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. 2020 Nov:181:114082.
doi: 10.1016/j.bcp.2020.114082. Epub 2020 Jun 7.

Pharmacological activity and NMR solution structure of the leech peptide HSTX-I

Kirsten L McMahon  1 , Bryan Tay  1 , Jennifer R Deuis  1 , Brian S Tanaka  2 , Steve Peigneur  3 , Ai-Hua Jin  1 , Jan Tytgat  3 , Stephen G Waxman  2 , Sulayman D Dib-Hajj  2 , Irina Vetter  4 , Christina I Schroeder  5
Affiliations

Pharmacological activity and NMR solution structure of the leech peptide HSTX-I

Kirsten L McMahon et al. Biochem Pharmacol. 2020 Nov.

Abstract

The role of voltage-gated sodium (NaV) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms NaV1.8 and NaV1.9, 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 NaV1.8 and mouse NaV1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant NaV 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 NaV1.8 and NaV1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.

Keywords: Chronic pain; Electrophysiology; Leech; NMR; Voltage-gated sodium channels.

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Conflict of interest statement

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.
Synthesis and oxidation of HSTX-I. A Schematic of HSTX-I oxidation protocol. B RP-HPLC and ESI-LCMS spectra (insert) after first cysteines were deprotected and oxidised (left) and RP-HPLC and ESI-LCMS spectra after removal of Acm protecting group (Mw 72), second cysteines oxidised and peptide purified (right). C 1D 1H NMR spectra of HSTX-I shows well dispersed peaks in the amide region.
Fig. 2.
Fig. 2.
HSTX-I inhibition of human NaV currents. A HSTX-I (90 μM) did not reduce peak currents in NaV1.7 expressing HEK93 cells, but B reduced peak current by a mean 20% in NaV1.8 expressing CHO cells at a concentration of 90 μM. C Voltage-clamp traces at NaV1.9 for baseline (black), HSTX-I (10 μM; red) and washout (green) periods. SCG neurons were held at −100 mV and stepped to −45 mV for 50 ms from a conditioning pulse of −120 mV for 1 sec. Depolarizing pulses were applied every 30 s until stable current responses were achieved. D NaV1.9 currents are blocked by HSTX-I at the 10 μM concentration by 19.3 ± 3.0% (n = 4), *P < 0.05 compared to baseline. E Stable NaV1.9 current during mock treatment with vehicle alone over 15 min period. F Superimposed current traces for the indicated Nav isoforms before (black) and after application of 20 μM HSTX-I (blue). Currents were elicited by depolarizing pulses to 0 mV. The asterisk indicates the peak current level in the presence of toxin; the dotted line indicates 0 current level.
Fig. 3.
Fig. 3.
Effect of HSTX-I in a mouse model of carrageenan-induced inflammation. A Mechanical threshold of wild type and NaV1.9−/− mice following intraplantar injection of carrageenan. B Effect of HSTX-I (10 mg/kg i.p) and oxycodone (1 mg/kg i.p) on mechanical thresholds following intraplantar injection of carrageenan C Heat thresholds of WT and NaV1.9−/− mice following intraplantar injection of carrageenan. D Effect of HSTX-I (10 mg/kg i.p) and oxycodone (1 mg/kg i.p) on heat thresholds following intraplantar injection of carrageenan. Data are presented as mean ± SEM. Statistical significance was determined using unpaired t-test or one-way ANOVA with Dunnett’s post-test as appropriate, *P < 0.05 compared to wild type or control.
Fig. 4.
Fig. 4.
NMR structural determination of HSTX-I. A Secondary Hα chemical shift deviations from random coil values calculated from 2D TOCSY NMR spectra. The structure was predicted to have an α-helix between residues A1–C8 (red helix) and two β-sheets between residues A10–C14 and I18–P21 (blue arrows). B Representation of the secondary structure antiparallel β-sheets in HSTX-I. Double headed arrows indicated observed NOEs and dashed lines represent putative hydrogen bonds and single-headed arrows represents a type I β-turn. Summary of local and medium range NMR data of HSTX-I with bar width representing the strength of the NOE. Open circles indicate slow exchange NH protons (> 4h). The α-helix and β-sheet regions are denoted by curved line and arrows, respectively. C The 20 lowest energy conformations of NMR solution structure as analysed by MOLMOL superimposed across C2 and C19. D The final 3D structure of HSTX-I following refinement in CNS is composed of a single α-helix between residues Y5–G9 (red helix) and two antiparallel β-sheet F13–I15 and I18–V20 (blue arrows), stabilised by two disulfide bridges (yellow).
Fig. 5.
Fig. 5.
Effect of HSTX-I against TRPA1. A NMR solution structure of HSTX-I compared to ProTx-I highlight structural similarities including alignment of β-sheets and disulfide bond (sticks) B 50 μM HSTX-I (blue circles) induces a delayed response compared to the DMSO buffer control (red squares) on TRPA1-expressing cells following 300 μM AITC-mediated calcium influx.
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