Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha1A-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 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.

Figure 1

Identification of AdTx1. (A) Ion-exchange chromatography of Dendroaspis angusticep crude venom. Labelled peaks were collected (13 fractions). (B) Reverse-phase chromatography of fraction E (arrow) on a Vydac C18 preparative column. Labelled peaks were collected (20 fractions). Fraction O (arrow) was eluted at around 32% acetonitrile (ACN). (C) Inhibition of ³H-prazosin (1 nM) binding by fraction O, quantified using a Bio-Rad protein assay on rat brain synaptosomes (200 µg).

Figure 2

(A) Analytical reverse-phase chromatography of fraction O (line a), reduced AdTx1 (line b) and oxidized AdTx1 (line c). Line d is the co-elution of fraction O and the oxidized synthetic AdTx1. *(B) Identification of the C-terminal part of the sequence of AdTx1. Fragmentation by electron capture dissociation of the m/z 863.37 ([M+4H]⁴⁺) ion obtained after trypsin digestion of fraction O. c-, z- and y-ion types used for the sequencing are indicated in brown, pink and blue respectively.

Figure 3

Inhibition binding curves for adrenoceptors and muscarinic receptors. (A) Inhibition curves for natural receptors from different tissues. Curves represent inhibition of ³H-prazosin (1 nM) binding to rat brain synaptosomes (RBS) (200 µg) with various concentrations of prazosin and AdTx1. The inhibition of ³H-rauwolscine (1 nM) binding to RBS (200 µg) by AdTx1 is also shown. Other data represent the non-displacement of ³H-CGP-12177 (1 nM) from heart membranes (100 µg), of ³H-CGP-12177 (1 nM) from lung membranes (100 µg) and of ³H-NMS (0.5 nM) from RBS (100 µg) by AdTx1. (B) Inhibition of ³H-prazosin (1 nM) in yeast membranes (20 µg) expressing human α_1A-adrenoceptor with prazosin and AdTx1. There was much weaker inhibition by AdTx1 of ³H-prazosin (1 nM) binding in yeast membranes expressing human-α_1B-adrenoceptor (30 µg) or rat α_1D-adrenoceptor (50 µg). (C) Kinetic analysis of the dissociation of ³H-prazosin bound to human α_1A-adrenoceptor. ³H-prazosin dissociation rate from α_1A-adrenoceptor (20 µg), induced by prazosin (1 µM) or AdTx1 (1 µM) or both.

Figure 4

Binding of ¹²⁵I-AdTx1 to human α_1A-adrenoceptor (20 µg). (A) Saturation binding assays for ¹²⁵IAdTx1 (0.1 to 2 nM) binding to human α_1A-adrenoceptor. (B) Displacement of ¹²⁵I-AdTx1 (0.2 nM) by various concentrations of prazosin and synthetic AdTx1. (C) Association of ¹²⁵I-AdTx1 (0.2 nM) on human α_1A-adrenoceptor. The AdTx1/α_1A-adrenoceptor complex is stable for at least 24 h. ¹²⁵I-AdTx1 (0.2 nM) dissociation, after 16 h of association with human α_1A-adrenoceptor, was induced by 1 µM AdTx1 alone or in addition to 1 µM prazosin.

Figure 5

Rabbit isolated prostate muscle. Cumulative concentration-response curves for phenylephrine (PHE) were obtained in prostatic strips after 30 min of incubation with vehicle, or 10 nM, 30 nM or 100 nM AdTx1. Contractile responses to phenylephrine are presented as mean ± SEM % of the maximal tension obtained with 30 µM of phenylephrine, n= 6.