Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the [alpha]1A-adrenoceptor

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 3H-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 (Ki= 0.35 nM) and high specificity for the human [alpha]1A-adrenoceptor subtype. We showed high selectivity and affinity (Kd= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (kon= 6 × 106·M-1·min-1) with an unusually stable [alpha]1A-adrenoceptor/AdTx1 complex (t1/2diss= 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. [PUBLICATION ABSTRACT]