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μ-Conotoxins are components of cone snail venom, wellknown 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' CysICysIV, 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, CysIIICysV), 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. [ABSTRACT FROM AUTHOR]

The role of voltage-gated sodium (Na V) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na V 1.8 and Na V 1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na V 1.8 and mouse Na V 1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na V isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na V 1.8 and Na V 1.9, and lacked analgesic efficacy in a murine model of inflammatory pain. [ABSTRACT FROM AUTHOR]

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx- IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (Na V) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following overwhelming evidence that Na V channel subtypes Na V 1.7–Na V 1.9 are involved in nociception. More recently, Na V 1.1, Na V 1.3 and Na V 1.6 have also been identified to be involved in pain pathways. Venom-derived disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads. However, few peptide-based leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drug candidates by downsizing these larger venom-derived Na V inhibitors into smaller, more "drug-like" molecules. Here, we use molecular engineering of small cyclic peptides to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across Na V subtypes. We designed a series of small, stable and novel Na V probes displaying Na V subtype selectivity and potency in vitro coupled with potent in vivo analgesic activity, involving yet to be elucidated analgesic pathways in addition to Na V subtype modulation. [ABSTRACT FROM AUTHOR]