AdTx1

ρ-Da1a toxin from eastern green mamba (Dendroaspis angusticeps) venom is a polypeptide of 65 amino acids with a strong affinity for the G-protein-coupled α(1A)-adrenoceptor. This neurotoxin has been crystallized from resolubilized lyophilized powder, but the best crystals grew spontaneously during lyophilization. The crystals belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 37.37, c = 66.05 Å, and diffracted to 1.95 Å resolution. The structure solved by molecular replacement showed strong similarities to green mamba muscarinic toxins.

Background and purpose:ρ-Da1a, a 65 amino-acid peptide, has subnanomolar affinity and high selectivity for the human α(1) (A) -adrenoceptor subtype. The purpose of this study was to characterize the pharmacological effects of ρ-Da1a on prostatic function, both in vivo and in vitro.Experimental approach:ρ-Da1a was tested as an antagonist of adrenaline-induced effects on COS cells transfected with the human α(1) (A) -adrenoceptor as well as on human isolated prostatic adenoma obtained from patients suffering from benign prostatic hyperplasia. Moreover, we compared the effects of ρ-Da1a and tamsulosin on phenylephrine (PHE)-induced increases in intra-urethral (IUP) and arterial pressures (AP) in anaesthetized rats, following i.v. or p.o. administration.Key results:On COS cells expressing human α(1) (A) -adrenoceptors and on human prostatic strips, ρ-Da1a inhibited adrenaline- and noradrenaline-induced effects. In anaesthetized rats, ρ-Da1a and tamsulosin administered i.v. 30 min before PHE significantly antagonized the effects of PHE on IUP. The pK(B) values for tamsulosin and ρ-Da1a for this effect were similar. With regards to AP, ρ-Da1a only reduced the effect of PHE on AP at the lowest dose tested (10 μg·kg(-1) ), whereas tamsulosin significantly reduced PHE effects at doses between 10 and 150 μg·kg(-1) .Conclusions and implications:ρ-Da1a exhibited a relevant effect on IUP and a small effect on AP. In contrast, tamsulosin antagonized the effects of PHE on both IUP and AP. We conclude that ρ-Da1a is more uroselective than tamsulosin. ρ-Da1a is the most selective peptidic antagonist for α(1A) -adenoceptors identified to date and could be a new treatment for various urological diseases.

Background and purpose:Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs.Experimental approach:We studied the interactions of mamba venom fractions with alpha(1)-adrenoceptors in binding experiments with (3)H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle.Key results:AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K(i)= 0.35 nM) and high specificity for the human alpha(1A)-adrenoceptor subtype. We showed high selectivity and affinity (K(d)= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k(on)= 6 x 10(6).M(-1).min(-1)) with an unusually stable alpha(1A)-adrenoceptor/AdTx1 complex (t(1/2diss)= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM.Conclusions and implications:AdTx1 is the most specific and selective peptide inhibitor for the alpha(1A)-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.