Acyl-coenzyme A: cholesterol O-acyltransferase 1 (ACAT1) [1].
EC₅₀: 9 nM (for human ACAT1) [1].
EC₅₀: 368 nM (for human ACAT2) [1].

In a cell-based fluorescence assay measuring cholesterol esterification, Nevanimibe potently and selectively inhibited human ACAT1 with an EC₅₀ of 9 nM, compared to ACAT2 with an EC₅₀ of 368 nM, demonstrating approximately 40-fold selectivity for ACAT1 [1].
In H295R human adrenocortical carcinoma cells, Nevanimibe (9 nM) treatment for 5 hours in the presence of exogenous cholesterol (45 μg/mL) increased intracellular free cholesterol levels by approximately 70% and decreased cholesteryl ester formation, consistent with ACAT1 inhibition. The free cholesterol to cholesteryl ester ratio shifted from 1.4:1 (cholesterol alone) to 5:1 (ATR-101 + cholesterol). Total intracellular cholesterol content was unchanged [1].
In H295R cells, Nevanimibe (30 nM) in the presence of cholesterol (45 μg/mL) induced apoptosis, as evidenced by increased caspase-3/7 activity (approximately 3-fold) and increased TUNEL-positive cells after 5-16 hours. In the absence of exogenous cholesterol, Nevanimibe at concentrations up to 3 μM showed no toxicity; only at 30 μM did it reduce viability by approximately 40% after 24 hours [1].
Co-incubation with exogenous cholesterol (45-60 μg/mL) markedly sensitized H295R cells to Nevanimibe, with 3 nM ATR-101 plus 60 μg/mL cholesterol reducing viability by approximately 60% after 24 hours [1].
The cholesterol transport inhibitor U18666A (100 nM) prevented Nevanimibe (30 nM)-induced caspase-3/7 activation, indicating that cholesterol trafficking to the endoplasmic reticulum is required for cytotoxicity [1].
Nevanimibe induced the unfolded protein response (UPR) in H295R cells. After 5 hours of treatment (30 nM ATR-101 + 45 μg/mL cholesterol), the following were observed: splicing of XBP-1 mRNA (XBP-1s), phosphorylation of PERK (indicated by a mobility shift on Western blot), and increased CHOP mRNA expression (approximately 2.5-fold) [1].
Inhibitors of endoplasmic reticulum calcium release (Xestospongin C, 10 μM; 2-APB, 100 μM), mitochondrial calcium uptake (Ruthenium Red, 15 μM), and mitochondrial membrane permeabilization (Cyclosporin A, 10 μM) all blocked Nevanimibe-induced caspase-3/7 activation in H295R cells [1].
Nevanimibe (30 nM + cholesterol) decreased mitochondrial membrane potential (TMRE fluorescence decreased by approximately 50% vs. control), an effect reversed by Cyclosporin A [1].
ACAT1 knockdown via shRNA in HAC15 adrenocortical cells (80% reduction in ACAT1 expression) mimicked the effects of Nevanimibe: increased free cholesterol (FC:CE ratio approximately 4:1 vs. 1:1 in control), decreased viability after 24 and 72 hours, and increased caspase-3/7 activity (approximately 3-4 fold) in the presence of exogenous cholesterol (60 μg/mL). These effects were blocked by U18666A, Ruthenium Red, and Cyclosporin A [1].

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The frequency of spleen was reduced by 60% when nervanibib hydrochloride (PD-132301 hydrochloride; ATR101 hydrochloride; 3 nM-30 μM) and cholesterol were depleted together. The toxicity was shown to rise in a dose-dependent way, with 3 nM nervanibib at 60 μg. 24 In the presence of cholesterol, all dosages of Nevanimibe (3 nM–30 μM) caused cytotoxicity, while treatment with cholesterol in the absence of Nevanimibe had no effect on cell viability. Influence [1].

