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


METHODS
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.


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
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.
Quinton
L
L
Laboratoire des Mécanismes Réactionnels, Ecole Polytechnique, Palaiseau, France.
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
D058668:Adrenergic alpha-1 Receptor Antagonists; D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation; D004546:Elapid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D017815:Elapidae; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D010843:Pichia; D011467:Prostate / Q000187:drug effects / Q000502:physiology; D011817:Rabbits; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1
C547713:ADRA1A protein, human; C549220:AdTx1 peptide, Dendroaspis angusticep; D058668:Adrenergic alpha-1 Receptor Antagonists; D004546:Elapid Venoms; D010455:Peptides; D018340:Receptors, Adrenergic, alpha-1