Ps-b02-14: discovery of a new drug for primary aldosteronism targeting the cyp11b2 and kcnj5 mutants

Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β;-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examing the detailed mechanism of action of the compounds. In addition, we would like to further experiment the effects of the compounds in vivo using hypertensive model mice, Task1 /Task3 double KO mice.