Cobatoxin 1 from Centruroides noxius scorpion venom: chemical synthesis, three-dimensional structure in solution, pharmacology and docking on K+ channels

Abstract

CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K⁺ channel subtypes (voltage-gated and Ca²⁺-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys³-Cys²², Cys⁸-Cys²⁷ and Cys¹²-Cys²⁹. The 3-D structure of CoTX1 (solved by ¹H-NMR) showed that it folds according to the common α/β scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD₅₀ value of 0.5 µg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca²⁺-activated (IKCa1) K⁺ channels showed potent inhibition of currents from rat K_v1.2 (K_d value of 27 nM). CoTX1 also weakly competed with ¹²⁵I-labelled apamin for binding to SKCa channels (small-conductance Ca²⁺-activated K⁺ channels) on rat brain synaptosomes (IC₅₀ value of 7.2 µM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg⁶ or Lys¹⁰, Arg¹⁴, Arg¹⁸, Lys²¹ (dyad), Ile²³, Asn²⁴, Lys²⁸ and Tyr³⁰ (dyad) residues of CoTX1 in its interaction with the rat K_v1.2 channel. In addition, a [Pro⁷,Gln⁹]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K_v channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K⁺ channels.