Discovery and Mode of Action of a Novel Analgesic β-toxin From the African Spider Ceratogyrus Darlingi

PLoS One. 2017 Sep 7;12(9):e0182848. doi: 10.1371/journal.pone.0182848. eCollection 2017.

Abstract

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn't act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1-1.2 and Nav1.7-1.8 (IC50 0.1-6.9 μM) but not Nav1.3-1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

MeSH terms

  • Analgesics / chemistry
  • Analgesics / pharmacology*
  • Animals
  • Binding Sites / drug effects
  • Calcium Channels, N-Type / metabolism
  • Electrophysiology
  • Fluorometry
  • Humans
  • Mice
  • NAV1.1 Voltage-Gated Sodium Channel / metabolism
  • NAV1.7 Voltage-Gated Sodium Channel / metabolism
  • Peptides / chemistry
  • Peptides / pharmacology
  • Rats
  • Spider Venoms / chemistry*
  • Spiders / chemistry*

Substances

  • Analgesics
  • Cacna1b protein, rat
  • Calcium Channels, N-Type
  • NAV1.1 Voltage-Gated Sodium Channel
  • NAV1.7 Voltage-Gated Sodium Channel
  • Peptides
  • Spider Venoms

Grant support

We acknowledge financial support from the Australian National Health and Medical Council (NHMRC Program Grant APP1072113 to RJL, PFA and GFK). SRS was supported by a University of Queensland International Research Tuition Award (UQIRS) PhD Scholarship. RJL, PFA and GFK were supported by NHMRC Principal Research Fellowships; IV was supported by an Australian Research Council Future Fellowship; GWZ was supported by grants from the Canadian Institutes of Health Research and holds a Canada Research Chair. CB was supported by a Studentship from Alberta Innovates Health Solutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors received no financial support for the research, authorship, and/or publication of this article.