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Development of TRPC6 channel specific inhibitor as a new prototypic anti-hypertensive drug.

开发 TRPC6 通道特异性抑制剂作为新的原型抗高血压药物。

基本信息

批准号：
14570079
负责人：
INOUE Ryuji
金额：
$ 1.92万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570079/
关键词：
TRP protein receptor-operated Ca channel vascular sympathetic nerve α1-adrenerigic receptor blood pressure specific inhbitor natural toxin screening new antihypertensives α_1アドレナリン受容体

项目摘要

Recent investigations including ours have suggested that a mammalian homologue of transient receptor potential (TRP) protein, TRPC6, is, the most abundantly expressed TRP protein in vascular smooth muscle tissues and plays a pivotal role in vascular tone regulation as a receptor-operated Ca^<2+> entry channel activated during the vascular sympathetic nerve excitation. Based on these findings, we have launched on the development of a new prototypic antihypertensive drug targeting TRPC6 by means of natural toxin screening which specifically inhibits its channel activities. In the first step of this project, we screened commercially available toxins extracted from both terrestrial and marine organisms which reportedly inhibit several voltage-dependent and fast ligand-gated channels, but none of them were found to be effective. In the next step, we collected crude toxin extracts from several snakes and tried to purify them to a single fraction containing TRPC6 channel-specific inhibitory a … More ctivities, by repeating the purification processes of the crude toxins with Sephadex chromatography and Ion exchange chromatography. As the result, two small subfractions which show potent and dose-dependent inhibitory actions on TRPC3 and TRPC7 (two closely related homologues of TRPC6) but not on TRPC6, have been isolated. The estimated molecular weight of toxins contained in the two subfractions is 65,000 and <1000KDa, respectively. Exploration of the site of actions of these subfractions by use of chimaeric TRP channels have revealed that the transmembrane region is involved in this inhibition. TRPC3, 6 and 7 channels constitute a subfamily amongst which more than 80% amino acid identity is found, but exhibit differential sensitivities to Ca^<2+>, flufenamate and mechanical stress imposed on the cell membrane. These findings strongly suggest that subtle differences in amino acid sequence, especially near the ion conductive pore region, would greatly change the sensitivity of these channels to various physiological modulators as well as pharmacological agents. In the subsequent period of research, we will put forward the purification process of the effective subfractions to yield single inhibitory peptides with computer-aided structural analysis, and simultaneously the identification of the sites of their actions, whereby the indispensable database for the development of specific TRP channel inhibitors is obtained, which would eventually lead to the discovery of a new generation of antihypertensive drug. Less

包括我们在内的最新研究表明，瞬时受体电位（TRP）蛋白的哺乳动物同源物TRPC 6是血管平滑肌组织中表达最丰富的TRP蛋白，在血管紧张度调节中起关键作用，作为血管交感神经兴奋过程中激活的受体操纵的Ca^2+进入通道。基于这些发现，我们通过天然毒素筛选启动了针对TRPC 6的新型原型降压药物的开发，该药物特异性抑制其通道活性。在本项目的第一步中，我们筛选了从陆地和海洋生物中提取的市售毒素，据报道这些毒素抑制几种电压依赖性和快速配体门控通道，但没有发现它们是有效的。下一步，我们收集了几种蛇的粗毒素提取物，并试图将它们纯化成含有TRPC 6通道特异性抑制剂的单一组分， ...更多信息通过重复Sephadex层析和离子交换层析纯化粗毒素的过程，结果，分离出了两个小的亚组分，它们对TRPC 3和TRPC 7（TRPC 6的两个密切相关的同系物）显示出有效的和剂量依赖性的抑制作用，但对TRPC 6没有。两个亚组分中毒素的分子量估计分别为65，000和<1000KDa。通过使用嵌合TRP通道探索这些亚组分的作用位点，揭示了跨膜区参与这种抑制。TRPC 3、6和7通道构成了一个亚家族，其中发现了超过80%的氨基酸同一性，但对Ca^2+、氟芬那酯和施加在细胞膜上的机械应力表现出不同的敏感性。这些发现强烈表明，氨基酸序列的细微差异，特别是在离子传导孔区域附近，将极大地改变这些通道对各种生理调节剂以及药理学试剂的敏感性。在后续的研究中，我们将提出有效亚组分的纯化过程，通过计算机辅助结构分析得到单一的抑制肽，同时鉴定其作用位点，从而获得开发特异性TRP通道抑制剂所必需的数据库，最终导致新一代抗高血压药物的发现。少