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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

最近的研究包括我们的研究表明，哺乳动物中瞬时受体潜力(TRP)蛋白的同源物TRPC6是血管平滑肌组织中表达最丰富的TRP蛋白，它作为受体操纵的钙通道在血管交感神经兴奋时激活，在血管张力调节中发挥关键作用。基于这些发现，我们已经开始开发一种新的针对TRPC6的原型降压药物，方法是通过筛选天然毒素来特异性地抑制其通道活性。在这个项目的第一步，我们筛选了从陆地和海洋生物中提取的商业可用的毒素，据报道，这些毒素抑制了几个电压依赖和快速配体门控通道，但没有一个被发现是有效的。在下一步中，我们收集了几种蛇的粗毒素提取物，并试图将它们提纯为含有TRPC6通道特异性抑制a…的单一组分通过Sephadex层析和离子交换层析重复粗毒素的纯化过程，以提高活性。结果，分离到两个对TRPC3和TRPC7(TRPC6的两个密切同系物)有强烈抑制作用且呈剂量依赖关系的小亚组分，但对TRPC6没有抑制作用。据估计，这两个亚组分所含毒素的分子量分别为65,000和1000 KDa。利用嵌合色氨酸通道探索这些亚组分的作用部位，发现跨膜区参与了这种抑制。TRPC3、6和7通道构成一个亚家族，其中80%以上的氨基酸具有同源性，但对Ca~(2+)、氟氰菊酯和施加在细胞膜上的机械应力具有不同的敏感性。这些发现有力地表明，氨基酸序列的细微差异，特别是在离子传导孔区附近，将极大地改变这些通道对各种生理调节剂以及药物的敏感性。在接下来的研究中，我们将提出有效亚组分的纯化过程，利用计算机辅助结构分析得到单一的抑制性多肽，同时鉴定它们的作用部位，从而获得开发特定的Trp通道抑制剂所必需的数据库，最终导致新一代降压药物的发现。较少