Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α_1A‐adrenoceptor

Abstract

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 a1-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 (Ki = 0.35 nM) and high specificity for the human a1A-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 ¥ 10) with an unusually stable a1A-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 a1A-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.

An Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation

“…One gram of Dendroaspis angusticeps venom (Latoxan, Valence, France) was separated into 13 fractions by ion exchange (2×15 cm) on Source 15S using the protocol previously described [26]. Fraction B (2 mg) was purified by reverse-phase chromatography (Waters 600) on a semipreparative column (C 18 , 15 μm, 1.6 cm, 20 cm; Vydac, Sigma-Aldrich, Saint Quentin Fallavier, France) using a linear gradient from 10% to 50% acetonitrile and 0.1% trifluoroacetic acid in 50 min at 4 mL/min (Eluent A was 0.1% TFA+5% ACN and Eluent B=0.1% TFA in ACN).…”

Orthosteric Binding of ρ-Da1a, a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

“…The modes of action of these peptide ligands on ARs are not clear. In equilibrium binding experiments, neither ρ-Da1a nor ρ-Da1b fully inhibits radioligand binding [9],[10]. In addition, in isolated prostatic muscle, ρ-Da1a acts as an insurmountable antagonist [9], and cell-based assays indicate that ρ-Da1b is a non-competitive antagonist at the human α 2A -AR [10].…”

“…We suspected that animal venoms are a potential source of novel GPCR binding agents, and developed a screening strategy, initially focused on the binding of green mamba venom to ARs. This screening led to the isolation of two novel snake toxins from Dendroaspis angusticeps : ρ-Da1a, previously called AdTx1, which is highly selective for the α 1A -AR [9], and ρ-Da1b, selective for α 2 -ARs [10]. ρ-Da1a and ρ-Da1b are peptides of 65 and 66 residues, respectively, reticulated by four disulfide bridges, and are members of the three-finger-fold toxin family.…”

Peptide and Protein Neurotoxin Toolbox in Research on Nicotinic Acetylcholine Receptors

“…We also would like to mention here the recent discovery of three-finger neurotoxins which interact with another group of GPCR, namely with the adrenoreceptors [54,55]. These toxins are most similar to muscarinic toxins and were also isolated from the eastern green mamba Dendroaspis angusticeps.…”