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Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxin KIIIA disulfide isomers

Open AccessPublished:February 12, 2022DOI:https://doi.org/10.1016/j.jbc.2022.101728
      μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (NaV) subtypes, including NaV1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a ‘native’ CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at NaV1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human NaV channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits NaV subtypes with a rank order of potency of NaV1.4 > 1.2 > 1.7 and Isomer 2 in the order of NaV1.4≈1.2 > 1.7, while the native isomer inhibited NaV1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNaV1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant NaV subtypes.

      Keywords

      Abbreviations:

      Acm (acetamidomethyl), ACN (acetonitrile), Fmoc (9-fluorenylmethoxycarbonyl), Mebzl (4-methylbenzyl), Mob (4-methoxybenzyl), Msbh (4,4′-dimethylsulfinylbenzhydryl), NaV (voltage-gated sodium channel), PDB (Protein data bank), RP-HPLC (reverse-phase HPLC), Trt (S-triphenylmethyl)
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      Conotoxins from the venom of marine cone snails are considered prospective drug leads because of their potency, NaV subtype selectivity, and analgesic efficacy in animal and human trials (
      • Dib-Hajj S.D.
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      ). The μ-conotoxin super family comprises five branches, termed M-1 to M-5, based on the number of residues in the third cystine loop between the fourth and fifth cysteine residues (
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      ). With a framework including six cysteine residues, the μ-conotoxins can theoretically form 15 different disulfide bond isomers. However, μ-conotoxins isolated directly from cone snail venom are typically found to adopt a CysI-CysIV, CysII-CysV, CysIII-CysVI conformation, which has accordingly been accepted as the ‘native’ fold of the μ-conotoxin family (
      • Corpuz G.P.
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      ). However, if isolated peptide material is not available for coelution with synthetic material, for example, when peptide sequences are identified from cDNA venom duct libraries, using proteomics or transcriptomics, or when the peptide is not present, or expressed at very low levels in the venom, it can be challenging to ascertain the native fold of the peptide. Accordingly, some recent reports have shown that it is not always the ‘native’ conotoxin peptide fold that is the more potent biologically active peptide, and conotoxins with non-native disulfide bond connectivity can outperform the presumed ‘native’ counterpart (
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      ,
      • Tietze A.A.
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      ,
      • Dutton J.L.
      • Bansal P.S.
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      ). For example, synthetic μ-PIIIA, a μ-conotoxin identified from the cDNA of the venom duct of the cone Conus purpurascens (
      • Shon K.-J.
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      • Yoshikami D.
      μ-Conotoxin PIIIA, a new peptide for discriminating among tetrodotoxin-sensitive Na channel subtypes.
      ), formed three active isomers under thermodynamic oxidative folding conditions with the non-native isomer of PIIIA (CysI-CysV, CysII-CysVI, CysIII-CysIV) being slightly more potent than the PIIIA isomer comprising the native μ-conotoxin disulfide-bond arrangement (
      • Tietze A.A.
      • Tietze D.
      • Ohlenschlager O.
      • Leipold E.
      • Ullrich F.
      • Kuhl T.
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      • Buntkowsky G.
      • Gorlach M.
      • Heinemann S.H.
      • Imhof D.
      Structurally diverse μ-conotoxin PIIIA isomers block sodium channel NaV 1.4.
      ). A similar peculiarity has been observed within the α-conotoxin family, where a non-native disulfide bond isomer of the 15-residue α-conotoxin AuIB (comprising two disulfide bonds) was 10-fold more potent than the native fold of the peptide (
      • Dutton J.L.
      • Bansal P.S.
      • Hogg R.C.
      • Adams D.J.
      • Alewood P.F.
      • Craik D.J.
      A new level of conotoxin diversity, a non-native disulfide bond connectivity in α-conotoxin AuIB reduces structural definition but increases biological activity.
      ). Discoveries such as these emphasize the importance, but also the potential, of further investigation into the activity of different disulfide bond connectivities of these small, highly disulfide-rich peptides and how different disulfide connectivities of the same peptide can influence not only potency but also receptor subtype selectivity. Herein, we focus our investigation on three different disulfide-bond isomers of μ-KIIIA, one of the most extensively investigated μ-conotoxins targeting NaV1.7 (
      • Wilson M.J.
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      • Azam L.
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      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
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      • Adams D.J.
      Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics?.
      ).
      KIIIA is a 16-residue μ-conotoxin, identified from a cDNA venom duct library of Conus kinoshitai that acts by blocking the pore of NaVs (
      • Zhang M.M.
      • Han T.S.
      • Olivera B.M.
      • Bulaj G.
      • Yoshikami D.
      μ-Conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.
      ,
      • Bulaj G.
      • West P.J.
      • Garrett J.E.
      • Watkins M.
      • Zhang M.M.
      • Norton R.S.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.
      ,
      • Stevens M.
      • Peigneur S.
      • Tytgat J.
      Neurotoxins and their binding areas on voltage-gated sodium channels.
      ,
      • King G.F.
      • Escoubas P.
      • Nicholson G.M.
      Peptide toxins that selectively target insect NaV and CV channels.
      ). KIIIA is the smallest μ-conotoxin identified to date (
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
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      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ), comprising only one residue in loop 1 of the peptide, compared to 4 to 6 for other μ-conotoxins (Table 1). Like other μ-conotoxins in the family, KIIIA has been reported to most potently inhibit rat NaV1.2 and rat NaV1.4 (
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • Knapp O.
      • McArthur J.R.
      • Adams D.J.
      Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics?.
      ,
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ). However, unlike most μ-conotoxins, KIIIA also inhibits the peripheral analgesic target NaV1.7 with nanomolar affinity (
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • Knapp O.
      • McArthur J.R.
      • Adams D.J.
      Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics?.
      ). Upon initial synthetic production, KIIIA was reported to form one major isomer during thermodynamic folding (
      • Bulaj G.
      • West P.J.
      • Garrett J.E.
      • Watkins M.
      • Zhang M.M.
      • Norton R.S.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.
      ), which was assumed to comprise the native μ-conotoxin disulfide connectivity (CysI-CysIV, CysII-CysV, CysIII-CysVI). A later study by Khoo et al. (
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ) reported two isomers during thermodynamic folding, and by carrying out direct mass spectrometric collision-induced dissociation fragmentation, it was identified that in fact neither of the two isomers observed during thermodynamic folding were KIIIA comprising the native μ-conotoxin fold (
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ). Instead, the two synthetically produced KIIIA isomers comprised CysI-CysV, CysII-CysIV, CysIII-CysVI connectivity (thermodynamic Isomer 1, referred to as Isomer 1-T) as the primary peak and CysI-CysVI, CysII-CysIV, CysIII-CysV connectivity (thermodynamic Isomer 2, referred to as Isomer 2-T) as the minor peak, with the minor isomer reported to be five times less potent than Isomer 1 against rNaV1.2 (
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ). Indeed, as of yet, there has been no studies on KIIIA comprising the native μ-conotoxin disulfide connectivity of CysI-CysIV, CysII-CysV, CysIII-CysVI.
      Table 1Sequences of peptides belonging to the μ-conotoxin family
      μ-conotoxinsSequencesReferences
      (
      • Bulaj G.
      • West P.J.
      • Garrett J.E.
      • Watkins M.
      • Zhang M.M.
      • Norton R.S.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.
      )
      (
      • Bulaj G.
      • West P.J.
      • Garrett J.E.
      • Watkins M.
      • Zhang M.M.
      • Norton R.S.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.
      ,
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      )
      (
      • Bulaj G.
      • West P.J.
      • Garrett J.E.
      • Watkins M.
      • Zhang M.M.
      • Norton R.S.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.
      ,
      • Wang C.Z.
      • Zhang H.
      • Jiang H.
      • Lu W.
      • Zhao Z.Q.
      • Chi C.W.
      A novel conotoxin from Conus striatus, μ-SIIIA, selectively blocking rat tetrodotoxin-resistant sodium channels.
      ,
      • Schroeder C.I.
      • Ekberg J.
      • Nielsen K.J.
      • Adams D.
      • Loughnan M.L.
      • Thomas L.
      • Adams D.J.
      • Alewood P.F.
      • Lewis R.J.
      Neuronally μ-conotoxins from Conus striatus utilize an α-helical motif to target mammalian sodium channels.
      )
      (
      • Schroeder C.I.
      • Ekberg J.
      • Nielsen K.J.
      • Adams D.
      • Loughnan M.L.
      • Thomas L.
      • Adams D.J.
      • Alewood P.F.
      • Lewis R.J.
      Neuronally μ-conotoxins from Conus striatus utilize an α-helical motif to target mammalian sodium channels.
      )
      (
      • West P.J.
      • Bulaj G.
      • Garrett J.E.
      • Olivera B.M.
      • Yoshikami D.
      μ-conotoxin SmIIIA, a potent inhibitor of tetrodotoxin-resistant sodium channels in amphibian sympathetic and sensory neurons.
      )
      (
      • Cruz L.J.
      • Gray W.R.
      • Olivera B.M.
      • Zeikus R.D.
      • Kerr L.
      • Yoshikami D.
      • Moczydlowski E.
      Conus geographus toxins that discriminate between neuronal and muscle sodium channels.
      )
      (
      • Cruz L.J.
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      Conus geographus toxins that discriminate between neuronal and muscle sodium channels.
      )
      (
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      • Zeikus R.D.
      • Kerr L.
      • Yoshikami D.
      • Moczydlowski E.
      Conus geographus toxins that discriminate between neuronal and muscle sodium channels.
      )
      (
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      • Adams D.
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      • Thomas L.
      • Adams D.J.
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      Neuronally μ-conotoxins from Conus striatus utilize an α-helical motif to target mammalian sodium channels.
      )
      (
      • Shon K.-J.
      • Olivera B.M.
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      • Floresca C.Z.
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      • Yoshikami D.
      μ-Conotoxin PIIIA, a new peptide for discriminating among tetrodotoxin-sensitive Na channel subtypes.
      ,
      • Tietze A.A.
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      • Ullrich F.
      • Kuhl T.
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      • Gorlach M.
      • Heinemann S.H.
      • Imhof D.
      Structurally diverse μ-conotoxin PIIIA isomers block sodium channel NaV 1.4.
      )
      (
      • Holford M.
      • Zhang M.M.
      • Gowd K.H.
      • Azam L.
      • Green B.R.
      • Watkins M.
      • Ownby J.P.
      • Yoshikami D.
      • Bulaj G.
      • Olivera B.M.
      Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus.
      )
      (
      • Holford M.
      • Zhang M.M.
      • Gowd K.H.
      • Azam L.
      • Green B.R.
      • Watkins M.
      • Ownby J.P.
      • Yoshikami D.
      • Bulaj G.
      • Olivera B.M.
      Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus.
      )
      (
      • Holford M.
      • Zhang M.M.
      • Gowd K.H.
      • Azam L.
      • Green B.R.
      • Watkins M.
      • Ownby J.P.
      • Yoshikami D.
      • Bulaj G.
      • Olivera B.M.
      Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus.
      )
      (
      • Zhang M.M.
      • Fiedler B.
      • Green B.R.
      • Catlin P.
      • Watkins M.
      • Garrett J.E.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      Structural and functional diversities among μ-conotoxins targeting TTX-resistant sodium channels.
      )
      (
      • Zhang M.M.
      • Fiedler B.
      • Green B.R.
      • Catlin P.
      • Watkins M.
      • Garrett J.E.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      Structural and functional diversities among μ-conotoxins targeting TTX-resistant sodium channels.
      )
      (
      • Zhang M.M.
      • Fiedler B.
      • Green B.R.
      • Catlin P.
      • Watkins M.
      • Garrett J.E.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      Structural and functional diversities among μ-conotoxins targeting TTX-resistant sodium channels.
      )
      (
      • Zhang M.M.
      • Fiedler B.
      • Green B.R.
      • Catlin P.
      • Watkins M.
      • Garrett J.E.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      Structural and functional diversities among μ-conotoxins targeting TTX-resistant sodium channels.
      )
      (
      • McMahon K.L.
      • Tran H.N.T.
      • Deuis J.R.
      • Lewis R.J.
      • Vetter I.
      • Schroeder C.I.
      Discovery, pharmacological characterisation and NMR structure of the novel μ-conotoxin SxIIIC, a potent and irreversible NaV channel inhibitor.
      )
      ∗, C-terminal amidation; Z, pyroglutamate; O, hydroxyproline; KIIIA highlighted in gray; Cys residues highlighted in yellow.
      KIIIA and other small μ-conotoxins have been shown to exhibit analgesic properties in mouse inflammatory pain assays (
      • Zhang M.M.
      • Han T.S.
      • Olivera B.M.
      • Bulaj G.
      • Yoshikami D.
      μ-Conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.
      ,
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ) and it is believed that the size and framework of μ-conotoxins, coupled with their high affinity and unique NaV channels selectivity, make them prospective therapeutic agents suitable for the development of novel analgesic drugs (
      • Terlau H.
      • Olivera B.M.
      Conus venoms: A rich source of novel ion channel-targeted peptides.
      ,
      • Corpuz G.P.
      • Jacobsen R.B.
      • Jimenez E.C.
      • Watkins M.
      • Walker C.
      • Colledge C.
      • Garrett J.E.
      • McDougal O.
      • Li W.
      • Gray W.R.
      • Hillyard D.R.
      • Rivier J.
      • McIntosh J.M.
      • Cruz L.J.
      • Olivera B.M.
      Definition of the M-conotoxin superfamily: Characterization of novel peptides from molluscivorous Conus venoms.
      ,
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • Barton M.E.
      • White H.S.
      The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on kindling acquisition and expression.
      ,
      • Cruz L.J.
      • Kupryszewski G.
      • LeCheminant G.W.
      • Gray W.R.
      • Olivera B.M.
      • Rivier J.
      μ-Conotoxin GIIIA, a peptide ligand for muscle sodium channels: Chemical synthesis, radiolabeling, and receptor characterization.
      ,
      • Green B.R.
      • Bulaj G.
      • Norton R.S.
      Structure and function of μ-conotoxins, peptide-based sodium channel blockers with analgesic activity.
      ,
      • Kim K.
      • Seong B.L.
      Peptide amidation: Production of peptide hormones in vivo and in vitro.
      ,
      • McIntosh J.M.
      • Hasson A.
      • Spira M.E.
      • Gray W.R.
      • Li W.
      • Marsh M.
      • Hillyard D.R.
      • Olivera B.M.
      A new family of conotoxins that blocks voltage-gated sodium channels.
      ,
      • Miljanich G.P.
      Ziconotide: Neuronal calcium channel blocker for treating severe chronic pain.
      ,
      • Sharpe I.A.
      • Palant E.
      • Schroeder C.I.
      • Kaye D.M.
      • Adams D.J.
      • Alewood P.F.
      • Lewis R.J.
      Inhibition of the norepinephrine transporter by the venom peptide χ-MrIA. Site of action, Na+ dependence, and structure-activity relationship.
      ,
      • Staats P.S.
      • Yearwood T.
      • Charapata S.G.
      • Presley R.W.
      • Wallace M.S.
      • Byas-Smith M.
      • Fisher R.
      • Bryce D.A.
      • Mangieri E.A.
      • Luther R.R.
      • Mayo M.
      • McGuire D.
      • Ellis D.
      Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: A randomized controlled trial.
      ). To better understand the structure and activity of KIIIA isomers and the influence of different disulfide connectivity on NaV affinity and subtype selectivity, we chemically produced three isomers of KIIIA including Native, Isomer 1, and Isomer 2 using a regioselective oxidation strategy. Our results revealed differences in NaV subtype selectivity and potency between the three disulfide isomers that could partially, albeit not wholly, be explained by structural studies and molecular modeling.

