Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the alpha(2)-adrenoceptors

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

EXPERIMENTAL APPROACH In binding experiments with 3H-rauwolscine, we studied the interactions of green mamba venom fractions with alpha(2)-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, rho-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human alpha(2)-adrenoceptors expressed in mammalian cells.

KEY RESULTS rho-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of 3H-rauwolscine binding to the three alpha(2)-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on alpha(2A)-adrenoceptor demonstrated that rho-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA(2) values of 5.93 and 5.32 for yohimbine and rho-Da1b, respectively.

CONCLUSIONS AND IMPLICATIONS rho-Da1b is the first toxin identified to specifically interact with alpha(2)-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of alpha(2)-adrenoceptor subtypes.