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Design, synthesis, and mechanism of action of novel μ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Na_v1.7.

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.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC10074208 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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

Zhao Zitong Z Pan Teng T Chen Shen S Harvey Peta J PJ Zhang Jinghui J Li Xiao X Yang Mengke M Huang Linhong L Wang Shoushi S Craik David J DJ Jiang Tao T Yu Rilei R

The Journal of biological chemistry 20230224 4

μ-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Nav isoforms, including Nav1.7, 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 patt ...[more]

PMID: 36842500

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Project description: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 collision-induced dissociation 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 Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance 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 μ-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and 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 of further expanding the snail's repertoire of active peptides.

Project description:The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.

Dataset Information

Design, synthesis, and mechanism of action of novel μ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Na_v1.7.

Publications

Design, synthesis, and mechanism of action of novel μ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Na<sub>v</sub>1.7.

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