      Results

      Synthesis

      KIIIA and analogs were assembled using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide chemistry. Unprotected KIIIA was thermodynamically oxidized in 0.1 M NH4HCO3, pH 8, 100 eq GSH, and 10 eq GSSG producing two distinct isomers (Isomer 1-T and Isomer 2-T, where T stands for thermodynamic) (Fig. 1A) that showed similar relative retention times and peak height ratios as those observed by Khoo et al. (
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ) The two thermodynamically folded isomers, Isomer 1-T and Isomer 2-T, were readily isolated by reverse-phase HPLC (RP-HPLC). To ensure correct disulfide bond-formation, regioselective synthesis of each isomer producing the desired connectivity was carried out (Isomer 1-R and Isomer 2-R, where R stands for regioselective). However, directing the disulfide connectivity of KIIIA proved challenging (Table S1 and Figs. S1–S3) and after trialing a variety of regioselective protecting group strategies including 4-methyltrityl/dimethylphosphinyl/S-acetamidomethyl (Acm), S-triphenylmethyl (Trt)/p-methoxybenzyl (Mob)/Acm, Trt/Acm/4-methylbenzyl (Mebzl), and Trt/Acm/Mob, we found that only the combination of using Cys with protecting groups Trt to form the first disulfide bond, Acm to form the second disulfide bond followed by 4,4′-dimethylsulfinylbenzhydryl (Msbh) to form the final third disulfide bond, successfully produced Native KIIIA (Table S1 and Fig. 2). Using this orthogonal cysteine-protecting group strategy, we were subsequently able to produce Isomer 1-R (CysI-CysV, CysII-CysIV, CysIII-CysVI), Isomer 2-R (CysI-CysVI, CysII-CysIV, CysIII-CysV), and Native (CysI-CysIV, CysII-CysV, CysIII-CysVI) KIIIA with ∼10% yield from crude peptides (Table S2 and Fig. S4).
      Figure thumbnail gr1
      Figure 1Analytical RP-HPLC traces showing disulfide bond isomers of KIIIA obtained through thermodynamic oxidation and isomers obtained through step-wise directed folding conditions. A, Isomer 1-T and Isomer 2-T formed in thermodynamic condition (black), directed Isomer 1-R (blue), Isomer 2-R (green), and Native (red) conformation. B, Isomer 1-T (black) coeluted with Isomer 1-R (CysI-CysV, CysII-CysIV, CysIII-CysVI) (blue). C, Isomer 2-T coeluted with both directed Isomer 2-R (CysI-CysVI, CysII-CysIV, CysIII-CysV) (green) and Native (CysI-CysIV, CysII-CysV, CysIII-CysVI) isomer (red) and was ultimately assigned as Isomer 2-R based on NMR. RP-HPLC, reverse-phase HPLC.
      Figure thumbnail gr2
      Figure 2Regioselective synthesis of Native KIIIA (CysI-CysIV, CysII-CysV, CysIII-CysVI), forming disulfide bonds in the order of CysIII-CysVI, CysII-CysV, and CysI-CysIV. A, synthetic regioselective oxidation scheme showing reaction conditions for each step. B, analytical RP-HPLC traces corresponding to the product obtained by each step of the synthesis including the linear starting peptide, following the formation of first, second, and third disulfide bond. C, observed mass fragmentation corresponding to each folding step acquired by LC-MS. ∗ denotes peptides of interest. RP-HPLC, reverse-phase HPLC.

