SUN-064 High-Throughput Screening Identifies Potential Therapeutic Agents for Cushing Disease

Abstract Disclosure: T. Hakata: None. I. Yamauchi: None. A. Sakurai: None. S. Kimura: None. D. Kosugi: None. T. Sugawa: None. K. Okamoto: None. Y. Ueda: None. D. Taura: None. D. Yabe: None. Objective: Cushing disease is a life-threatening disorder caused by the autonomous secretion of adrenocorticotropic hormone (ACTH) from pituitary neuroendocrine tumors (PitNETs). Few drugs are currently available for the treatment of inoperable Cushing disease. To identify potential therapeutic agents that regulate ACTH secretion in PitNETs, we conducted high-throughput screening (HTS).Materials and Methods: HTS was performed using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. We developed a live-cell ACTH reporter assay to enable high-throughput evaluation of ACTH secretion. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, allowing high-throughput fluorescence image acquisition. AtT-20 cells were treated with various compounds, and ACTH concentrations in the conditioned media were measured using the ACTH reporter assay. Cell viability was concurrently assessed for AtT-20 cells treated with each compound.Results: Out of 2,480 screened compounds, 254 inhibited ACTH secretion by more than 50%. There was a significant correlation between changes in ACTH secretion and cell viability (r = 0.92, p < 0.001). Specifically, 171 compounds reduced both ACTH secretion and cell viability by over 50%. The top five classes of these agents included 10 histone deacetylase inhibitors, 8 topoisomerase inhibitors, 7 cyclin-dependent kinase inhibitors, 7 heat shock protein 90 inhibitors, and 7 phosphoinositide-3 kinase inhibitors. Conversely, 83 compounds reduced ACTH secretion by more than 50% without reducing cell viability by the same margin. Among these, the top five classes were 14 calcium channel blockers (12 L-type and 2 T-type), 7 dopamine receptor antagonists, 6 antihistamines, 5 muscarinic receptor antagonists, and 5 tricyclic antidepressants. Discussion: Using an original ACTH reporter assay, we conducted HTS and identified numerous candidate compounds that may contribute to the treatment of Cushing disease. Most compounds that reduced both ACTH secretion and cell viability have been reported to exert antitumor effects on ACTH-secreting PitNETs. On the other hand, compounds that specifically reduced ACTH secretion without significantly affecting cell viability have been rarely reported. Further studies are needed to investigate these compounds in more detail. Presentation: Sunday, July 13, 2025