Endocrine Abstracts

Introduction: Major advances have been made in understanding the pathophysiology of primary aldosteronism (PA). A breakthrough was the identification of mutations in the potassium channel KCNJ5, which drive aldosterone overproduction in aldosterone-producing adenomas and familial hyperaldosteronism type III. Our objective was to identify small molecules that selectively target mutated KCNJ5 channels to broaden the therapeutic strategies for PA.

Methods: We conducted a virtual screening of over 6 million small molecules to identify those that potentially bind to KCNJ5 channels. The functionality of these candidate molecules was evaluated in vitro using human adrenocortical cells. Assessment included cell viability assays, flow cytometry, confocal microscopy, qPCR, and mass spectrometry for steroid measurements.

Results: The virtual screening identified 108 candidate molecules. Each was tested for antagonist activity by evaluating their ability to rescue mutant KCNJ5-induced cell death. A positive correlation was observed between multiple aromatic rings in the candidate compounds and increased cell death rescue. Among these molecules, compound-81 (C-81), effectively rescued adrenal cell death induced by mutated KCNJ5 in both monolayer and spheroid cultures. The specificity of C-81 for mutated vs. wild-type KCNJ5 was demonstrated by a reduction in aldosterone synthase (CYP11B2) gene expression induced by different KCNJ5 mutations (69% - 88% reduction for KCNJ5 L168R, T158A, and G151R), with negligible effects on angiotensin II-stimulated CYP11B2 expression in unmutated cells. Consistently, C-81 led to a 73% decrease in aldosterone secretion from cells expressing KCNJ5-L168R without significantly affecting cells overexpressing wild-type KCNJ5. Mutated KCNJ5 substantially increased cortisol production, while C-81 treatment reduced cortisol secretion by 75%.

Conclusion: High-throughput virtual screening is a valid approach for the discovery of novel therapeutics that selectively target aldosterone overproduction driven by a KCNJ5 mutation. C-81 or its structural analogs are putative small molecule antagonists targeting pathological aldosterone secretion in both sporadic and familial forms of PA.