Identification of a novel small-molecule compound that suppresses depolarization-induced CYP11B2 expression and aldosterone production in KCNJ5 mutant adrenocortical cells

Primary aldosteronism (PA) is one of the most common causes of secondary hypertension, with aldosterone-producing adenomas (APAs) accounting for approximately half of cases. In most APAs, somatic mutations in ion channel or pump genes constitutively activate Ca 2+ signaling through membrane depolarization, driving overexpression of the aldosterone synthase CYP11B2 and excess aldosterone production. Although KCNJ5 mutations represent the most prevalent somatic driver of APA pathogenesis, existing pharmacotherapies—including mineralocorticoid receptor antagonists (MRAs) and CYP11B2 enzyme inhibitors—have limitations in efficacy and selectivity, and are associated with notable adverse effects, indicating an urgent need for novel strategies that suppress CYP11B2 expression at the transcriptional level through a distinct mechanism of action. To address this need, we performed high-throughput screening of a large compound library using a KCl stimulation-dependent CYP11B2 reporter system, with multi-step filtering incorporating cytotoxicity evaluation and validation in doxycycline-inducible KCNJ5-L168R mutant-expressing cells. This approach identified compound 35 (2-benzylidene-5-allyladamantan-1-ol) as a lead candidate. Compound 35 suppressed CYP11B2 mRNA expression and aldosterone secretion in KCNJ5-L168R mutant cells without inhibiting KCl-induced Ca 2+ influx, demonstrating that its mechanism of action lies downstream of Ca 2+ entry. Compound 35 also reduced NURR1 and NGFIB expression, pointing to a mechanism upstream of NR4A-dependent CYP11B2 induction. Notably, compound 35 showed no significant effect on CYP11B1 mRNA expression, suggesting selectivity at the transcriptional level that is mechanistically distinct from the structural similarity-based selectivity challenges faced by CYP11B2 enzyme inhibitors. These results identify compound 35 as a chemically tractable lead with a mechanism of action distinct from MRAs, Ca 2+ channel blockers, and CYP11B2 enzyme inhibitors, providing a starting point for the development of transcriptional suppressors of CYP11B2 as a novel therapeutic strategy for KCNJ5 mutation-associated PA.