ACAT-1/acyl-CoA:cholesterol acyltransferase 1 (IC50 = 0.45 μM)

K-604's effectiveness and selectivity against human ACAT-1 and ACAT-2 were investigated. K-604's selectivity for ACAT-1 is 229 times greater than that of ACAT-2, as evidenced by its IC50 values of 0.45 μM for human ACAT-1 and 102.85 μM for human ACAT-2. With a Ki value of 0.378 μM, kinetic studies demonstrated that the inhibition was competitive with oleoyl-CoA. K-604 has an IC50 value of 68 nM, which efficiently inhibits the esterification of cholesterol in human macrophages[1]. K604 at 0.5 µM significantly reduced ACAT1 enzyme activity in cell-free biochemical experiments, but not ACAT2 enzyme activity. N2a cells were treated with K604 to see if inhibiting ACAT1 would promote autophagy in neuronal cells. At doses of 0.1 to 1 µM, K604 was found to suppress ACAT activity by 60–80%. After adding K604 to N2a cells for 24 hours at a dose of 0.1 to 1 µM, the levels of LC3 were measured using Western blotting. The findings demonstrated that K604 enhanced, in a dose-dependent manner, the LC3-II/LC3-I ratio, a trustworthy indicator of autophagosome production. Following treatment with K604, N2a cells showed a substantial increase in the quantity of fluorescent LC3 puncta. K604 dramatically lowered the amounts of p62 in N2a cells by Western blotting [2].

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Competitive inhibition of ACAT-1 by K-604 [1]
Immunoblotting analysis showed that the microsomal fractions obtained from CHO-ACAT-1 and CHO-ACAT-2 cells contained human ACAT-1 and ACAT-2 protein, respectively (Fig. 2A). Kinetic analyses showed that Vmax values of [14C]oleoyl-CoA for human ACAT-1 and human ACAT-2 were 21.8 and 4.0 nmol/min/mg protein, respectively. The Km values of [14C]oleoyl-CoA were 4.62 and 9.99 μmol/L for ACAT-1 and ACAT-2, respectively. Next, we characterized the enzymatic inhibition of ACAT-1 by K-604. Double reciprocal plots clearly showed that ACAT-1 inhibition by K-604 is competitive rather than non-competitive (Fig. 3A). K-604 competitively inhibited the enzyme activity with oleoyl-CoA with a Ki value of 0.378 μmol/L (Fig. 3B).

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Selectivity of K-604 to ACAT-1 [1]
We examined the potency and selectivity of K-604 for human ACAT-1 and ACAT-2. ACAT-1 activity was inhibited more efficiently by K-604 than by CI-1011 (Fig. 4A), whereas ACAT-2 activity was inhibited more efficiently by CI-1011 than by K-604 (Fig. 4B). The IC50 value of K-604 for ACAT-1 was 0.45 μmol/L and that for ACAT-2 was 102.85 μmol/L, indicating that K-604 is 229-fold more selective for ACAT-1 than ACAT-2. In contrast to K-604, the IC50 value of CI-1011 for ACAT-2 was indistinguishable from that for ACAT-1 (Table 1). Thus, K-604 was regarded as a potent and highly selective ACAT-1 inhibitor.

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K-604 suppresses cholesterol esterification in human monocyte-derived macrophages [1]
We examined the effects of K-604 on cholesterol esterification (metabolic incorporation of exogenously added [14C]oleate into intracellular cholesteryl [14C]oleate generated by ACAT reaction) in cultured human monocyte-derived macrophages. Human macrophages were incubated for 22 h with AcLDL followed by an additional incubation for 2 h with K-604 in the presence of [14C]oleate. Fig. 5 shows that K-604 efficiently inhibited cholesterol esterification in human macrophages with IC50 value of 68 nmol/L.

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K-604 enhances cholesterol efflux from THP-1 cells [1]
We examined the effects of K-604 on cholesterol efflux from THP-1 cell-derived macrophages. THP-1 macrophages were pretreated for 48 h with AcLDL labeled with [3H]cholesterol to induce cholesteryl ester accumulation. Subsequently, cells were incubated for 24 h with 200 μg/mL HDL3 in the presence of various concentrations of K-604. As shown in Fig. 6, K-604 enhanced HDL3-mediated efflux of cellular [3H]cholesterol in a dose-dependent manner. Enhancement of cholesterol efflux by K-604 became significant at 0.1 μmol/L. The effect of K-604 on apoA-I-mediated cholesterol efflux was also examined. Fig. 7 shows that apo-A-I-mediated efflux of cellular [3H]cholesterol was significantly enhanced by K-604 at concentrations of 0.1 μmol/L or higher.