Canine Study (Pharmacodynamics and Toxicity): Three male beagle dogs were administered Nevanimibe orally once daily at 3 mg/kg/day for 7 days followed by 30 mg/kg/day for an additional 7 days. After 14 days of treatment, histologic examination of adrenal glands revealed cortical atrophy, vacuolation, degeneration/necrosis, and mononuclear cell infiltration primarily affecting the zona fasciculata. Approximately 60% of cortical cells in each representative field stained positive for TUNEL, indicating ongoing apoptosis. No abnormalities were observed in the adrenal medulla [1].
Steroidogenesis Inhibition: In the same dogs, ACTH-stimulated cortisol levels decreased by 62% after 7 days of 3 mg/kg/day and by 71% after 14 days of 30 mg/kg/day compared to baseline. ACTH-stimulated levels of corticosterone, 17-hydroxyprogesterone, 11-deoxycortisol, and 11-deoxycorticosterone were also significantly reduced. Pre-ACTH levels of adrenal androgens (androstenedione, DHEA-S) were significantly lower after 14 days of treatment [1].
Adrenal Cholesterol Content: Adrenal glands of treated dogs showed a marked decrease in cholesteryl ester content (191 μg/mg protein) compared to an untreated dog (606 μg/mg protein), consistent with ACAT1 inhibition. Free cholesterol levels were unchanged (37 vs. 38 μg/mg protein) [1].

ACAT1/ACAT2 Fluorescent Cell-Based Assay: ACAT-deficient Chinese hamster ovary (AC29) cells were transfected with constructs expressing human ACAT1 or ACAT2. Esterification of a fluorescent cholesterol analog (22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol) was measured. Cells were incubated with increasing concentrations of Nevanimibe, and esterification was determined to calculate EC₅₀ values [1].

Cytotoxicity Assay (MTT): H295R cells were plated in 96-well plates and treated with Nevanimibe in the presence or absence of water-soluble cholesterol (cholesterol-methyl-β-cyclodextrin complex) for 24 hours. MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) was added at 0.5 mg/mL for 2 hours. Formazan dye was extracted with DMSO, and absorbance was read at 570 nm [1].
Crystal Violet Staining: Cells were treated similarly, fixed with 4% paraformaldehyde, stained with 0.4% crystal violet in 10% ethanol, and after solubilization with 1% SDS, absorbance was measured at 570 nm [1].
Caspase-3/7 Assay: Cells were treated for 5 hours, and caspase-3/7 activity was measured using a luminescence-based kit. Caspase detection reagent was added, and luminescence was measured with a luminometer [1].
TUNEL Staining: Cells on chamber slides were treated for 16 hours, and TUNEL staining was performed using a fluorometric TUNEL system. Cells were counterstained with DAPI, and images were captured [1].
Mitochondrial Membrane Potential Measurement: Cells were treated for 5 hours, then TMRE (tetramethyl rhodamine) was added at 2 μM for 15 minutes. Live-cell TMRE fluorescence was measured on a plate reader [1].
Free and Esterified Cholesterol Measurement: Cells were treated for 5 hours, lipids extracted with hexane/isopropanol (3:2), and free and total cholesterol were measured using a fluorometric assay. Esterified cholesterol was calculated by subtracting free cholesterol from total cholesterol [1].
Quantitative RT-PCR: RNA was extracted, reverse transcribed, and CHOP mRNA expression was quantified using SYBR Green PCR and normalized to GAPDH. XBP-1 splicing was detected by PCR with primers spanning the splice site [1].
Western Blotting: PERK phosphorylation was detected by immunoblotting using an anti-PERK antibody. Activated PERK was identified by an upward mobility shift [1].
ACAT1 Knockdown: HAC15 cells were transduced with lentiviral shRNA targeting SOAT1 (human ACAT1). Stable knockdown cells were selected with puromycin, and ACAT1 expression was confirmed by qPCR (80% reduction) [1].

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Cytotoxicity assay [1]
Cell Types: H295R and HAC clone 15 (HAC15) Human ACC cell line
Tested Concentrations: 3 nM-30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: 3 nM-3 μM non-toxic, while 30 μM treatment improved survival diminished by approximately 40% in 24 hrs (hours).

Canine Toxicology and Pharmacodynamics Study: Three male beagle dogs (approximately 10-12 kg) were administered Nevanimibe orally once daily at 3 mg/kg/day for 7 days followed by 30 mg/kg/day for an additional 7 days. The compound was formulated in 0.5% hydroxypropylmethylcellulose at a dose volume of 5 mL/kg. Blood samples were collected for serum cortisol and steroid analysis before and 1 hour after ACTH stimulation (5 μg/kg, IV) on days -3, 1, 3, 7, 8, 10, and 14. On day 14, animals were euthanized, and adrenal glands were collected for histology (H&E and TUNEL staining), and for free and esterified cholesterol analysis [1].
Canine Tissue Distribution Study: Three female beagle dogs (approximately 19 months old, 7-10 kg) were administered Nevanimibe orally once daily at 3 mg/kg/day for 7 days in 0.5% hydroxypropylmethylcellulose (5 mL/kg). On the last day, blood was collected 4 hours post-dose, and animals were euthanized. Tissues (adrenal glands, kidney, liver, skeletal muscle, sc fat, ovaries, and CSF) were collected for determination of ATR-101 content by LC-MS/MS [1].

