Small cyclic sodium channel inhibitors

Abstract

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_V1.7-Na_V1.9 are involved in nociception. More recently, Na_V1.1, Na_V1.3 and Na_V1.6 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.

Keywords: Cone snail toxin; Cyclic peptide; Nociception; Pain; Spider toxin; Voltage gated sodium channel.

Fig. 1.

Sequence alignment of PnTx1, μ-KIIIA and the previously designed peptide Pn [24]. Key residues for Na_V channel inhibition are shown in green [5,10,14,24] and cysteines are shown in blue. The asterisk indicates amidation. Orange lines (dashed and solid) signify disulfide bonds for KIIIA and Pn. Disulfide connectivity is not known for PnTx1. The sequence for PnTx1 continues after the … (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Electrophysiological characterization of PnCS1[W4K] across Na_V channel subtypes Na_V1.1–1.8. Representative whole-cell current traces in control (black) and toxin (red) conditions are shown. The dotted line indicates the zero-current level. The arrow marks steady-state current traces after application of 1 μM peptide. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Antinocicpetive effects of PnCs1, PnCS1[W4K], PnCS1[R6E] and PnCS1 [W7Y] in a mouse model of Na_V1.7 mediated nociception. Local intraplantar injection of 10 μM PnCs1 and PnCs1[R6E] partially reduced OD1 induced pain behaviours (n = 5 per group) while intraplantar injection of 100 μM of all four peptides reduced pain behaviours (n = 5 per group). Vehicle administration did not cause significant pain (1.3 ± 0.5% of OD1 control). Data are expressed as mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s post-test; *, p < 0.05; **, p < 0.01, ***, p < 0.001 compared to OD1 control.

Fig. 4.

Antinociceptive effect of (A) PnCS1, (B) PnCS1[W4K], (C) PnCS1[R6E] and (D) PnCS1[W7Y] upon intraplantar injection in mice.

Fig. 5.

Exclusion of a possible systemic effect of (A) PnCS1, (B) PnCS1[W4K], (C) PnCS1[R6E] and (D) PnCS1[W7Y].

Fig. 6.

Electrophysiology analysis of PnCS1 MAPS library on Na_V1.2, 1.4, 1.5 and 1.6. IC₅₀ were calculated for each peptide and compared to the activity of parent peptide PnCS1 (Na_V1.2: 1.0 ± 0.1 μM; Na_V1.4: 0.6 ± 0.2 μM; Na_V1.5: 2.8 ± 0.6 μM; Na_V1.6: 0.7 ± 0.2 μM). Working concentrically from the centre, segments correspond to native peptide sequence (navy), effects of Ala substitution, effects of Lys substitution, effects of Glu substitution and effects of Tyr substitution. Colours and shading represent effect of substitute on IC₅₀, equal to PnCS1 (orange plain), equal to PnCS1 and > 95% inhibition (orange chequered) and > 95% inhibition (grey chequered) and no change (grey). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)