title: ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs.
abstract: ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.
authors.affiliation: Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: LaPensee
authors.affiliation: Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104.
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Mann
authors.affiliation: Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104.
authors.forename: William E
authors.initials: WE
authors.lastname: Rainey
authors.affiliation: Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104.
authors.forename: Valentina
authors.initials: V
authors.lastname: Crudo
authors.affiliation: Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Hunt
authors.affiliation: Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Hammer
mesh_terms: D000302:Adrenal Cortex / Q000187:drug effects* / Q000378:metabolism; D000306:Adrenal Cortex Neoplasms / Q000378:metabolism / Q000473:pathology; D018268:Adrenocortical Carcinoma / Q000378:metabolism / Q000473:pathology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004285:Dogs; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010671:Phenylurea Compounds / Q000494:pharmacology*; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D010671:Phenylurea Compounds; D002785:Sterol O-Acyltransferase; C493492:sterol O-acyltransferase 1; D053148:Caspase 3; C079534:N-(2,6-bis(1-methylethyl)phenyl)-N'-((1-(4-(dimethylamino)phenyl)cyclopentyl)methyl)urea hydrochloride