) from Conus bullatus was shown to block NaV1.3 with submicromolar potency (Kd = 0.2 μm), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of μ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure–activity studies. In the present study, we report the synthesis and characterization of μ-BuIIIB analogs incorporating a disulfide-deficient, diselenide-containing scaffold designed to simplify synthesis and facilitate structure–activity studies directed at identifying amino acid residues involved in NaV1.3 blockade. Our results indicate that, similar to other μ-conotoxins, the C-terminal residues (Trp16, Arg18 and His20) are most crucial for NaV1 blockade. At the N-terminus, replacement of Glu3 by Ala resulted in an analog with an increased potency for NaV1.3 (Kd = 0.07 μm), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ-carboxyglutamate replacement, decreased potency (Kd = 0.33 μm), whereas replacement with positively-charged 2,4-diamonobutyric acid increased potency (Kd = 0.036 μm). These results provide a foundation for the design and synthesis of μ-BuIIIB-based analogs with increased potency against NaV1.3.








disulfide-deficient selenocysteine-containing BuIIIB




the α-subunit of the voltage-gated sodium channel subtype 1, cloned from rat


structure–activity relationship


trifluoroacetic acid


μ-conotoxin BuIIIB


The μ-conotoxins are a family of venom-derived peptides that block the α-subunit of the voltage-gated sodium channels (NaV1), several of which are implicated in various pain pathways. These peptides act by binding at neurotoxin receptor site 1 on the outer vestibule of the Na+ conductance pore [1]. The μ-conotoxins are characterized by a six-cysteine framework (CICII(Xn)CIII(Xn)CIV(Xn)CVCVI) cross-linked by three disulfide bridges. The earliest reports identified μ-conotoxins, specifically μ-GIIIA and μ-PIIIA, that were selective for the skeletal muscle subtype (NaV1.4) [2, 3]. More recent studies have focused on μ-conotoxins such as μ-KIIIA and μ-SIIIA, which preferentially block neuronal subtypes (NaV1.2) over skeletal (NaV1.4) and cardiac (NaV1.5) muscle subtypes. These peptides have attracted considerable interest because of their potent analgesic activity in animal models of pain [4], although nonselective blockade of other NaV1 subtypes has hindered their development as therapeutics. In an attempt to engineer in subtype selectivity, structure–activity relationship (SAR) studies were conducted to determine critical amino acid residues and subsequently identify potential sites for chemical modification. To date, detailed SAR studies have been carried out on a limited number of these peptides, including μ-GIIIA, μ-PIIIA, μ-KIIIA and μ-SIIIA [3-7].

Recently, several new μ-conotoxins were identified in the venom of Conus bullatus [8]. μ-conotoxin BuIIIB (μ-BuIIIB) exhibited significant differences from previously described μ-conotoxins, particularly with respect to the primary sequence at the N-terminus (Fig. 1A). Initial studies revealed that μ-BuIIIB almost completely blocked the sodium current in the NaV1.4 (skeletal muscle) subtype, with near irreversibility [8]. More recently, Wilson et al. [9] showed that μ-BuIIIB also blocked the NaV1.3 subtype with relatively high potency (Kd = 0.2 μm), whereas μ-KIIIA and μ-SIIIA exhibited only modest blockade of this subtype (IC50 = 8 and 11 μm, respectively). The importance of NaV1.3 with respect to neuropathic pain lies in its increased expression after axotomy, where the expression of other subtypes such as NaV1.8 and NaV1.9 is decreased [10, 11]. To further examine the role of NaV1.3 in neuropathic pain, potent and selective inhibitors of this channel are needed. However, such ligands are currently unavailable.

Figure 1.

(A) Sequence alignment of μ-BuIIIB with other μ-conotoxins. These toxins are characterized by a shared disulfide-framework and the ability to block voltage-gated sodium channel subtypes. μ-BuIIIB is distinguished from other members of this class by an extended N-terminus, in addition to increased length of inter-cysteine loop 1. (B) Description of the ddSecBuIIIB scaffold. Solution structure of μ-BuIIIB, with disulfide connectivity, is shown (Protein Data Bank code: 2LO9) (19). Diselenide replacement of the Cys5-Cys17 bridge (blue) facilitated cyclization between these two residues independent of disulfide bridge formation. Removal of the Cys6-Cys23 bridge (red), by replacement with Ala, reduced the number of possible folding species to three for each ddBuIIIB analog shown. The BuIIIB[C5U,C17U,C6A,C23A] analog (i.e. ddSecBuIIIB) was selected as a framework to carry out Ala-replacement studies based on the ‘native-like’ blockade of NaV1.3 (Kd = 0.2 μm). The ddSecBuIIIB scaffold was employed to assess the importance of each noncysteine residue within the primary sequence against NaV1.3 and NaV1.4.

