Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α1A-adrenoceptor
Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α1A-adrenoceptor
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DOI：
10.1111/j.1476-5381.2009.00532.x
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发表时间：
2010-01-01
影响因子：
7.3
通讯作者：
Gilles, N.
中科院分区：
文献类型：
--
作者：
Quinton, L.;Girard, E.;Gilles, N.
背景和目的：毒液是离子通道配体的丰富来源，但关于它们调节G蛋白偶联受体（GPCR）活性的能力知之甚少。我们开发了一种策略，以确定新的毒素靶向GPCRs.Experimental方法：我们研究了在结合实验中与H-3-哌唑嗪的α（1）-肾上腺素能受体的曼巴蛇毒馏分的相互作用。通过Edman降解和质谱片段化对活性肽（AdTx 1）进行测序。它的合成同源物的特点是结合实验，使用克隆受体和功能实验兔离体前列腺smoothmuscles.Key结果：AdTx 1，一个65个氨基酸的肽稳定的四个二硫键，属于三指折叠肽家族。它对人α（1A）-肾上腺素受体亚型具有亚纳摩尔亲和力（Ki = 0.35 nM）和高特异性。我们在直接结合实验中显示了放射性标记的AdTx 1的高选择性和亲和力（Kd = 0.6 nM），并揭示了与异常稳定的α（1A）-肾上腺素受体/AdTx 1复合物（t（1/2diss）= 3.6 h）的缓慢缔合常数（k（on）= 6 x 10（6）.M-1.min（-1））。AdTx 1在体外（兔离体前列腺肌）的苯肾上腺素的行动显示出强大的不可逾越的拮抗作用，浓度为10至100 nM.Conclusions和影响：AdTx 1是最具体的和选择性的肽抑制剂的α（1A）-肾上腺素能受体确定的日期。它表现出不可克服的拮抗作用，作为一种有效的平滑肌松弛剂。它的肽性质可以被利用来开发新工具，例如放射性标记的AdTx 1或荧光标记的AdTx 1。因此，AdTx 1的鉴定为开发治疗良性前列腺增生的新药提供了新的前景。英国药理学杂志（2010）159，316-325; doi：10.1111/j.1476-5381.2009.00532.x; 2009年12月15日在线发表
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 H-3-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. British Journal of Pharmacology (2010) 159, 316-325; doi: 10.1111/j.1476-5381.2009.00532.x; published online 15 December 2009