      Assignment of disulfide bond connectivity by coelution

      Linear KIIIA formed two isomers during thermodynamic oxidation, as shown by analytical HPLC (Fig. 1A). The major peak, Isomer 1-T, coeluted with regioselectively synthesized Isomer 1-R (CysI-CysV, CysII-CysIV, CysIII-CysVI) KIIIA, confirming the results obtained by RP-HPLC and NMR (Figs. 1B and S5). In addition, the secondary Hα chemical shifts of Isomer 1-R superimposed well with chemical shifts from the structure of KIIIA published by Khoo et al. (Fig. S5B) (
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
      • Zhang M.M.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ), confirming that Isomer 1-T corresponds to the previously described structure with connectivity of CysI-CysV, CysII-CysIV, CysIII-CysVI.
      In our hands, the minor peak obtained from thermodynamic folding, Isomer 2-T, coeluted with both Isomer 2-R (CysI-CysVI, CysII-CysIV, CysIII-CysV) and Native KIIIA (CysI-CysIV, CysII-CysV, CysIII-CysVI) produced regioselectively (Fig. 1C). However, using NMR, we could delineate the differences in their structures via 1D 1H NMR spectra and secondary Hα chemical shifts (Fig. S5). 1D 1H NMR spectra showed good dispersion of peaks in the order of Isomer 1 > Isomer 2 > Native. Isomer 1 was found to produce the highest quality spectrum with sharp peaks. Compared to Isomer 2, the Native isomer NMR spectra were not of similar sharpness and dispersion (Fig. S5A).

      Activity and selectivity of KIIIA isomers at human NaV channels

      Evaluation of inhibitory activity and subtype selectivity of the three KIIIA isomers across hNaV1.2, hNaV1.4, and hNaV1.7 by automated whole-cell patch-clamp electrophysiology (Fig. 3) provided insights into differences in activity displayed by the three different peptides. All three isomers inhibited hNaV1.2, hNaV1.4, and hNaV1.7 with distinct potency and subtype selectivity (Fig. 3). Across hNaV channels tested, Isomer 1 was the most potent inhibitor overall, followed by Native KIIIA and Isomer 2 (Table 2). However, the selectivity profiles of the three isomers showed preferential inhibition of NaV1.4 over NaV1.2 and NaV1.7 by Isomer 1 (NaV1.4 > NaV1.2 > NaV1.7), the Native isomer displayed selectivity for NaV1.4 > NaV1.7 ≈ NaV1.2, and Isomer 2 was approximately equipotent at NaV1.4 and NaV1.2 displaying diminished activity at NaV1.7 (NaV1.4 ≈ NaV1.2 > NaV1.7).
      Figure thumbnail gr3
      Figure 3Pharmacology of KIIIA isomers at hNaV channels. A, representative current trace before and after the addition of Isomer 1 (blue), Isomer 2 (green), and Native KIIIA (red) at hNaV1.2, hNaV1.4, and hNaV1.7. BD, concentration-response curve showing inhibitory activity of KIIIA isomers at hNav1.2, hNaV1.4, and hNaV1.7 respectively, the data are presented as mean ± SEM. E, selectivity profile of KIIIA Isomer, Isomer 2, and Native acquired using automated whole-cell patch-clamp electrophysiology on HEK293 cells overexpressing hNaV1.2, hNaV1.4, or hNaV1.7 in combination with β1-subunits. The data are presented as mean ± SD, with n = 5 cells per data point. 2-way ANOVA significance ∗ p value < 0.0332, ∗∗ p value < 0.0021, ∗∗∗ p value < 0.0002, ∗∗∗∗ p value < 0.0001. NaV, voltage-gated sodium channel.
      Table 2Activity of KIIIA isomers on hNaV1.2, hNaV1.4, and hNaV1.7 evaluated using automated electrophysiology
      Sodium channel subtypesIsomer 1Isomer 2Native KIIIA
      hNaV1.2124 ± 34 nM1371 ± 403 nM875 ± 129 nM
      hNaV1.465 ± 15 nM2051 ± 482 nM472 ± 94 nM
      hNaV1.7413 ± 71 nM5388 ± 547 nM887 ± 295 nM
      The data are presented as mean ± SD, with n = 5 cells per data point.
      Compared to Isomer 1, KIIIA Isomer 2 was more than 10-fold less active on NaV1.2 (p < 0.0002), 31-fold less potent on NaV1.4 (p < 0.0002), and 13-fold less active on hNaV1.7 (p < 0.0001) (Table 2 and Fig. 3). On the other hand, Native KIIIA was more than 7-fold less active on NaV1.2 and 10-fold less potent on NaV1.4 while only being 2-fold less active on hNaV1.7 compared to Isomer 1 (Table 2 and Fig. 3). These results demonstrate that disulfide connectivity can affect both potency and subtype selectivity and that the presumed native fold may not always be the most potent isoform at nonprey-specific pharmacological targets, or display the most desirable selectivity profile at human NaV channels.

      3D NMR structure of KIIIA isomers

      To better understand factors driving the distinct potency and selectivity of the three KIIIA isomers, we investigated the 3D structures of Isomer 1, Isomer 2, and Native KIIIA (Fig. 4, AC) using homonuclear 1H NMR. TOCSY and NOESY spectra were used to assign individual spin systems and the sequential walk (
      • Wüthrich K.
      NMR of Proteins and Nucleic Acids.
      ) in Ccpnmr (
      • Vranken W.F.
      • Boucher W.
      • Stevens T.J.
      • Fogh R.H.
      • Pajon A.
      • Llinas M.
      • Ulrich E.L.
      • Markley J.L.
      • Ionides J.
      • Laue E.D.
      The CCPN data model for NMR spectroscopy: Development of a software pipeline.
      ). Intra-, inter-, and long-range NOEs were assigned for individual peptides, and an initial 20 structures were calculated using the AUTO function in Cyana followed by refinement in a watershell in CNS (
      • Brunger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      Crystallography & NMR system: A new software suite for macromolecular structure determination.
      ). Structural statistics for the three isomers were evaluated using Molmol (
      • Koradi R.
      • Billeter M.
      • Wuthrich K.
      MOLMOL: A program for display and analysis of macromolecular structures.
      ) and MolProbity (
      • Chen V.B.
      • Arendall 3rd, W.B.
      • Headd J.J.
      • Keedy D.A.
      • Immormino R.M.
      • Kapral G.J.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MolProbity: All-atom structure validation for macromolecular crystallography.
      ) and a family of 20 structures with the lowest energy and best MolProbity scores were chosen to represent each of the peptides (Table S3). The solution structures of Native KIIIA and KIIIA Isomer 2 have been submitted to the Protein Data Bank (KIIIA Native PDB ID: 7SAV and KIIIA Isomer 2 PDB ID: 7SAW) and the BioMagnetic Resonance Bank (KIIIA Native BMRB: 30953 and KIIIA Isomer 2 BMRB: 30954). All three isomers maintained the characteristic α-helix in the central loop of the peptide. Isomer 1 exhibited a very compact structure for μ-conotoxins, with a typical α-helical turn from Ser6 to Ser13 (
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
      • Zhang M.M.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ,
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ). Native KIIIA also produced a very tight structure though its α-helix was observed to be shorter compared to Isomer 1, stretching from Ser6 to His12 (Fig. 4). Across the α-helical segment, the 3D structures of Isomer 1 and Native KIIIA were well structured with RMDS′ of 0.35 ± 0.1 Å and 0.4 ± 0.13 Å, respectively. The main difference between the two structures is noted in the N- and C-termini, which are oriented in different directions due to the constraint brought about by the cystine connectivity. Isomer 2 also displays an α-helical turn stretching from residue Ser6 to His12, though this helix (RMSD 0.54 ± 0.23 Å) exhibits more flexibility than Isomer 1 or Native, corresponding with fewer hydrogen bonds being observed across this stretch of amino acids for Isomer 2 (Table S3). Sidechain orientations residues which are responsible for interaction with the channels (
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ), such as Arg14, Ser13, His12, Asp11, Arg10 in Isomer 1 and Native KIIIA are similar, though these sidechains are orientated differently in Isomer 2 (Fig. 4, AC). Overall, these structural differences, in particular, the shorter and more flexible helical structure of Isomer 2 (four hydrogen bonds in region Ser5–His12) compared to the compact α-helical structure of Isomer 1 and Native KIIIA (six hydrogen bonds each in the Ser6–His12 region), and the resultant changes in structural flexibility and sidechain positioning, could explain the relative decrease in potency observed for Isomer 2 in this study.
      Figure thumbnail gr4
      Figure 4Three-dimensional structure of KIIIA isomers used in this study and docking studies with KIIIA-hNaV1.2 complex (Pan et al. (PDB 6J8E) (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      )). A, KIIIA Isomer 1 (PDB ID: 2XLG) (blue). B, KIIIA Isomer 2 (green) (this study). C, KIIIA Native (red) (this study). D, docked KIIIA Isomer 1 (blue) and Native KIIIA (red) compared to cryo-EM of KIIIA (Isomer 1, teal) in complex with hNaV1.2. E, docked Isomer 2 (green) of KIIIA compared to cryo-EM of KIIIA (Isomer 1, teal) in complex with hNaV1.2. F, putative interactions between KIIIA isomers and hNaV1.2 compared to cryo-EM of KIIIA (Isomer 1) in complex with hNaV1.2 (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ). NaV, voltage-gated sodium channel.