In the present study, we describe the development of a disulfide-deficient, diselenide-bridge containing analog of μ-conotoxin BuIIIB (disulfide-deficient selenocysteine-containing BuIIIB; ddSecBuIIIB) as the basis for an alanine scan to identify residues that contribute to blockade of NaV1.3 (Fig. 1B). The advantages of this approach over a more traditional Ala-scan using the peptide containing three disulfide bridges are a simplification of the oxidative folding pathway and unambiguous assignment of the disulfide connectivity. μ-BuIIIB was chosen as a model system for the present study because of the particular challenges faced by oxidative folding of this peptide. By reducing the number of potential folding intermediates through diselenide replacement of the Cys5-Cys17 pairing, and by elimination of the Cys6-Cys23 disulfide bridge, we created a peptide scaffold in which the individual contributions of noncysteine amino acid residues could be studied. Disulfide deletion and diselenide-bridge incorporation have previously been used, either individually or in combination, to facilitate oxidative folding studies of other conotoxins such as μ-SIIIA, μ-KIIIA and ω-GVIA [12-14], although these strategies have yet to be employed to facilitate SAR studies of disulfide-rich conotoxins. This approach could also be employed to rapidly determine amino acid residues critical for biological activity in other cysteine-rich peptides.


Chemical synthesis

The synthesis, purification and oxidative folding steps are often the greatest impediments to SAR studies of disulfide-rich conotoxins. Oxidation of such conotoxins can result in multiple folding isoforms, yielding limited quantities of the properly folded peptide for pharmacological characterization. Furthermore, determination of the disulfide connectivities of the active isoform can be labor intensive and often requires sophisticated analysis methods, such as selective reduction-alkylation, direct MS fragmentation or NMR methods [15, 16]. Before embarking on SAR studies of μ-BuIIIB, we first determined the contributions of individual disulfide bridges in μ-BuIIIB to NaV1.3 inhibition.

An initial series of disulfide-deficient analogs was constructed where we systematically replaced the pair of cysteines in each disulfide with a pair of alanines (Table 1). Removal of the Cys5-Cys17 bridge led to a slight increase in kon (0.17 versus 0.085 μm−1·min−1) and a much greater increase in koff compared to the unmodified peptide (2.6 versus 0.07 min−1). These values translated to a significant decrease in potency for BuIIIB[C5A,C17A] (Kd = 15.7 versus 0.2 μm), indicating that this bridge is important for NaV1.3 inhibition. The Cys5-Cys17 bridge was therefore selected for replacement by a redox-favored diselenide bridge. Removal of either the Cys6-Cys23 or Cys13-Cys24 bridges yielded similar results, with kon values of 0.23 and 0.34 μm−1·min−1, respectively. The koff values were also similar, and closer to that of the unmodified peptide (0.059 and 0.038 min−1, respectively). These values translated to potencies in blocking NaV1.3 equal to or greater than that of wild-type μ-BuIIIB (Kd of 0.26 and 0.11 μm for BuIIIB[C6A,C23A] and BuIIIB[C13A,C24A], respectively) (Table 1). The solution structure of μ-BuIIIB shows that the N-terminal helix is anchored to the core of the molecule by the Cys5-Cys17 and Cys6-Cys23 disulfide bridges [16]. Removal of either the Cys6-Cys23 or Cys13-Cys24 bridges would be expected to have structural consequences, although it appears that those changes do not significantly affect the peptide's activity against NaV1.3.

Table 1. Structural and pharmacological characterization of ddBuIIIB and ddSecBuIIIB scaffolds. Analogs were screened against r-NaV1.3. U, selenocysteine. ΔKd, ratio of each analog to wild-type μ-BuIIIB
PeptideMr (calculated/observed)Correct isomerkonm·min)−1koff (min)−1Kdm)ΔKd
  1. a

    Masses determined by MALDI-TOF.

  2. b

    Masses determined by ESI-MS.

μ-BuIIIB2761.20/2761.19a16%0.085 ± 0.010.017 ± 0.0070.20 ± 0.02 
BuIIIB[C5A,C17A]2700.25/2700.24a32%0.16 ± 0.062.6 ± 0.8615.7 ± 2.679
BuIIIB[C6A,C23A]2700.25/2700.24a37%0.23 ± 0.020.059 ± 0.0040.26 ± 0.031.3
BuIIIB[C13A,C24A]2700.25/2700.80a35%0.34 ± 0.030.038 ± 0.0030.11 ± 0.0130.55
BuIIIB[C5U,C17U,C6A,C23A]2796.13/2796.13b51%0.12 ± 0.010.026 ± 0.0040.2 ± 0.04 
BuIIIB[C5U,C17U,C13A,C24A]2796.13/2796.14b43%0.19 ± 0.020.08 ± 0.0050.42 ± 0.052.1

Using the information obtained from disulfide-deficient BuIIIB analogs, two additional analogs, BuIIIB[C5U,C17U,C6A,C23A] and BuIIIB[C5U,C17U,C13A,C24A], were constructed to identify the optimal disulfide-deficient, selenoconotoxin scaffold as a basis for SAR studies. Slight differences in biological activity were observed between the disulfide-deficient analogs and the ddSecBuIIIB scaffolds. BuIIIB[C5U,C17U,C6A,C23A] exhibited a faster kon and koff than BuIIIB[C5U,C17U,C13A,C24A], resulting in a scaffold that exhibited ‘native-like’ potency for NaV1.3 (Kd = 0.2 μm) (Table 1).

