Design of Bioactive Peptides from Naturally Occurring μ-Conotoxin Structures

Stevens, Miguel; Peigneur, Steve; Dyubankova, Natalia; Lescrinier, Eveline; Herdewijn, Piet; Tytgat, Jan

doi:10.1074/jbc.m112.375733

Cited by 31 publications

(31 citation statements)

References 53 publications

(82 reference statements)

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“…There are several excellent reviews available describing the Na V channel binding sites and their interaction with toxins in detail (Catterall, 2012;Stevens et al, 2012). The amino acid residues that form the neurotoxin receptor site 1 are primarily located in the pore loop which is formed by the membrane dipping part of the connecting loop between S5 and S6 of each domain (Catterall et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms

et al. 2015

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Section: Introductionmentioning

confidence: 99%

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms

et al. 2015

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“…21,23–27 It was further supported by the observation that two-disulfide analogues of μ-KIIIA containing just the [C2-C15] and [C4-C16] bridges showed a similar structure and similar activity profile to those of μ-KIIIA itself. 22,28,29 …”

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confidence: 99%

Distinct Disulfide Isomers of μ-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels

et al. 2012

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In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for μ-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA/μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.

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“…Our study showed no significant differences between the three-disulfide bridged [Ala 7 ]KIIIA and the disulfide-depleted [Ala 1,7,9 ]KIIIA in potencies and residual currents in blocking Na v 1.2 [33], while recently described disulfide-depleted analogues of KIIIA lacking Cys 1 and Cys 9 exhibited a potent block of Na v 1.2 [31]. Interestingly, while KIIIA was previously reported to share the disulfide pattern typical of the µ-conotoxin family (i.e., 1 st Cys to 4 th Cys, 2 nd to 5 th 3 rd to 6 th ) [32, 33], the disulfide connectivity of the synthetic peptide was recently shown as: 1 st to 5 th 2 nd to 4 th 3 rd to 6 th [34, 35].…”

Section: Resultsmentioning

confidence: 63%

“…Promising in this regard is the observation that a W8E replacement in KIIIA produces a 200-fold decrease in activity against Na v 1.4 [5]. In addition, recent work of Stevens and colleagues [31] showed that chimeric analogues of KIIIA and another µ-conotoxin, BuIIIC, varied in their selectivities for different subtypes of sodium channels. Our work and the recent SAR studies on KIIIA [4, 5, 31] establish the groundwork for next generation KIIIA analogues with improved selectivity profiles and efficacies.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, recent work of Stevens and colleagues [31] showed that chimeric analogues of KIIIA and another µ-conotoxin, BuIIIC, varied in their selectivities for different subtypes of sodium channels. Our work and the recent SAR studies on KIIIA [4, 5, 31] establish the groundwork for next generation KIIIA analogues with improved selectivity profiles and efficacies. For example, exploring combinations of peptoid analogues of KIIIA in position 7 in the context of mutations at Trp 8 or Arg 14 may lead to favorable selectivity profiles for future KIIIA-derived drug leads.…”

Section: Resultsmentioning

confidence: 99%

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Expanding chemical diversity of conotoxins: Peptoid–peptide chimeras of the sodium channel blocker μ-KIIIA and its selenopeptide analogues

Walewska

Han

Zhang

et al. 2013

European Journal of Medicinal Chemistry

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The µ-conotoxin KIIIA is a three disulfide-bridged blocker of voltage-gated sodium channels (VGSCs). The Lys 7 residue in KIIIA is an attractive target for manipulating the selectivity and efficacy of this peptide. Here, we report the design and chemical synthesis of µ-conopeptoid analogues (peptomers) in which we replaced Lys 7 with peptoid monomers of increasing side-chain size: N-methylglycine, N-butylglycine and N-octylglycine. In the first series of analogues, the peptide core contained all three disulfide bridges; whereas in the second series, a disulfidedepleted selenoconopeptide core was used to simplify oxidative folding. The analogues were tested for functional activity in blocking the Na v 1.2 subtype of mammalian VGSCs exogenously expressed in Xenopus oocytes. All six analogues were active, with the N-methylglycine analogue, [Sar 7 ]KIIIA, the most potent in blocking the channels while favoring lower efficacy. Our findings demonstrate that the use of N-substituted Gly residues in conotoxins show promise as a tool to optimize their pharmacological properties as potential analgesic drug leads.

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Design of Bioactive Peptides from Naturally Occurring μ-Conotoxin Structures

Cited by 31 publications

References 53 publications

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms

Distinct Disulfide Isomers of μ-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels

Expanding chemical diversity of conotoxins: Peptoid–peptide chimeras of the sodium channel blocker μ-KIIIA and its selenopeptide analogues

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