      KIIIA isomers docking with hNaV1.2

      To begin to understand how these relatively minor structural differences might contribute to the observed functional differences, we further investigated the interactions of Isomer 1, Isomer 2, and Native KIIIA with hNaV1.2 using the structure of hNaV1.2 solved in combination with KIIIA, displaying a CysI-CysV, CysII-CysIV, CysIII-CysVI disulfide bond connectivity (Isomer 1-T), published by Pan et al. (PDB 6J8E) (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ). The three isomers from this study were docked onto hNaV1.2 containing a β2-subunit using Autodock VINA (
      • Trott O.
      • Olson A.J.
      AutoDock vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.
      ) and docking models with the lowest energy were chosen for further analysis. These docking studies confirmed that our Isomer 1-R and Native KIIIA (Fig. 4D), but not Isomer 2 (Fig. 4E), superimposed well with KIIIA in the hNaV1.2 cryo-EM complex. Possible interactions between the residues of KIIIA isomers and hNaV1.2 were identified by PDBsum (
      • Laskowski R.A.
      • Jablonska J.
      • Pravda L.
      • Varekova R.S.
      • Thornton J.M.
      PDBsum: Structural summaries of PDB entries.
      ) and are summarized in Figure 4F and Fig. S6. We first compared interactions in the Lys7–Ser13 region of docked Isomer 1 and the cryo-EM complex (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ). There were multiple similarities between the two complexes, that is, Lys7 of KIIIA forms a hydrogen bond with Glu945 on hNaV1.2, Trp8 interacts via nonbonded interactions with Tyr363, Arg10 interacts with Arg922 and Asp1426, Asp11 with Arg922, His12 with Ile914, Ser915, and Asp916, and Ser13 with Asn916, validating our docking study. Furthermore, when comparing the interactions of Isomer 1, Isomer 2, and the Native conformation of KIIIA with hNaV1.2, we observed that Isomer 1 and the Native conformation KIIIA shared many interactions (Figs. 4F and S6). In contrast, Isomer 2 only shared two interactions with Isomer 1 and Native KIIIA, including His12 and Ser13 with Asn916. These differences and similarities between Native and Isomer 1/Isomer 2 could explain why Native KIIIA is more potent than Isomer 2 but less potent than Isomer 1 across the subtypes evaluated in this study. We also compared the interactions between residues in the termini of Isomer 1 and the Native KIIIA conformation with hNaV1.2. Compared to the Native conformation of KIIIA, we noticed that Isomer 1 had two and three more interactions with the channel at the N- and C-terminus, respectively. These observations could explain why Isomer 1 is slightly more active at hNaV1.2 than Native KIIIA. The results from these docking studies will need to be experimentally validated through structure-activity relationship studies of the peptide in combination with corresponding channel mutants and docking studies with NaV1.4 and NaV1.7 structures which may identify other important interactions.

      Discussion

      μ-Conotoxins, in particular KIIIA, have long been of interest as leads for novel analgesics due to their small size, chemical stability, and pharmacological profile (
      • Zhang M.M.
      • Han T.S.
      • Olivera B.M.
      • Bulaj G.
      • Yoshikami D.
      μ-Conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.
      ,
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ). Like most μ-conotoxins, KIIIA displays activity at the neuronal NaV1.2 and the skeletal muscle isoform NaV1.4, which could be associated with adverse effects. However, KIIIA is one of only a few known μ-conotoxins exhibiting nanomolar potency at hNaV1.7 (
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ,
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ,
      • McMahon K.L.
      • Tran H.N.T.
      • Deuis J.R.
      • Lewis R.J.
      • Vetter I.
      • Schroeder C.I.
      Discovery, pharmacological characterisation and NMR structure of the novel μ-conotoxin SxIIIC, a potent and irreversible NaV channel inhibitor.
      ,
      • Tran H.N.T.
      • Tran P.
      • Deuis J.R.
      • Agwa A.J.
      • Zhang A.H.
      • Vetter I.
      • Schroeder C.I.
      Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: Creating double-knotted peptides with improved sodium channel NaV1.7 inhibition.
      ,
      • Knuhtsen A.
      • Whiting R.
      • McWhinnie F.S.
      • Whitmore C.
      • Smith B.O.
      • Green A.C.
      • Timperley C.M.
      • Kinnear K.I.
      • Jamieson A.G.
      μ-Conotoxin KIIIA peptidomimetics that block human voltage-gated sodium channels.
      ). The KIIIA sequence was initially identified from a cDNA venom duct library from C. kinoshitai (
      • Bulaj G.
      • West P.J.
      • Garrett J.E.
      • Watkins M.
      • Zhang M.M.
      • Norton R.S.
      • Smith B.J.
      • Yoshikami D.
      • Olivera B.M.
      Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.
      ) and was subsequently chemically synthesized and folded thermodynamically, resulting in the formation of two disulfide bond isomers (Isomer 1; CysI-CysV, CysII-CysVI, CysIII-CysVI and Isomer 2; CysI-CysVI, CysII-CysIV, CysIII-CysV) as reported by Khoo et al. (
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ) However, it remains unclear which isomer(s) of KIIIA exist in C. kinoshitai venom (if it is present at all), what the ‘real native’ disulfide connectivity of the venom peptide is, and what machinery assists the snail with the folding of the peptide. Here, we report the regioselective synthesis of three KIIIA isomers, including for the first time KIIIA with the ‘native’ μ-conotoxins disulfide connectivity, their activity, selectivity, and their structure and interactions with human NaVs.