Oxidative folding

Crude peptide was removed from the solid support resin using modified reagent K cleavage mixture containing 2,2′-dithiobis(5-nitropyridine) (DTNP) as described previously [17, 18]. Briefly, the cleavage mixture was supplemented with 1.3 equivalents DTNP to remove the p-methoxybenzyl protecting group from selenocysteine, resulting in a selenocysteine–2-thio-5-nitropyridine adduct. This adduct was then removed by treatment for 2 h with 50 mm dithiothreitol, leading to spontaneous formation of the diselenide bridge between residues 5 and 17 (Sec5-Sec17) [19]. Deprotection and cyclization steps were performed directly on the crude mixture to minimize losses from additional purification steps. As such, quantitative yields of deprotection were not determined. However, analysis of the crude, Se-Se containing BuIIIB[C5U,C17U,C6A,C23A] analog revealed a single major product comprising 22% of the total mixture based on analytical HPLC peak area. The diselenide-containing peptides were then purified in parallel by C18 solid-phase extraction. Finally, formation of either the Cys13-Cys24 or Cys6-Cys23 bridge was achieved using the solid support oxidant CLEAR-Ox® (Peptides International, Louisville, KY, USA). For each of the ddSecBuIIIB analogs, a well-separated major peak corresponding to the fully folded peptide was observed (Fig. 2). Oxidative folding resulted in greater accumulation of the desired products, as determined by peak area using analytical HPLC, for both BuIIIB[C5U,C17U,C6A,C23A] (51%) and BuIIIB[C5U,C17U,C13A,C24A] (43%) compared to 16% for the unmodified μ-BuIIIB (Fig. 2B,E and Table 1).

Figure 2.

Comparison of wild-type μ-BuIIIB with ddSecBuIIIB. (A) Sequence of the unmodified peptide showing experimentally-determined disulfide connectivity. (B) HPLC folding profiles of linear peptide (top) and folding mixture (bottom); asterisks indicate peaks tested for functional activity. (C) Representative time course of the blockade of rNaV1.3 by 10 μm peptide (bar indicates when peptide was present). Inset shows current traces obtained before (green) and during (red) exposure of the peptide. (D) Sequence of the ddSecBuIIIB scaffold showing the positions of the selenocysteine (U) and alanine replacements. (E) HPLC profile of linear peptide (top) and folding mixture (bottom); asterisks indicate peak tested for functional activity. The sample profile illustrates the efficiency of the folding pathway. (F) Representative time course of the blockade of NaV1.3 (for comparison of kinetic constants, see Table 1). Inset: current traces obtained before (green) and during (red) exposure to peptide.

1H NMR of ddSecBuIIIB

The conformation of BuIIIB[C5U,C17U,C6A,C23A] was compared with that of the native peptide using NMR spectroscopy. After optimization of the pH and temperature (Fig. 3), one-dimensional (1D) and two-dimensional (2D) 1H NMR spectra were acquired at pH 3.0 and 35 °C, 1H chemical shifts were determined by standard sequential assignment (Tables S1 and S2; BMRB ID #19923) and plots of deviations from random coil chemical shifts [20] for ddSecBuIIIB and μ-BuIIIB were constructed. The 1D 1H spectrum for ddSecBuIIIB was less well dispersed than that of μ-BuIIIB (Fig. 4A). Hα chemical shift deviations from random coil (Fig. 4) suggested that the N-terminus of ddSecBuIIIB was largely unstructured, with some degree of helicity near the C-terminus between residues Gly14 and His20. Hα chemical shifts were generally similar to μ-BuIIIB, whereas the HN shifts showed greater differences compared to the native peptide (Fig. 4B). The solution structure of native μ-BuIIIB contains helices at both the N- and C-termini [16] and it is clear that the C-terminal helix is partially preserved in ddSecBuIIIB but the N-terminal region is less structured. These results are of interest because it was reported previously that functionally important residues in μ-KIIIA reside in the C-terminal helical region of that peptide [21].

Figure 3.

