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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.

SUBMITTER: Quinton L

PROVIDER: S-EPMC2825353 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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

Quinton L L Girard E E Maiga A A Rekik M M Lluel P P Masuyer G G Larregola M M Marquer C C Ciolek J J Magnin T T Wagner R R Molgó J J Thai R R Fruchart-Gaillard C C Mourier G G Chamot-Rooke J J Ménez A A Palea S S Servent D D Gilles N N

British journal of pharmacology 20091215 2

<h4>Background and purpose</h4>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.<h4>Experimental approach</h4>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 fragmentat ...[more]

PMID: 20015090

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Project description:?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the ?1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the ?1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the ?1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the ?1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the ?1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human ?1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the ?1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Project description:BACKGROUND AND PURPOSE: This study investigated whether deletion of the alpha1A-adrenoceptor gene influences contractile responses of mouse prostate to noradrenaline. Responses of mouse prostate to noradrenaline are known to be mediated by alpha1L-adrenoceptors, which are thought to be a functional phenotype of alpha1A-adrenoceptor. EXPERIMENTAL APPROACH: Prostate tissues from alpha1A-adrenoceptor knockout mice which were homozygous (alpha1A -/-) and heterozygous (alpha1A +/-) for the disrupted alpha1A-adrenoceptor gene, as well as wild-type (alpha1A +/+) littermates were mounted in glass-isolated organ baths. Electrical field stimulation of nerves and exogenous application of noradrenaline were used to investigate the effects of alpha1A-adrenoceptor disruption on prostate contractility. KEY RESULTS: Frequency-response curves to electrical field stimulation (0.5 ms pulse duration, 60 V, 0.1-20 Hz) yielded frequency-dependent contractions. At frequencies of 10 and 20 Hz, prostates from alpha1A -/- mice elicited an approximately 30% decreased response compared with prostates from alpha(1A)+/+ mice. Prazosin (0.3 muM) attenuated responses to electrical field stimulation in prostates from alpha1A +/+ and alpha1A +/- mice but not from alpha1A -/- mice. Increasing concentrations of exogenously administered noradrenaline (10 nM-1 mM) produced mean concentration-response curves in prostates from alpha1A +/+ and alpha1A +/- mice, which were not different. Maximum responses to noradrenaline were decreased by approximately 80% in prostates from alpha1A -/- mice compared with alpha1A +/+ mice. Prazosin attenuated responses to noradrenaline in all genotypes. CONCLUSIONS AND IMPLICATIONS: alpha1L-Adrenoceptor-mediated responses in mouse prostate are abolished in alpha1A -/- mice, demonstrating that the alpha1A-adrenoceptor gene is essential to the manifestation of the prostatic alpha1L-adrenoceptor phenotype. This implies that alpha1L-adrenoceptors are indeed a functional phenotype of alpha1A-adrenoceptor.

Dataset Information

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

Publications

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

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