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.

ATR‐101 inhibits cholesterol efflux and cortisol secretion by ATP‐binding cassette transporters, causing cytotoxic cholesterol accumulation in adrenocortical carcinoma cells

“…ATR-101 administration reduces the levels of circulating steroids in mice with ACC cell xenografts and in dogs stimulated with ACTH (Cheng et al, 2016;Lapensee et al, 2016). Some MDR1 inhibitors reduced circulating corticosteroid levels in clinical trials, suggesting that MDR1 inhibition can reduce steroid secretion in man (Guthrie et al, 1983;Shapiro et al, 1990;Kobayashi et al, 1996).…”

“…Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibition was proposed to cause cholesterol accumulation and cytotoxicity in ACC cell lines (Sbiera et al, 2015;Lapensee et al, 2016). Most ACAT inhibitors do not cause cholesterol accumulation or cytotoxicity under normal cell culture conditions, and differences in the ACAT inhibitory activities of different compounds do not correlate with differences in cytotoxicity (Junquero et al, 2001;Rodriguez and Usher, 2002;An et al, 2008;Pokhrel et al, 2012).…”

“…MDR1 is required to maintain normal circulating corticosterone levels in mice and for steroid secretion by mouse adrenocortical cells (Altuvia et al, 1993;Muller et al, 2003). It is not known if the effects of MDR1 on cholesterol levels and on steroid secretion are direct and independent or each other or if these effects are indirect consequences of a single molecular function of MDR1.Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibition was proposed to cause cholesterol accumulation and cytotoxicity in ACC cell lines (Sbiera et al, 2015;Lapensee et al, 2016). Most ACAT inhibitors do not cause cholesterol accumulation or cytotoxicity under normal cell culture conditions, and differences in the ACAT inhibitory BJP V E Burns and T K Kerppola 3316 British Journal of Pharmacology (2017) 174 3315-3332 activities of different compounds do not correlate with differences in cytotoxicity (Junquero et al, 2001;Rodriguez and Usher, 2002;An et al, 2008;Pokhrel et al, 2012).…”

Biochemical mechanism of the o,p’-DDD effect on the adrenal cortex

“…Treatment of dogs with ATR-101 decreased the formation of corticosteroids and induced adrenocorticocytes apoptosis, followed by a decrease in the adrenal cortex. The authors suggested that the use of ATR-101 (the ACAT inhibitor) may be promising in the treatment of adrenalcortical cancer [47]. In H295 cells, o,p'-DDD dramatically increased the expression of reticular stress marker CHOP (transcription factor encoded by the ddIT3 gene) [44].…”

“…ATR-101 treatment of H295R cells led to the accumulation of free fatty acids and cholesterol that induces ER stress [47]. In so doing the increased caspase 3/7 activity and activation of apoptosis, assessed by terminal deoxynucleotidyl transferase nick end labe-…”