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

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.

The NaV1.7 Channel Subtype as an Antinociceptive Target for Spider Toxins in Adult Dorsal Root Ganglia Neurons

“…This test has the advantages of being less invasive and more sensitive than neuropathic and inflammatory tests. Indeed, only a low amount of toxin candidate to be evaluated is necessary to relieve pain, as exemplified by GpTx-I, Cd1a, m3-HwTx-IV and Pn3a ( Deuis et al, 2016 ; Cardoso et al, 2017 ; Rahnama et al, 2017 ; Sousa et al, 2017 ). However, the question of lack of physiological relevance of this test may be raised.…”

Heterodimeric insecticidal peptide provides new insights into the molecular and functional diversity of ant venoms

“…Insecticidal activity was evaluated injection of peptides into the ventro-lateral thoracic region of sheep blowflies (Lucilia cuprina; mass 22.4-31.7 mg) as previously described [60].…”

Modulation of voltage-gated CaV2.2 Ca2+ channels by newly identified interaction partners

“…Cumulative inactivation [69]; b - [70]; c - [71]; d - [11,12]; e - [13]; f - [72]; g - [61]; h - [30,31]; i - [62]; j - [63] less prominent [19,38,39]. It has been suggested that all three putative interaction sites together form a G-protein binding pocket (GPBP) [40].…”

“… a – [ 69 ]; b – [ 70 ]; c – [ 71 ]; d – [ 11 , 12 ]; e – [ 13 ]; f – [ 72 ]; g – [ 61 ]; h – [ 30 , 31 ]; i – [ 62 ]; j – [ 63 ] …”