      Synthesis

      While the development of an extensive repertoire of Cys-protecting groups has expanded the toolbox for regioselective oxidation of complex disulfide-rich peptides (
      • Eliasen R.
      • Andresen T.L.
      • Conde-Frieboes K.W.
      Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin.
      ,
      • Wu F.
      • Mayer J.P.
      • Gelfanov V.M.
      • Liu F.
      • DiMarchi R.D.
      Synthesis of four-disulfide insulin analogs via sequential disulfide bond formation.
      ,
      • Gali H.
      • Sieckman G.L.
      • Hoffman T.J.
      • Owen N.K.
      • Mazuru D.G.
      • Forte L.R.
      • Volkert W.A.
      Chemical synthesis of Escherichia coli ST(h) analogues by regioselective disulfide bond formation: Biological evaluation of an (111)In-DOTA-Phe(19)-ST(h) analogue for specific targeting of human colon cancers.
      ,
      • Boulegue C.
      • Musiol H.-J.
      • Prasad V.
      • Moroder L.
      Synthesis of cystine-rich Pp.
      ,
      • Vetter I.
      • Dekan Z.
      • Knapp O.
      • Adams D.J.
      • Alewood P.F.
      • Lewis R.J.
      Isolation, characterization and total regioselective synthesis of the novel μO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels.
      ,
      • Jin A.H.
      • Dekan Z.
      • Smout M.J.
      • Wilson D.
      • Dutertre S.
      • Vetter I.
      • Lewis R.J.
      • Loukas A.
      • Daly N.L.
      • Alewood P.F.
      Conotoxin Φ-MiXXVIIA from the superfamily G2 employs a novel cysteine framework that mimics granulin and displays anti-apoptotic activity.
      ,
      • Deuis J.R.
      • Dekan Z.
      • Inserra M.C.
      • Lee T.H.
      • Aguilar M.I.
      • Craik D.J.
      • Lewis R.J.
      • Alewood P.F.
      • Mobli M.
      • Schroeder C.I.
      • Henriques S.T.
      • Vetter I.
      Development of a μO-conotoxin analogue with improved lipid membrane interactions and potency for the analgesic sodium channel NaV1.8.
      ,
      • Dekan Z.
      • Mobli M.
      • Pennington M.W.
      • Fung E.
      • Nemeth E.
      • Alewood P.F.
      Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.
      ), complete orthogonal production of a peptide like KIIIA, containing three disulfide bonds (37.5% overall Cys content) is still not always straightforward. Directed synthesis of up to four disulfide bonds in a cysteine-rich peptide has been accomplished by using various protecting-group schemes with either Boc (tert-butoxycarbonyl) or Fmoc chemistry (
      • Dekan Z.
      • Mobli M.
      • Pennington M.W.
      • Fung E.
      • Nemeth E.
      • Alewood P.F.
      Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.
      ); most commonly with combinations of Trt, Acm, Mebzl, Mob, Msbh, tertbutyl, or S-tertbutylthio groups (
      • Eliasen R.
      • Andresen T.L.
      • Conde-Frieboes K.W.
      Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin.
      ,
      • Wu F.
      • Mayer J.P.
      • Gelfanov V.M.
      • Liu F.
      • DiMarchi R.D.
      Synthesis of four-disulfide insulin analogs via sequential disulfide bond formation.
      ,
      • Gali H.
      • Sieckman G.L.
      • Hoffman T.J.
      • Owen N.K.
      • Mazuru D.G.
      • Forte L.R.
      • Volkert W.A.
      Chemical synthesis of Escherichia coli ST(h) analogues by regioselective disulfide bond formation: Biological evaluation of an (111)In-DOTA-Phe(19)-ST(h) analogue for specific targeting of human colon cancers.
      ,
      • Boulegue C.
      • Musiol H.-J.
      • Prasad V.
      • Moroder L.
      Synthesis of cystine-rich Pp.
      ,
      • Vetter I.
      • Dekan Z.
      • Knapp O.
      • Adams D.J.
      • Alewood P.F.
      • Lewis R.J.
      Isolation, characterization and total regioselective synthesis of the novel μO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels.
      ,
      • Jin A.H.
      • Dekan Z.
      • Smout M.J.
      • Wilson D.
      • Dutertre S.
      • Vetter I.
      • Lewis R.J.
      • Loukas A.
      • Daly N.L.
      • Alewood P.F.
      Conotoxin Φ-MiXXVIIA from the superfamily G2 employs a novel cysteine framework that mimics granulin and displays anti-apoptotic activity.
      ,
      • Deuis J.R.
      • Dekan Z.
      • Inserra M.C.
      • Lee T.H.
      • Aguilar M.I.
      • Craik D.J.
      • Lewis R.J.
      • Alewood P.F.
      • Mobli M.
      • Schroeder C.I.
      • Henriques S.T.
      • Vetter I.
      Development of a μO-conotoxin analogue with improved lipid membrane interactions and potency for the analgesic sodium channel NaV1.8.
      ,
      • Dekan Z.
      • Mobli M.
      • Pennington M.W.
      • Fung E.
      • Nemeth E.
      • Alewood P.F.
      Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.
      ). Whereas the robustness of the Trt, Acm, and Mebzl or Mob protecting groups is established, the removal of tertbutyl groups often results in the formation of side products and low yields (
      • Kluver E.
      • Schulz-Maronde S.
      • Scheid S.
      • Meyer B.
      • Forssmann W.G.
      • Adermann K.
      Structure-activity relation of human β-defensin 3: Influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.
      ,
      • Schulz A.
      • Kluver E.
      • Schulz-Maronde S.
      • Adermann K.
      Engineering disulfide bonds of the novel human β-defensins hBD-27 and hBD-28: Differences in disulfide formation and biological activity among human β-defensins.
      ,
      • Szabo I.
      • Schlosser G.
      • Hudecz F.
      • Mezo G.
      Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH3SiCl3 mixture for the synthesis of α-conotoxin GI.
      ), and the removal of S-tertbutylthio groups by reducing agents has been observed to be sequence dependent (
      • Eliasen R.
      • Andresen T.L.
      • Conde-Frieboes K.W.
      Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin.
      ,
      • Denis B.
      • Trifilieff E.
      Synthesis of palmitoyl-thioester T-cell epitopes of myelin proteolipid protein (PLP). Comparison of two thiol protecting groups (StBu and Mmt) for on-resin acylation.
      ).
      After trialing several different Cys-protecting groups following the preferential order of disulfide bond formation previously described (
      • Norton R.S.
      • Pallaghy P.K.
      The cystine knot structure of ion channel toxins and related polypeptides.
      ,
      • Pallaghy P.K.
      • Nielsen K.J.
      • Craik D.J.
      • Norton R.S.
      A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.
      ,
      • Agwa A.J.
      • Tran P.
      • Mueller A.
      • Tran H.N.T.
      • Deuis J.R.
      • Israel M.R.
      • McMahon K.L.
      • Craik D.J.
      • Vetter I.
      • Schroeder C.I.
      Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7.
      ) (Table S1), we successfully produced the three KIIIA isomers pursued in this study by regioselective chemical synthesis. By following a process described by Dekan et al. (
      • Dekan Z.
      • Mobli M.
      • Pennington M.W.
      • Fung E.
      • Nemeth E.
      • Alewood P.F.
      Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.
      ), using a combination of Trt/Acm/Msbh-protecting groups, we synthesized all three desired isomers with a yield of ∼10% calculated from the crude peptide. In general, regioselective oxidation methods are highly sequences dependent, meaning they may work for one peptide, but this may not always be transferable and work for other similar peptides.

