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 Ca2+ 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 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 Ca2+ influx, demonstrating that its mechanism of action lies downstream of Ca2+ 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, Ca2+ 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.