Optimization of NMR conditions for ddSecBuIIIB. (A) 1D 1H NMR spectra of BuIIIB[C5U,C17U,C13A,C24A] and BuIIIB[C5U,C17U,C6A,C23A] versus μ-BuIIIB. Peptide samples were 160 μm in 10 mm phosphate buffer containing 5% 2H2O (pH 5.5). The water signal was suppressed by excitation sculpting. Spectra were recorded at 22 °C over 128 scans at 600 MHz at a spectral width of 11 p.p.m. The amide/aromatic region has been shown to illustrate the decreased spectral dispersion of the disulfide-deficient, diselenide bridge-containing analogs compared to the wild-type μ-BuIIIB. (B) 1D 1H NMR spectra of BuIIIB[C5U,C17U,C6A,C23A] (ddSecBuIIIB) at different temperatures in 5% 2H2O. The sample pH was 4.8 and spectra were acquired using a Bruker 600 MHz spectrometer using 128 scans at a spectral width of 12 p.p.m. (C) pH titration of ddSecBuIIIB. 1D 1H NMR spectra were collected in 100% 2H2O, the pH was adjusted to with NaO2H and spectra were acquired at 25 °C. Spectra were collected using 32 scans at a spectral width of 16 p.p.m. The amide/aromatic region is shown.

Figure 4.

Structural comparisons of μ-BuIIIB and ddSecBuIIIB. (A) 1D 1H NMR spectra of ddSecBuIIIB scaffold (red) and wild-type μ-BuIIIB (black) at 35 °C, pH 3.0 and 600 MHz. (B) Deviation from random coil shifts of the Hα, Hβ and HN resonances of μ-BuIIIB (black) and ddSecBuIIIB (red) at 35 °C and pH 3.0 [20]. Random coil shift values for oxidized Cys were used to estimate chemical shift deviation from random coil for selenocysteines. HN and Hα chemical shift differences between ddSecBuIIIB and μ-BuIIIB are also plotted.

Structure–activity studies on ddSecBuIIIB

Although NMR suggested conformational differences between ddSecBuIIIB and μ-BuIIIB, structure–activity studies were carried out to identify amino acid residues in ddSecBuIIIB that contributed to NaV1.3 blockade. These studies identified a number of residues, particularly near the C-terminus, that were important for NaV1.3 potency (Table 2). Specifically, Ala-replacement of Trp16 or His 20 resulted in slow on-rate kinetics, which prevented steady-state binding from being achieved within the experimental time window at peptide concentrations near the expected Kd or IC50. These analogs had an estimated 150-fold lower potency compared to μ-BuIIIB or ddSecBuIIIB (Kd > 30 μm). Ala-replacement of Arg15, Arg18 or Arg22 also led to deleterious effects on NaV1.3 blockade (Kd = 1.84, 15.7 and 3.81 μm, respectively) (Fig. 5 and Table 2). These results were interesting because earlier studies identified a crucial role for basic residues at the equivalent position to Arg15 in μ-BuIIIB [5]. Although mutations at this position decreased potency, it is clear that the other positively-charged residues in this region play more pronounced roles in NaV1.3 inhibition. These results were consistent with those reported by McArthur et al. [22], which showed three basic residues (Arg12, Arg14 and Lys17) near the C-terminus of μ-PIIIA contributing to the blockade of NaV1.2 despite sharing a high degree of sequence homology with NaV1.4-selective μ-GIIIA. Our results highlight the importance of basic residues at the C-terminus of ddSecBuIIIB for blockade of NaV1.3 (Fig. 5 and Table 2).

Table 2. Summary of the ability of μ-BuIIIB and Ala-scan analogs of ddSecBuIIIB to block rNaV1.3 and rNaV1.4 expressed in X. laevis oocytes, as determined by two-electrode voltage-clamp measurements
konm·min)−1koff (min)−1Kdm)konm·min)−1koff (min)−1Kdm)
  1. Rate constants were calculated from at least three independent experiments using prism software (GraphPad Software Inc., San Diego, CA, USA). Kd = koff/kon. NA, Kd value was not available as a result of slow kinetics, which precluded steady state from being achieved at concentrations tested within the experimental time frame. Kinetic data for μ-BuIIIB are from Wilson et al. [9].

  2. a

    Analogs possessing the ddSecBuIIIB scaffold (BuIIIB[C5U,C17U,C6A,C23A]).

