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

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 Ca_v2.2 (IC₅₀ 2.6 μM) but not Ca_v1.3 or Ca_v3.1 (IC₅₀ > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Ca_v2.2 with similar potency (IC₅₀ 3 μM) without influencing the voltage dependence of Ca_v2.2 activation gating, suggesting that Cd1a doesn’t act on Ca_v2.2 as a classical gating modifier toxin. The Cd1a binding site on Ca_v2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Ca_v2.2mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Na_v1.1–1.2 and Na_v1.7–1.8 (IC₅₀ 0.1–6.9 μM) but not Na_v1.3–1.6 (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.