      Activity

      μ-Conotoxins have previously been reported to mainly target NaV1.4 and NaV1.2 and only a few peptides, specifically KIIIA, SxIIIC, and CnIIIC, have been shown to target the therapeutically relevant isoform hNaV1.7 (
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ,
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ,
      • McMahon K.L.
      • Tran H.N.T.
      • Deuis J.R.
      • Lewis R.J.
      • Vetter I.
      • Schroeder C.I.
      Discovery, pharmacological characterisation and NMR structure of the novel μ-conotoxin SxIIIC, a potent and irreversible NaV channel inhibitor.
      ,
      • Tran H.N.T.
      • Tran P.
      • Deuis J.R.
      • Agwa A.J.
      • Zhang A.H.
      • Vetter I.
      • Schroeder C.I.
      Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: Creating double-knotted peptides with improved sodium channel NaV1.7 inhibition.
      ,
      • Knuhtsen A.
      • Whiting R.
      • McWhinnie F.S.
      • Whitmore C.
      • Smith B.O.
      • Green A.C.
      • Timperley C.M.
      • Kinnear K.I.
      • Jamieson A.G.
      μ-Conotoxin KIIIA peptidomimetics that block human voltage-gated sodium channels.
      ,
      • Markgraf R.
      • Leipold E.
      • Schirmeyer J.
      • Paolini-Bertrand M.
      • Hartley O.
      • Heinemann S.H.
      Mechanism and molecular basis for the sodium channel subtype specificity of μ-conopeptide CnIIIC.
      ). In this study, we therefore focused our attention on these three sodium channel subtypes in order to investigate the activity and selectivity of Isomer 1, Isomer 2, and Native KIIIA. Previous selectivity and activity of KIIIA (Isomer 1) has been reported mainly for rat (
      • Zhang M.M.
      • Han T.S.
      • Olivera B.M.
      • Bulaj G.
      • Yoshikami D.
      μ-Conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.
      ,
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
      • Zhang M.M.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ,
      • Van Der Haegen A.
      • Peigneur S.
      • Tytgat J.
      Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity.
      ,
      • Catterall W.A.
      From ionic currents to molecular mechanisms: The structure and function of voltage-gated sodium channels.
      ,
      • He B.
      • Soderlund D.M.
      Functional expression of rat NaV1.6 voltage-gated sodium channels in HEK293 cells: Modulation by the auxiliary β1 subunit.
      ) and mouse NaV subtypes (
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • Van Der Haegen A.
      • Peigneur S.
      • Tytgat J.
      Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity.
      ), as well as some human NaV isoforms expressed in Xenopus laevis oocytes, with or without β subunits (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ,
      • Knuhtsen A.
      • Whiting R.
      • McWhinnie F.S.
      • Whitmore C.
      • Smith B.O.
      • Green A.C.
      • Timperley C.M.
      • Kinnear K.I.
      • Jamieson A.G.
      μ-Conotoxin KIIIA peptidomimetics that block human voltage-gated sodium channels.
      ,
      • Van Der Haegen A.
      • Peigneur S.
      • Tytgat J.
      Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity.
      ).
      The rank order of potency at human NaV subtypes has not previously been reported, however Khoo et al. (
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
      • Zhang M.M.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ,
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ) and Wilson et al. (
      • Knapp O.
      • McArthur J.R.
      • Adams D.J.
      Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics?.
      ) observed that Isomer 1 of KIIIA inhibits NaVs with a rank order of rNaV1.2 > rNaV1.4 > mNaV1.7 ≥ rNaV1.1 > rNaV1.3 > rNaV1.5. Thus, the subtype selectivity preference for Isomer 1 of KIIIA across the human NaV subtypes tested in this study is broadly consistent with the activity reported by these previous works. Although rat and human NaV α-subunits are >95% identical at the level of amino acid sequence (
      • Goldin A.L.
      • Barchi R.L.
      • Caldwell J.H.
      • Hofmann F.
      • Howe J.R.
      • Hunter J.C.
      • Kallen R.G.
      • Mandel G.
      • Meisler M.H.
      • Netter Y.B.
      • Noda M.
      • Tamkun M.M.
      • Waxman S.G.
      • Wood J.N.
      • Catterall W.A.
      Nomenclature of voltage-gated sodium channels.
      ), species-specific activity differences have previously been reported for μ-conotoxins (
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ,
      • West P.J.
      • Bulaj G.
      • Garrett J.E.
      • Olivera B.M.
      • Yoshikami D.
      μ-conotoxin SmIIIA, a potent inhibitor of tetrodotoxin-resistant sodium channels in amphibian sympathetic and sensory neurons.
      ,
      • Keizer D.W.
      • West P.J.
      • Lee E.F.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structural basis for tetrodotoxin-resistant sodium channel binding by μ-conotoxin SmIIIA.
      ) and may account for some of the potency differences observed in this study compared to the literature.
      Furthermore, coexpression of different β-subunits in heterologous expression systems can significantly affect affinity and efficacy of toxins, including μ-conotoxins (
      • Gilchrist J.
      • Das S.
      • Van Petegem F.
      • Bosmans F.
      Crystallographic insights into sodium-channel modulation by the β4 subunit.
      ,
      • Namadurai S.
      • Yereddi N.R.
      • Cusdin F.S.
      • Huang C.L.
      • Chirgadze D.Y.
      • Jackson A.P.
      A new look at sodium channel β subunits.
      ). Specifically, the on-rates (kon) of μ-conotoxins at several NaV channel isoforms can be increased in the presence of β1 and β3 subunits, while they are decreased by β2 and β4 subunits (
      • Zhang M.M.
      • Wilson M.J.
      • Azam L.
      • Gajewiak J.
      • Rivier J.E.
      • Bulaj G.
      • Olivera B.M.
      • Yoshikami D.
      Co-expression of NaV β subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins.
      ). In contrast, all four β-subunits increase the kon of μO-conotoxin MrVIB (a gating-modifier inhibitor) at NaV1.8 channels (
      • Wilson M.J.
      • Yoshikami D.
      • Azam L.
      • Gajewiak J.
      • Olivera B.M.
      • Bulaj G.
      • Zhang M.M.
      μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.
      ). Furthermore, coexpression of β2-or β4-subunits protects tetrodotoxin-sensitive NaV1.1 to 1.7 subtypes against block by an analog of μO§-conotoxin GVIIJ (
      • Gajewiak J.
      • Azam L.
      • Imperial J.
      • Walewska A.
      • Green B.R.
      • Bandyopadhyay P.K.
      • Raghuraman S.
      • Ueberheide B.
      • Bern M.
      • Zhou H.M.
      • Minassian N.A.
      • Hagan R.H.
      • Flinspach M.
      • Liu Y.
      • Bulaj G.
      • et al.
      A disulfide tether stabilizes the block of sodium channels by the conotoxin μO section sign-GVIIJ.
      ). For KIIIA, coexpression of all rNaV isoform except NaV1.5 with β1 increased the rate of inactivation of INa (
      • Knapp O.
      • McArthur J.R.
      • Adams D.J.
      Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics?.
      ).
      Thus, to permit direct comparison of pharmacological activities of the three KIIIA isomers, we performed automated patch-clamp electrophysiology at human NaV isoforms 1.2, 1.4, and 1.7 co-expressed in HEK cells with the β1-subunit. Consistent with the principal activity of many μ-conotoxins at NaV1.4 and NaV1.2, the KIIIA isomers were potent across these two isoforms. However, Native KIIIA displayed an improved relative selectivity for NaV1.7 compared with Isomer 1 and Isomer 2 driven by a loss of activity at NaV1.4 and NaV1.2. As NaV subtypes are characterized by a high degree of sequence identity (hNaV1.4 cf. hNaV1.7: 60.33% identical; hNaV1.2 cf. hNaV1.7: 77.11% identical; hNaV1.2 cf. hNaV1.4: 63.02% identical), similar functional characteristics, and a typical overall structure (
      • Vetter I.
      • Deuis J.R.
      • Mueller A.
      • Israel M.R.
      • Starobova H.
      • Zhang A.
      • Rash L.D.
      • Mobli M.
      NaV1.7 as a pain target - from gene to pharmacology.
      ), these subtle shifts in potency and selectivity, driven by relatively modest shifts in the 3D peptide structure, remain difficult to explain. Recent advances in our understanding of the structure of NaV channels, including a cryo-EM structure of KIIIA bound to human NaV1.2/β2 (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ) are particularly valuable in this regard and were thus used for docking studies to visualize putative interactions between key residues of different isomers of KIIIA with NaV channels.

      NMR and docking studies

      Our experimentally determined Native KIIIA structure was highly similar across the α-helix part of the peptide compared to the structure of Isomer 1 (PDB ID: 2LXG) calculated by Khoo et al. (
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
      • Zhang M.M.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ,
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ), consistent with the high degree of similarity between these two isomers. Due to the small size of KIIIA, the different disulfide connectivities of the three isomers had minimal effect on backbone of the structure, apart from across the N- and the C-terminus of KIIIA Isomer 2 structure (this study). However, the direction of the sidechains differed greatly, thus likely contributing to the different potency and selectivity profiles we observed.
      Our docking studies also confirmed several critical interactions between KIIIA isomers and NaV1.2 that were reported by previous studies (
      • Zhang M.M.
      • Han T.S.
      • Olivera B.M.
      • Bulaj G.
      • Yoshikami D.
      μ-Conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.
      ,
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ,
      • Van Der Haegen A.
      • Peigneur S.
      • Tytgat J.
      Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity.
      ,
      • Zhang M.M.
      • Green B.R.
      • Catlin P.
      • Fiedler B.
      • Azam L.
      • Chadwick A.
      • Terlau H.
      • McArthur J.R.
      • French R.J.
      • Gulyas J.
      • Rivier J.E.
      • Smith B.J.
      • Norton R.S.
      • Olivera B.M.
      • Yoshikami D.
      • et al.
      Structure/function characterization of μ-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels.
      ). Overall, the most pharmacologically similar isomers (Isomer 1 and Native KIIIA) had more interactions in common compared to Isomer 2, which was also considerably less potent across all NaV subtypes studied. However, it is worth noting that these docking studies could be associated with some limitations. Firstly, both peptide NMR and the NaV1.2/β2 cryo-EM structures may not reflect the full range of physiologically relevant conformations, which could contribute to subtle differences in activity. For example, our pharmacological activity studies were conducted in HEK293 cells coexpressing the β1-subunit, while the KIIIA-hNaV1.2 cryo-EM structure was obtained in the presence of the β2-subunit (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ). Additionally, the KIIIA-NaV1.2/β2 cryo-EM structure was obtained with Biotin-(AEEA)2-KIIIA (Isomer 1) at very high peptide concentrations (25 mM) (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ). Interestingly, although several interactions identified in the cryo-EM structure could explain preferential inhibition of NaV1.2 over the tetrodotoxin-resistant isoforms NaV1.5, NaV1.8, and NaV1.9, the even greater potency of KIIIA (Isomer 1) at hNaV1.4, as well as potent activity at hNaV1.7 observed by us, cannot be fully explained by this structure given that the majority of critical interacting residues are conserved across these subtypes.
      We propose that the peptide N- and C-termini also contribute to differential interactions with NaV subtypes, in addition to the helical region of KIIIA that has previously been identified to hold key residues accounting for μ-conotoxin activity at NaV channels (
      • Zhang M.M.
      • Han T.S.
      • Olivera B.M.
      • Bulaj G.
      • Yoshikami D.
      μ-Conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.
      ,
      • McArthur J.R.
      • Singh G.
      • McMaster D.
      • Winkfein R.
      • Tieleman D.P.
      • French R.J.
      Interactions of key charged residues contributing to selective block of neuronal sodium channels by μ-conotoxin KIIIA.
      ,
      • Van Der Haegen A.
      • Peigneur S.
      • Tytgat J.
      Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity.
      ,
      • Zhang M.M.
      • Green B.R.
      • Catlin P.
      • Fiedler B.
      • Azam L.
      • Chadwick A.
      • Terlau H.
      • McArthur J.R.
      • French R.J.
      • Gulyas J.
      • Rivier J.E.
      • Smith B.J.
      • Norton R.S.
      • Olivera B.M.
      • Yoshikami D.
      • et al.
      Structure/function characterization of μ-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels.
      ). This is supported by previous work showing that both termini of KIIIA contribute to the interaction with NaV1.7 and are sensitive to alterations (
      • Tran H.N.T.
      • Tran P.
      • Deuis J.R.
      • Agwa A.J.
      • Zhang A.H.
      • Vetter I.
      • Schroeder C.I.
      Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: Creating double-knotted peptides with improved sodium channel NaV1.7 inhibition.
      ). Specifically, the amidated C-terminus has proven critical for Isomer 1 KIIIA activity at hNaV1.7, as both six amino acid C-terminal extension and deamidation resulted in complete loss of activity (
      • Tran H.N.T.
      • Tran P.
      • Deuis J.R.
      • Agwa A.J.
      • Zhang A.H.
      • Vetter I.
      • Schroeder C.I.
      Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: Creating double-knotted peptides with improved sodium channel NaV1.7 inhibition.
      ). In addition, an extension of the N-terminus of Isomer 1 KIIIA by a poly-Gly tail can either increase or decrease the potency of the peptide (
      • Khoo K.K.
      • Gupta K.
      • Green B.R.
      • Zhang M.M.
      • Watkins M.
      • Olivera B.M.
      • Balaram P.
      • Yoshikami D.
      • Bulaj G.
      • Norton R.S.
      Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
      ,
      • Tran H.N.T.
      • Tran P.
      • Deuis J.R.
      • Agwa A.J.
      • Zhang A.H.
      • Vetter I.
      • Schroeder C.I.
      Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: Creating double-knotted peptides with improved sodium channel NaV1.7 inhibition.
      ). Contribution of the termini to the biological activity of KIIIA would also be consistent with the high degree of structural similarity of the isomers in the α-helical region, although this remains to be explored in greater detail at the practical level.