μ-BuIIIB0.085 ± 0.010.017 ± 0.0070.2 ± 0.0864.20 ± 0.7600.015 ± 0.0050.004 ± 0.001
ddSecBuIIIB0.13 ± 0.070.026 ± 0.0020.2 ± 0.027.15 ± 0.0360.009 ± 0.0020.001 ± 0.000
[V1A]a0.17 ± 0.0150.038 ± 0.0010.22 ± 0.020.940 ± 0.0110.026 ± 0.0020.027 ± 0.002
[G2A]a0.087 ± 0.0130.042 ± 0.0030.48 ± 0.081.64 ± 0.2580.054 ± 0.0070.033 ± 0.007
[E3A]a0.29 ± 0.0470.02 ± 0.0010.07 ± 0.01241.6 ± 2.9730.030 ± 0.0020.001 ± 0.000
[R4A]a0.026 ± 0.0050.028 ± 0.0031.1 ± 0.230.78 ± 0.0810.022 ± 0.0010.028 ± 0.003
[K7A]a0.07 ± 0.0050.05 ± 0.0050.71 ± 0.0881.73 ± 0.1870.010 ± 0.0010.006 ± 0.001
[N8A]a0.062 ± 0.0070.016 ± 0.0010.26 ± 0.0321.45 ± 0.2200.005 ± 0.0000.003 ± 0.001
[G9A]a0.068 ± 0.0130.027 ± 0.0010.4 ± 0.081.03 ± 0.1000.012 ± 0.0010.012 ± 0.001
[K10A]a0.050 ± 0.0040.051 ± 0.0041.02 ± 0.112.05 ± 0.2760.008 ± 0.0010.004 ± 0.001
[R11A]a0.033 ± 0.0020.037 ± 0.0051.12 ± 0.151.11 ± 0.1900.010 ± 0.0000.009 ± 0.002
[G12A]a0.098 ± 0.0190.033 ± 0.0040.34 ± 0.085.12 ± 0.0680.008 ± 0.0010.002 ± 0.000
[G14A]a0.190 ± 0.0020.070 ± 0.0060.230 ± 0.1075.53 ± 0.2620.014 ± 0.0010.003 ± 0.000
[R15A]a0.060 ± 0.0080.240 ± 0.0121.84 ± 0.3850.410 ± 0.0350.105 ± 0.0050.260 ± 0.026
[W16A]aNA1.69 ± 0.180> 30NA1.68 ± 0.1232.07 ± 0.284
[R18A]a0.06 ± 0.0080.78 ± 0.07515.7 ± 0.2961.26 ± 0.1240.147 ± 0.0080.13 ± 0.009
[D19A]a0.22 ± 0.0120.73 ± 0.0730.54 ± 0.02722.5 ± 3.0050.144 ± 0.0090.009 ± 0.001
[H20A]aNA1.01 ± 0.119> 300.03 ± 0.0040.219 ± 0.02310.3 ± 2.448
[S21A]a0.09 ± 0.0030.02 ± 0.0030.37 ± 0.0593.41 ± 0.0780.018 ± 0.0020.005 ± 0.001
[R22A]a0.06 ± 0.0020.24 ± 0.0123.81 ± 0.194NA0.174 ± 0.0070.28 ± 0.005
Figure 5.

Effects of Ala-substitution of noncysteine residues in the ddSecBuIIIB scaffold. Bar graphs compare the potencies (∆pKds) of ddSecBuIIIB Ala-walk analogs with that of wild-type μ-BuIIIB (∆pKd = 0) against NaV1.3 (A) and NaV1.4 (B). Substitutions that increased potency are shown in blue, those that dramatically decreased potency are shown in red, and those that produced mild to moderate effects are shown in black. (C) Sequence alignment of the P-loop regions of NaV1.3 and NaV1.4, which is the site of interaction of all μ-conotoxins, including μ-BuIIIB. Amino acid differences between subtypes in this region are indicated in red; residues comprising the selectivity filter (D, E, K and A) are underlined.

Replacement of Val1, Asn8, Gly9, Gly12, Gly14 or Ser21 with Ala had little effect on NaV1.3 potency (Table 2). These positions in the sequence are therefore potential sites of modification for peptide engineering to improve the physicochemical and/or pharmacological properties of μ-BuIIIB. Similar approaches were applied previously, resulting in potent and/or selective analogs of the μ-conotoxins KIIIA and SIIIA [3, 23]. In the context of the ddSecBuIIIB scaffold, mutations made to the basic residues in loop 1 (Lys7, Lys10 and Arg11) led to moderate effects on NaV1.3 inhibition (Kd = 0.71, 1.02 and 1.12 μm, respectively) and little or no effect on NaV1.4 inhibition (Fig. 5).

The most striking outcome of these studies was the improved potency obtained through substitution of Glu3 with Ala. This replacement actually improved NaV1.3 potency by nearly three-fold (Kd = 0.07 μm) (Fig. 5 and Table 2). To further investigate the effects of amino acid replacement of the acidic residue, a positional scan of Glu3 was conducted.

Positional scanning Glu3

Removal of the negatively-charged residue at position 3 led to increased potency against NaV1.3. This was consistent with findings by Ekberg et al. [24], who showed that the charge state of μ-conotoxins was important for interaction with the negatively-charged voltage-gated sodium channel pore region. To further explore the effects of charge at this position, analogs were constructed where Glu3 was replaced with either γ-carboxyglutamate (Gla; −2 charge) or l-2,4-diaminobutyric acid (Dab; +1 charge) (Fig. 6). Increasing the negative charge at position 3 reduced NaV1.3 potency by more than 1.5-fold (Kd = 0.33 μm), whereas reversal of the negative charge increased NaV1.3 potency (Kd = 0.038 μm) beyond that of the (neutral) Ala substitution (Fig. 6).

Figure 6.