Tissue Distribution in Dogs: After 7 days of oral dosing at 3 mg/kg/day, Nevanimibe showed preferential distribution to the adrenal glands. When normalized to plasma concentration, only the adrenal glands had concentrations approaching equivalence to plasma. No other tissues had concentrations exceeding 30% of plasma concentration. ATR-101 was not detectable in cerebrospinal fluid (CSF) [1].
Plasma Concentrations: Plasma concentrations of Nevanimibe after dosing at 3 mg/kg/day and 30 mg/kg/day were reported to approximate those previously seen in toxicology studies (data shown in Supplemental Figure 1) [1].

Adrenal-Specific Toxicity in Dogs: After 14 days of oral Nevanimibe treatment (3 mg/kg/day for 7 days, then 30 mg/kg/day for 7 days), histologic examination revealed adrenal cortical atrophy, vacuolation, degeneration/necrosis, and mononuclear cell infiltration, primarily in the zona fasciculata. Approximately 60% of cortical cells stained TUNEL-positive. No abnormalities were observed in the adrenal medulla [1].
No Effects on Other Tissues: A separate 28-day toxicity study in male dogs showed no effects on testes or macrophages (data not shown) [1].
Steroidogenesis Impairment: Nevanimibe treatment resulted in progressive reductions in both pre- and post-ACTH-stimulated levels of glucocorticoids, mineralocorticoids, and adrenal androgens, consistent with reduced adrenal function and cell death [1].
In vitro Toxicity: In H295R cells, Nevanimibe was non-toxic up to 3 μM in the absence of exogenous cholesterol; only at 30 μM did it reduce viability by ~40% after 24 hours [1].

[1]. 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. Endocrinology. 2016 May;157(5):1775-88.

Nevanimibe (also known as PD 132301-2) is a selective and potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum that catalyzes the esterification of free cholesterol [1].
The compound was originally developed as a potential treatment for hypercholesterolemia but was abandoned due to adrenal-specific toxicity in animal models. This adrenal-specific toxicity led to its repurposing for adrenocortical carcinoma (ACC) [1].
The mechanism of action involves inhibition of ACAT1, leading to accumulation of free cholesterol in the endoplasmic reticulum. This triggers ER stress (unfolded protein response), release of ER calcium stores, mitochondrial calcium uptake, loss of mitochondrial membrane potential, and ultimately apoptosis [1].
Nevanimibe is currently in Phase I clinical development for the treatment of adrenocortical cancer [1].
The compound is highly lipophilic, which may contribute to its selective distribution to the adrenal cortex, possibly via lipoprotein uptake or centripetal adrenal blood flow [1].

C27H40CLN3O

458.079006195068

457.286

C, 70.79; H, 8.80; Cl, 7.74; N, 9.17; O, 3.49

133825-81-7

Nevanimibe;133825-80-6

131678

White to off-white solid powder

528.1ºC at 760 mmHg

273.2ºC

3.05E-11mmHg at 25°C

7.712

3

2

7

32

543

0

Cl.O=C(NC1C(=CC=CC=1C(C)C)C(C)C)NCC1(C2C=CC(=CC=2)N(C)C)CCCC1

SDOOGTHIDFZUNM-UHFFFAOYSA-N

InChI=1S/C27H39N3O.ClH/c1-19(2)23-10-9-11-24(20(3)4)25(23)29-26(31)28-18-27(16-7-8-17-27)21-12-14-22(15-13-21)30(5)6;/h9-15,19-20H,7-8,16-18H2,1-6H3,(H2,28,29,31);1H

1-[[1-[4-(dimethylamino)phenyl]cyclopentyl]methyl]-3-[2,6-di(propan-2-yl)phenyl]urea;hydrochloride

PD-132301 hydrochloride; N-(2,6-bis(1-methylethyl)phenyl)-N'-((1-(4-(dimethylamino)phenyl)cyclopentyl)methyl)urea hydrochloride; RefChem:165610; Nevanimibe hydrochloride; 133825-81-7; ATR-101 HCl; PD132301 hydrochloride; PD 132301 hydrochloride; ATR-101 hydrochloride;ATR 101 hydrochloride;ATR101 hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~41.67 mg/mL (~90.97 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)