      Conclusion

      We produced three different disulfide bond isomers of KIIIA using a combination of thermodynamic and step-wise regioselective protocols evaluating a range of different Cys-protecting groups and disulfide bond formation order. We found that the combination and order of Cys(Trt), Cys(Acm), and Cys(Msbh) was the only strategy employed in this study, which was able to produce the desired folded peptides. Isomer 1, Isomer 2, and Native KIIIA were distinguishable via NMR, and although all three isomers share a similar overall 3D structure, comprising an α-helical turn locating from Lys7 to His12, they displayed different potency and selectivity profiles across hNaV1.2, hNaV1.4, and hNaV1.7. Docking of the 3D NMR structures revealed a series of interactions between the different KIIIA isomers and a hNaV1.2/β2 complex that could provide an explanation for their distinct bioactivity. The docking study showed that while the α-helical turn and a series of hydrogen bonds are critical, both the N- and C-termini of KIIIA also appear to contribute to the interaction with hNaV subtypes. Further experimental validation of such interactions may lead to the rational design of potent and selective hNaV1.7 inhibitors with therapeutic potential.

      Experimental procedures

      Unless otherwise stated, all chemicals, solvents, and reagents were purchased from Sigma Aldrich (Sigma Aldrich). Amino acids were purchased from Iris Biotech GmbH.

      Peptide synthesis

      Peptides were manually assembled on Fmoc-Rink amide polystyrene resins on a 0.125 mmol scale (RAPP Polymer, 0.69 mmol/g). 9-fluorenylmethoxycarbonyl-protected amino acid couplings were performed in dimethylformamide using 4 eq of amino acid/0.5 M HCTU (O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)/N,N-diisopropylamine (1:1:1) relative to resin substitution (2 × 10 min). 9-fluorenylmethoxycarbonyl removal was accomplished by treatment with 30% piperidine/dimethylformamide (2 × 2 min).
      Cleavage from the resin and simultaneous removal of sidechain-protecting groups was accomplished by treatment with 95% TFA/2.5% triisopropylsilane/2.5% H2O for 2 h at room temperature. Following the filtration of cleavage solution, ice-cold diethyl ether was added to precipitate peptides. Crude peptides were centrifuged 3 × 5 min at 5000g, washed with diethyl ether, dissolved in 0.1% TFA/50% acetonitrile (ACN)/H2O, and lyophilized.

      Reverse-phase HPLC

      Preparative and analytical RP-HPLC Shimadzu LC-20AT systems equipped with an SPD-20A Prominence UV/VIS detector and a SIL-20AHT autoinjector were used for purification and analysis. An Eclipse XDB–C18 column (Agilent; 7 μm, 21.2 cm × 250 mm, 80 Å, flow rate 8 ml/min) was used for peptide purification. A Zobrax 300SB–C18 column (Agilent; 5 μm, 2.1 × 150 mm, 300 Å, flow rate 1 ml/min) was used to monitor oxidation and analyze the peptide purity. All samples were run from 0 to 60% B in 30 min (solvent A: 0.05% TFA and solvent B: 90% ACN/0.05% TFA). Absorbance was recorded at 214 nm and 280 nm.

      Peptide oxidative folding

      Linear KIIIA was thermodynamically folded as described previously (
      • Tran H.N.T.
      • Tran P.
      • Deuis J.R.
      • Agwa A.J.
      • Zhang A.H.
      • Vetter I.
      • Schroeder C.I.
      Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: Creating double-knotted peptides with improved sodium channel NaV1.7 inhibition.
      ). Briefly, linear unprotected KIIIA was dissolved in 0.1 M NH4HCO3, pH 8, at a concentration of 0.3 mg/ml with 100 eq GSH/10 eq GSSG and allowed to stir at room temperature for 24 h.
      General procedures for removing cysteine-protecting groups and disulfide bond formation have been previously described by Dekan et al. (Figs. 1A and S1–S3). (
      • Dekan Z.
      • Mobli M.
      • Pennington M.W.
      • Fung E.
      • Nemeth E.
      • Alewood P.F.
      Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.
      ) Briefly, dithiol-containing peptide was dissolved in AcOH (2 mg/ml) following by the dropwise addition of 1 eq of aqueous iodine (I2) in MeOH and kept stirring for 15 min to form the first disulfide bond. Subsequently, water and HCl were added to the above solution so that the final volume was 50% AcOH/50% H2O/0.1% HCl. 8 eq of I2 (dissolved in MeOH) was added to the peptide solution and stirred for 30 min to remove Acm group and form the second disulfide bond. The reactions were monitored by RP-HPLC and mass spectrometry. To stop the reaction, aqueous ascorbic acid was added to quench the I2 until the solution became colorless. The product was isolated by RP-HPLC and lyophilized. The lyophilized bis(Cys(Msbh))-containing peptide was dissolved in TFA (1 mg/ml) and cooled to 0 °C (in an ice bath). Dimethyl sulfide (1% v/v) was added to the stirred solution, followed by NaI (5 eq/sulfoxide group). The solution gradually became yellow. After 15 min, the solution was poured into an ice-cold solution of 10 mM ascorbic acid in H2O (15 × volume of TFA) and the product was isolated by RP-HPLC.

      Liquid chromatography/mass spectrometry

      A Q-Star Pulsar mass spectrometer (SCIEX) equipped with an auto-injector (Agilent Technologies Inc) was used for high-resolution mass analysis. A Zobrax 300SB – C18 column (Agilent; 3.5 μm, 2.1 × 100 mm, 300 Å, flow rate 0.3 ml/min) was used to run all samples. All runs were conducted in solvent A (0.1 % formic acid in H2O) and solvent B (aqueous ACN/0.1 % formic acid).

      Cell culture and automated patch-clamp electrophysiology

      Automated whole-cell patch-clamp electrophysiology (QPatch-16X; Sophion A/S) was used to examine the activity of KIIIA isomers at hNaV1.2, hNaV1.4, and hNaV1.7 as previously described (
      • McMahon K.L.
      • Tran H.N.T.
      • Deuis J.R.
      • Lewis R.J.
      • Vetter I.
      • Schroeder C.I.
      Discovery, pharmacological characterisation and NMR structure of the novel μ-conotoxin SxIIIC, a potent and irreversible NaV channel inhibitor.
      ). hNaV subtypes 1.2, 1.4, and 1.7 (α and β1-subunits) were stably overexpressed in HEK293 cells (SB Drug Discovery). The cells were maintained in Minimum Essential Medium Eagle supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) GlutaMAX, 0.004 mg/ml blasticidin, and 0.6 mg/ml geneticin.
      The intracellular solution contained (in mM) 140 CsF, 1 EGTA, 5 CsOH, 10 Hepes, 10 NaCl, pH 7.3 and adjusted to 320 mOsm with sucrose. The extracellular solution comprised (in mM) 2 CaCl2, 1 MgCl2, 10 Hepes, 4 KCl, 145 NaCl, pH 7.4 and adjusted to 305 mOsm with sucrose. Voltage-clamp experiments were performed in a single-hole configuration mode. KIIIA isomers were diluted in extracellular solution with 0.1% bovine serum albumin. Na+ currents were sampled at 25 kHz and filtered at 8 kHz. The same cells were exposed sequentially to multiples concentrations. Each peptide concentration was incubated for 5 min and the peak current was compared to buffer control. Concentration-response curves were acquired using a holding potential of −90 mV and a 50 ms pulse to −20 mV every 20 s (0.05 Hz). Peak current postpeptide addition (I) was normalized to buffer control (I0). IC50s were determined by plotting difference in peak current (I/I0) and log peptide concentration. Calculated IC50 were compared across subtypes and statistical differences determined by ordinary one-way ANOVA. Concentration-response curves were fitted using the log (inhibitor) versus response-variable slope (four parameters) and analyzed in Prism 8 (GraphPad Software).

