Discovery and mode of action of a novel analgesic β-toxin from the African spider Ceratogyrus darlingi

摘要

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 Na_v1.7 (IC₅₀ 16 nM) and produced a 29 mV depolarising shift in Na_v1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Na_v1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Ca_v2.2 inactivation gating, associated with the Ca_v2.2 α-subunit pore, while altering the activation gating of Na_v1.7. Cd1a was inactive at some of the Na_v and Ca_v 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.