Exploration of charge effects at position 3 using the ddSecBuIIIB scaffold. (A) Representative electrophysiology traces of ddSecBuIIIB analogs that reveal the consequences of different charges at position 3. (B) Table of kinetics of the blockade of NaV1.3 by ddSecBuIIIB analogs. Each value represents the mean of at least three different peptide concentrations tested in triplicate. BuIIIB[C5U,C17U,C6A,C23A,E3Dab] was almost three-fold more potent than μ-BuIIIB in blocking NaV1.3. Current traces obtained before (green) and during (red) exposure of the peptide.

The steric effects of basic residues at this position were also examined (Fig. 7). A series of positively-charged analogs was synthesized and tested that replaced Glu3 with l-Dap, Orn, Lys, Arg or His. All analogs containing basic residues at position 3 exhibited improved NaV1.3 potency. These studies revealed an optimal size for the side chain at this position, with groups smaller (e.g. Dap) or larger (e.g. ornithine, lysine, arginine and histidine) than Dab resulting in decreased potency compared to BuIIIB[C5U,C17U,C6A,C23A,E3Dab] (Fig. 7). Similar to what was seen in experiments using NaV1.3, the [E3Dab] mutant showed increased potency against NaV1.4, with kon = 32.1 μm·min−1, koff = 0.017 ± 0.001 min−1 and Kd = 0.0005 ± 0.0001 μm (data not shown). The SAR data suggested that any changes in NaV1.3 potency were closely mirrored by changes in potency against NaV1.4. As such, the other analogs were assayed against NaV1.3 only because the focus of the present study was on potency against NaV1.3 rather than subtype selectivity.

Figure 7.

Exploration of steric effects at position 3 in the ddSecBuIIIB scaffold. (A) Representative electrophysiology traces of ddSecBuIIIB analogs that reveal the consequences of increased steric bulk of residue side-chains at position 3. (B) Table of kinetics of the blockade of NaV1.3 by ddSecBuIIIB analogs. Each value represents the mean of at least three experiments tested in triplicate. Analogs with increased or decreased steric bulk, relative to that of BuIIIB[C5U,C17U,C6A,C23A,E3Dab], had greater potency than μ-BuIIIB in blocking NaV1.3, although not to the extent that BuIIIB[C5U,C17U,C6A,C23A,E3Dab] did. Current traces obtained before (green) and during (red) exposure of the peptide.


We have created an analog of the μ-conotoxin BuIIIB, ddSecBuIIIB, which folds efficiently during oxidative folding to a single major product with well-defined disulfide/diselenide connectivity. This stands in marked contrast to the native peptide, where, under optimized glutathione-assisted folding conditions, the biologically-active isoform comprised only 16% of the total folding mixture (Fig. 2 and Table 1).

1H NMR spectra of the ddSecBuIIIB scaffold showed reduced amide peak dispersion compared to μ-BuIIIB. The differences in structure presumably resulted from greater flexibility of the molecule caused by removal of the Cys6-Cys23 disulfide bridge. It is intriguing, therefore, that this analog retained native-like potency for NaV1.3. These results are not without precedent because Tietze et al. [25] showed that less-structured forms of the conotoxin μ-PIIIA also exhibited activity against the muscle subtype NaV1.4, albeit with lower potency. Closer examination of chemical shift plots (Fig. 4) shows that ddSecBuIIIB retains some degree of α-helical content near the C-terminus, which is known to be important for NaV1-subtype blockade. In light of the conformational differences between the two peptides in solution, we suggest that the ddSecBuIIIB scaffold should be considered as a new blocker of this channel rather than simply as a proxy for native μ-BuIIIB. This analog therefore represents a useful new pharmacological tool for studies of NaV1 channels, one that, importantly, refolds very efficiently to a well-defined product.

Structure–activity studies using ddSecBuIIIB identified the critical roles of aromatic and basic residues near the C-terminus of ddSecBuIIIB (Fig. 5). These results were largely consistent with previous SAR studies of other μ-conotoxins, presumably as a result of the high degree of homology amongst the neuronal-subtype preferring μ-conotoxins at the C-terminus. This region is considered to contribute to general voltage-gated sodium channel target specificity, whereas structural diversity at the N-termini is considered to aid in discrimination amongst voltage-gated sodium channel subtypes [26]. μ-BuIIIB differs from previously described μ-conotoxins in having a short, helical extension of the N-terminus. The ddSecBuIIIB [V1A] and [G2A] analogs exhibited native-like potency for NaV1.3 (Kd = 0.2 and 0.48 μm, respectively). This differs slightly from the results for N-terminally substituted analogs of μ-BuIIIB described by Kuang et al. [16], which showed a modest decrease in potency against NaV1.3 upon Ala-replacement of Val1 (Kd = 0.71 ± 0.3 μm) [16]. The differences between NaV1.3 potencies for ddSecBuIIIB and μ-BuIIIB likely arise from greater flexibility in the N-terminus resulting from removal of the stabilizing effects of the Cys6-Cys23 bridge. In further support of this, previous NMR studies showed that the conformation of the N-terminus of μ-BuIIIB could be further constrained through replacement of Gly2 with D-Ala, resulting in increased NaV1.3 potency above that of wild-type μ-BuIIIB [16]. Similar improvement to NaV1.3 blockade was not, however, seen with the [G2DA] replacement on the ddSecBuIIIB background, where the results more closely reflected those of unmodified ddSecBuIIIB (data not shown).