      NMR and 3D structure calculation

      Peptides were dissolved in 500 μl MilliQ water (MilliPore) and 50 μl D2O (Cambridge isotopes). A Bruker 600 MHz Avance III spectrometer equipped with a cryoprobe (Bruker) was used to acquire NMR spectra, as described by Agwa et al. (
      • Agwa A.J.
      • Blomster L.V.
      • Craik D.J.
      • King G.F.
      • Schroeder C.I.
      Efficient enzymatic ligation of inhibitor cystine knot spider venom peptides: Using sortase a to form double-knottins that probe voltage-gated sodium channel NaV1.7.
      ) 1D 1H, and 2D 1H-1H TOCSY (80 ms mixing time) and 1H-1H NOESY (200 ms mixing time), natural abundance 1H−15N HSQC and D2O exchange 2D 1H-1H TOCSY and 1H−13C HSQC were collected (
      • Braunschweiler L.
      • Ernst R.R.
      Coherence transfer by isotropic mixing - application to proton correlation spectroscopy.
      ,
      • Jeener J.
      • Meier B.H.
      • Bachmann P.
      • Ernst R.R.
      Investigation of exchange processes by two-dimensional NMR spectroscopy.
      ). Spectra were processed using TopSpin 3.5 (Bruker) and CCPNMR Analysis 2.4.1 (CCPN, University of Cambridge) (
      • Wüthrich K.
      NMR of Proteins and Nucleic Acids.
      ,
      • Vranken W.F.
      • Boucher W.
      • Stevens T.J.
      • Fogh R.H.
      • Pajon A.
      • Llinas M.
      • Ulrich E.L.
      • Markley J.L.
      • Ionides J.
      • Laue E.D.
      The CCPN data model for NMR spectroscopy: Development of a software pipeline.
      ). The chemical shift of water at 4.76 ppm was used as a reference (
      • Gottlieb H.E.
      • Kotlyar V.
      • Nudelman A.
      NMR chemical shifts of common laboratory solvents as trace impurities.
      ).
      Dihedral angles were identified using TALOS-N (
      • Shen Y.
      • Bax A.
      Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks.
      ), and initial 3D structures were calculated using the AUTO and ANNEAL functions in CYANA (
      • Gottstein D.
      • Kirchner D.K.
      • Guntert P.
      Simultaneous single-structure and bundle representation of protein NMR structures in torsion angle space.
      ) followed by refinement in a watershell using CNS (
      • Brunger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      Crystallography & NMR system: A new software suite for macromolecular structure determination.
      ,
      • Brunger A.T.
      Version 1.2 of the crystallography and NMR system.
      ). Additional H-bond restraints were included derived from temperature coefficient experiments in combination with D2O exchange data (
      • Baxter N.J.
      • Williamson M.P.
      Temperature dependence of 1H chemical shifts in proteins.
      ). Fifty structures were calculated and the best 20 structures (based on energy and MolProbity scores (
      • Chen V.B.
      • Arendall 3rd, W.B.
      • Headd J.J.
      • Keedy D.A.
      • Immormino R.M.
      • Kapral G.J.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MolProbity: All-atom structure validation for macromolecular crystallography.
      )) were kept as the final 3D structure. Molmol (
      • Koradi R.
      • Billeter M.
      • Wuthrich K.
      MOLMOL: A program for display and analysis of macromolecular structures.
      ) was used for visualization and RMSD calculations.

      Docking and protein–protein interaction analysis

      Autodock VINA software (
      • Trott O.
      • Olson A.J.
      AutoDock vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.
      ) assisted by MGLTools (
      • Morris G.M.
      • Huey R.
      • Lindstrom W.
      • Sanner M.F.
      • Belew R.K.
      • Goodsell D.S.
      • Olson A.J.
      AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility.
      ) was used for molecular docking of KIIIA isomers in human NaV1.2 β2 cryo-EM structure (PDB 6J8E) (
      • Pan X.
      • Li Z.
      • Huang X.
      • Huang G.
      • Gao S.
      • Shen H.
      • Liu L.
      • Lei J.
      • Yan N.
      Molecular basis for pore blockade of human Na(+) channel NaV1.2 by the μ-conotoxin KIIIA.
      ). To define the search space of the hNaV1.2 structure, a grid box with the following dimensions: center x = 143.432, center y = 136.254, and center z = 155.036 was used. The size of the grid box for all the docking in hNaV1.2 was as follows: size x = 30, size y = 30, and size z = 30. The exhaustiveness for the search was set to 8. The lowest energy models were submitted and analyzed by PBDSum (
      • Laskowski R.A.
      • Jablonska J.
      • Pravda L.
      • Varekova R.S.
      • Thornton J.M.
      PDBsum: Structural summaries of PDB entries.
      ) for protein–protein interactions. PyMol was used for visualization.

      Data availability

      Supporting information includes orthogonal peptide oxidation, statistical analysis of NMR solution structures of Isomer 2 and Native KIIIA, regioselective oxidation, 1D and 2D 1H NMR on Isomer 1, Isomer 2, and Native KIIIA, and putative-binding interactions between NaV1.2 and Isomer 1, Isomer 2 and Native KIIIA can be found online. NMR coordinates for Isomer 2, and Native KIIIA solution structures have been submitted to the Protein Data Bank (KIIIA Isomer 2 PDB ID: 7SAW and KIIIA Native PDB ID: 7SAV) and the BioMagnetic Resonance Bank (KIIIA Isomer 2 BMRB: 30954 and KIIIA Native BMRB: 30953). All other data are included in the main article.

      Supporting information

      This article contains supporting information (
      • Khoo K.K.
      • Feng Z.P.
      • Smith B.J.
      • Zhang M.M.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      • Norton R.S.
      Structure of the analgesic μ-conotoxin KIIIA and effects on the structure and function of disulfide deletion.
      ,
      • Laskowski R.A.
      • Jablonska J.
      • Pravda L.
      • Varekova R.S.
      • Thornton J.M.
      PDBsum: Structural summaries of PDB entries.
      ,
      • Dekan Z.
      • Mobli M.
      • Pennington M.W.
      • Fung E.
      • Nemeth E.
      • Alewood P.F.
      Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.
      ,
      • Norton R.S.
      • Pallaghy P.K.
      The cystine knot structure of ion channel toxins and related polypeptides.
      ,
      • Pallaghy P.K.
      • Nielsen K.J.
      • Craik D.J.
      • Norton R.S.
      A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.
      ,
      • Agwa A.J.
      • Tran P.
      • Mueller A.
      • Tran H.N.T.
      • Deuis J.R.
      • Israel M.R.
      • McMahon K.L.
      • Craik D.J.
      • Vetter I.
      • Schroeder C.I.
      Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7.
      ,
      • Gottlieb H.E.
      • Kotlyar V.
      • Nudelman A.
      NMR chemical shifts of common laboratory solvents as trace impurities.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      This work was funded by the Australian National Health and Medical Research Council (NMHRC) through Project and Ideas Grants ( APP1080405 , 2002860 ). The authors would like to thank Mr Zoltan Dekan from the Institute for Molecular Bioscience at the University of Queensland for invaluable advice and interesting peptide synthesis discussions and assistance with HF cleavage, Assoc Prof. Johan Rosengren from the School of Biomedical Sciences at the University of Queensland for assistance with NMR, and Ms Thao Ho from the Institute for Molecular Bioscience at the University of Queensland for advice with docking studies. This research was supported [in part] by the Intramural Research Program of the National Cancer Institute , Center for Cancer Research (Grant ZIA BC 012003 ).

      Author contributions

      H. N. T. T., I. V., and C. I. S. conceptualization; H. N. T. T. and C. I. S. methodology; H. N. T. T., K. L. M., J. R. D., and I. V. investigation; H. N. T. T. data curation; H. N. T. T. formal analysis; H. N. T. T. writing–original draft; H. N. T. T., I. V., and C. I. S. writing–review and editing; I. V. and C. I. S. supervision; I. V. and C. I. S. funding acquisition; I. V. and C. I. S. project administration.

      Funding and additional information

      This work was funded by a Career Development Fellowship ( APP1162503 ) awarded to I. V. and an Early Career Fellowship ( APP1139961 ) awarded to J. R. D. C. I. S was an Australian Research Council (ARC) Future Fellow (FT160100055). Australian Government Research Training Program Scholarships supported H. N. T. T. and K. L. M. through the University of Queensland .

      Supporting information

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