The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression
The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression
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DOI：
10.1016/j.bbrc.2020.11.020
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发表时间：
2021-01-01
影响因子：
3.1
通讯作者：
Sugawara，Akira
中科院分区：
文献类型：
--
作者：
Ito，Ryo;Morita，Masanobu;Sugawara，Akira
醛固酮在肾上腺中由醛固酮合成酶CYP 11B 2合成。虽然CYP 11B 2表达的控制对于维持矿物质稳态是重要的，但已报道由去极化诱导的钙（Ca 2+）信号激活诱导的CYP 11B 2过表达在原发性醛固酮增多症（PA）中增加醛固酮的合成。目前针对PA的药物主要集中在抑制CYP 11B 2的表达上，但由于CYP 11B 2通过Ca ~（2+）信号激活转录调控的分子机制尚不清楚，因此尚未开发出针对PA的药物。为了解决这个问题，我们试图揭示CYP 11B 2的转录调控机制，使用化学筛选。我们利用CRSPR/Cas9系统将Nanoluc基因作为报告基因插入H295 R肾上腺皮质细胞CYP 11B 2位点，构建了一个细胞系，并利用该细胞系建立了高通量筛选系统。然后，我们从经验证的化合物库（3399种化合物）中鉴定出9种抑制钾介导的去极化诱导的CYP 11B 2表达的化合物。特别是，他克莫司（一种磷酸酶钙调磷酸酶抑制剂）即使在10 nM时也能强烈抑制CYP 11 B2的表达。这些结果表明，该系统是有效的，在识别药物，抑制去极化诱导的CYP 11B 2的表达。因此，我们的筛选系统可能是一个有用的工具，用于开发新的药物对PA。
Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter intoCYP11B2locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.