In addition to the N-terminal extension, another distinguishing feature of μ-BuIIIB, as well as other μ-conotoxins from C. bullatus described to date (μ-BuIIIA and μ-BuIIIC), is an extended inter-cysteine loop between the second and third Cys ranging between five and eight residues (Fig. 1 and Table 3). This loop includes a characteristic Lys10-Arg11 dipeptide that is not observed in the other neuronal NaV1-preferring μ-conotoxins. μ-SmIIIA also possesses a basic dipeptide at the equivalent position (Arg7-Arg8) and exhibits potent blockade of NaV1.3, which may make loop 1 of interest for designing subtype-preferring peptide analogs [9]. Additionally, there appear to be differences in both the contributions and distribution of residues that confer mild to moderate effects on NaV1 inhibition within this region. Where previous studies of μ-conotoxins showed little to no effect of Ala-substitution of residues near the N-terminus, μ-BuIIIB appears more sensitive to modification in this region. This is illustrated by an increased number of residues for which a three- to five-fold decrease in NaV1 potency was observed after Ala-replacement (Table 3).

Table 3. Contributions of individual amino acids to activity in μ-conotoxins possessing three disulfide bridgesThumbnail image of

The structural basis for μ-conotoxin blockade of NaV1 channels has been investigated extensively, and models of these interactions have been constructed [22, 27, 28]. Because of the large amount of available SAR data, efforts have focused primarily on interactions between μ-conotoxin GIIIA and NaV1.4. The selectivity of μ-conotoxins for NaV1.3 or other NaV1 subtypes is most likely caused by interactions with the turret region (Fig. 5C), for which there are no similar sequences in databases, making modeling a challenging task (S. Kuyucak, personal communication).

Crystal structures of bacterial sodium channels have been reported recently [29, 30]. Unfortunately, the lack of tetrameric symmetry, coupled with differences in amino acid composition and selectivity filter loop size of the bacterial channels, have limited their use as proxies for mammalian NaV1 subtypes. Until accurate models of the mammalian channel are developed, μ-conotoxin analogs such as those described in the present study will prove useful as molecular tools for testing and refining models of NaV1.3.

The ddSec-strategy has proven valuable for facilitating the rapid identification of amino acid residues critical for NaV1.3 blockade, as well as those amenable to modification for potential improved pharmacological activity. It is also likely that the strategy employed in the present study will be more broadly applicable to other disulfide-rich peptides for which inefficient oxidative folding precludes detailed SAR studies.

Experimental procedures

Ethics statement

The use of animals in this study followed protocols approved by the University of Utah's Animal Care and Use Committee that conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Peptide synthesis

All analogs were synthesized at a 30-μmol scale using an Apex 396 automated peptide synthesizer (AAPPTec, Louisville, KY, USA) employing standard Fmoc-protocols. Peptides were constructed using pre-loaded Fmoc-Cys(Trt) Rink Amide MBHA resin (Peptides International, Louisville, KY, USA) (substitution = 0.32 meq·g−1). Fmoc-removal was accomplished with 20 min of deprotection using 20% (v/v) piperidine in dimethylformamide. Standard amino acids were purchased from AAPPTec and used without further purification. Fmoc-L-Sec(pMeOBzl)-OH was purchased from Chem-Impex International, Inc. (Wood Dale, IL, USA). Amino acid coupling was accomplished using one equivalent of 0.22 m benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate and two equivalents 2 m diisopropylethyl amine in N-methyl-2-pyrrolidone. Standard amino acids were coupled for 60 min in 10-fold excess and Fmoc-selenocysteine was coupled for 90 min in three-fold excess. Upon completion of peptide synthesis, crude peptides were cleaved from solid-support resin by 5 h of treatment with enriched reagent K [trifluoroacetic acid (TFA)/thioanisole/phenol/nH2O (90 : 2.5 : 7.5 : 2.5, v/v/v/v)] containing 1.3 equivalents of DTNP. DTNP was added to the mixture to facilitate removal of Mob-protecting groups from selenocysteine side-chains. Crude peptides were removed from resin by vacuum filtration and were precipitated overnight at −20 °C in chilled methyl-tert butyl ether. Precipitate was washed repeatedly with chilled methyl-tert butyl ether. 5-thionitropyridyl derivatives were removed from Sec side chains, with concomitant diselenide-bridge formation by 2 h of treatment with 50 mm dithiothreitol, 0.1 m Tris-HCl and 1 mm EDTA (pH 7.5) at room temperature.

RP-HPLC purification and oxidative folding

Crude peptide analogs containing the pre-formed diselenide bridge were purified, in parallel, by solid phase extraction using a Supelco Visiprep vacuum manifold (Supelco, Bellefonte, PA, USA). Briefly, 3-mL Supelco Discovery DSC-18 solid phase extraction columns were conditioned with five column volumes of solvent B90 [90% (v/v) acetonitrile (ACN); 10% (v/v) H2O; 0.1% (v/v) TFA] followed by equilibration with five column volumes of solvent A [0.1% (v/v) TFA; nH2O]. A gradient of increasing organic solvent concentration was applied to each column ranging from 5 to 35% solvent B90, and flow-through was checked for purity by analytical RP-HPLC equipped with a C18 column (catalog number 218TP54; Vydac, Hesperia, CA, USA) over a linear gradient ranging from 10 to 50% solvent B90 with 10 min of pre-equilibration at the initial conditions. Clean fractions were pooled and final yields were calculated by UV absorbance at 280 nm.

Formation of the Cys13-Cys24 disulfide bridge was performed in parallel using a 48 m equivalent excess (one equivalent = 2.768 μg resin·nmol peptide−1·number of disulfides oxidized−1) of the solid-support oxidant CLEAR-Ox® (Peptides International, Inc., Louisville, KY, USA). Resin was prepared by first swelling in 500 μL of dichloromethane for 30 min at room temperature. Excess dichloromethane was removed by vacuum filtration and the resin was washed once with 500 μL of each of: dimethylformamide, MeOH, 50% (v/v) ACN in H2O and 0.05 m Tris-HCl; 75% (v/v) ACN (pH 8.7). Peptide was dissolved in 0.05 m Tris-HCl; 75% (v/v) ACN (pH 8.7) to a final concentration of 3 mm and added directly to CLEAR-Ox® resin. Oxidation was allowed to proceed for 2 h at room temperature, after which folding was quenched by 100-fold dilution with solvent A. Folded analogs were purified by preparative RP-HPLC over a linear gradient ranging from 10 to 50% solvent B90 over 40 min with a 10-min pre-equilibration at the initial conditions. The folded peptide analogs were quantified by UV absorbance (λ = 280 nm). Molecular masses of the folded disulfide-depleted, diselenide-containing peptides were confirmed by ESI-MS (Table 1).

Two-electrode voltage clamp electrophysiology

Xenopus laevis oocytes were injected either with 2.5 ng·μL−1 NaV1.3 or 94 ng·μL−1 NaV1.4 cRNA. Although μ-conopeptide activity can be sensitive to co-expression of NaVβ-subunits [31], neither NaV1 was co-expressed with any NaVβ-subunits so that the results could be compared directly with those obtained previously [9]. Two-electrode voltage clamping was performed using microelectrodes containing 3 m KCl (< 0.5 mΩ). Sodium currents were acquired using a holding potential of −80 mV and stepping to 0 mV for 50 ms every 20 s. Peptides were applied to oocytes under static bath conditions. Off-rates (koff) were calculated from single exponential fits of the time course of recovery from block after washout. On-rates (kon) were calculated by linear regression of the slopes of plots of kobs versus peptide concentration. Dissociation constants (Kd) were determined from the ratio of koff/kon. Experiments were all conducted in triplicate (n = 3 oocytes) for each data point at room temperature.

NMR spectroscopy

Peptide samples were dried by lyophilization and dissolved to a final concentration of 500 μm (ddSecBuIIIB) or 80 μm (μ-BuIIIB) in 95% (v/v) H2O, 5% (v/v) 2H2O, and the pH was adjusted to 3.0. All spectra were acquired on a Bruker 600 MHz NMR spectrometer (Bruker Instruments, Inc., Bellerica, MA, USA). For 1D 1H NMR the water signal was suppressed using excitation sculpting [32, 33]. Experimental conditions were established through a series of temperature dependence and pH titration experiments, which showed better dispersion at elevated temperature and acidic pH (Fig. 3). Chemical shifts were referenced to water. 2D 1H NMR spectra were acquired with a mixing time of 200 ms (NOESY) and spin-lock times of 30 and 70 ms (TOCSY). Data were processed with nmr pipe ( and 1H chemical shifts were determined by standard sequential assignment of the NOESY and TOCSY spectra in ccpnmr analysis [34].


This project was supported by the National Institutes of Health Grant GM 48677. We thank Dr Joanna Gajewiak for critically reviewing the manuscript and for numerous discussions integral to the success of this work. RSN acknowledges fellowship support from the National Health and Medical Research Council of Australia.

Author Contribution

BRG, GB and RSN designed experiments. BRG performed the peptide synthesis and NMR analyses. MMZ and MJW performed the electrophysiology assays and DJ assisted with their analysis. SC and SDR assisted with acquisition and analysis of NMR spectra. AR assisted in the preparation of disulfide-deficient analogs. BMO, GB and RSN conceived the study and BRG and RSN wrote the paper with input from all authors.