The effects of K-604 on aortic lesion regions and plasma cholesterol levels were studied using F1B hamsters, an animal model susceptible to diet-induced hyperlipidemia and atherosclerosis. Administration of K-604 did not impact body weight or food consumption. Plasma cholesterol levels were roughly 12 times higher in fat-fed hamsters than in chow-fed hamsters, and K-604 significantly reduced plasma cholesterol levels only at the highest dose tested (30 mg/kg), but not at lower doses (1 -10 mg/kg ). Fatty streak lesions stained by Oil Red O were considerably induced by high-fat diet and significantly decreased following K-604 treatment. In addition, histological investigation of atherosclerotic lesions was undertaken. Fatty streak lesions in controls were defined by the presence of foamy macrophages in the subendothelial space. In contrast, the region occupied by foamy macrophages was dramatically reduced after injection of K-604 [1].

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K-604 suppresses atherosclerotic lesions without affecting plasma cholesterol levels in high fat-fed F1B hamsters [1]
Using F1B hamsters, an animal model susceptible to diet-induced hyperlipidemia and atherosclerosis, we assessed the effects of K-604 on aortic lesion areas and plasma cholesterol levels. Administration of K-604 did not affect body weight or food consumption. As shown in Fig. 7, the plasma cholesterol levels in fat-fed hamsters were ∼12-fold higher than those in chow-fed hamsters, which were significantly decreased by K-604 only at the highest dose tested (30 mg/kg) but not at lower doses (1–10 mg/kg) (Fig. 7).
The fatty streak lesions stained with oil red O were markedly induced by the high-fat diet (Fig. 8a), which was significantly reduced by administration of K-604 (Fig. 8b). Further, we performed histological analyses of the atherosclerotic lesions. The fatty streak lesions in the control group were characterized by accumulation of foamy macrophages in the subendothelial space (Fig. 8c). In contrast, the areas occupied by foamy macrophages were markedly reduced by administration of K-604 (Fig. 8d).

Assay for ACAT activity [1]
ACAT activity was determined by measuring the production of cholesteryl[14C]oleate as described previously. Microsomal fractions derived from CHO-ACAT-1 or CHO-ACAT-2 cells were diluted with 2.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in 0.5 mol/L KCl, 0.5 mmol/L EDTA, and 25 mmol/L Tris–HCl (pH 7.8) (buffer A) to a final protein concentration of 1.0 mg/mL. The microsomal fractions containing 9.2 μg of protein for ACAT-1 or 4.9 μg for ACAT-2 in 10 μL of buffer A were mixed with various concentrations of K-604 or CI-1011 in 5 μL of dimethylsulfoxide (DMSO), and reconstituted with 152.5 μL of mixed micelles containing 1.6 mmol/L cholesterol, 11.2 mmol/L phosphatidylcholine, and 9.3 mmol/L taurocholate in 25, 0.5 mmol/L EDTA, and Tris–HCl (pH 7.8). The reaction was started by adding 10 μL of 0.45 μmol/L [14C]oleoyl-CoA, 10 nmol/L fatty acid-free bovine serum albumin (fatty acid-free BSA) and 25 mmol/L Tris–HCl (pH 7.8). Reaction mixtures were incubated for 25 min at 37 °C and terminated by adding 150 μL of chloroform/methanol (2:1). The organic phase was dried under a stream of nitrogen. The lipids were resuspended with 150 μL of chloroform/methanol (2:1) and developed by thin layer chromatography (TLC) using hexane/diethyl ether/acetic acid (75:25:1). Radioactivities of cholesterol [14C]oleate were determined using a BAS 2500 image analyzer.

Cholesterol esterification in human monocyte-derived macrophages [1]
Cholesterol esterification rate was determined by the method of Wilde et al. Peripheral blood mononuclear cells (4.0 × 105 cells) obtained from healthy volunteers were suspended in 0.4 mL of RPMI1640 containing 10% human serum and seeded onto each well of 48-well plates. Cells were incubated for 7 days for differentiation into macrophages. The medium was replaced with 0.2 mL of RPMI1640 containing 10% human serum and 100 μg/mL acetylated low-density lipoprotein (AcLDL) follow by incubation for 22 h. Cells were further incubated for an additional 2 h after adding various concentrations of K-604 in 2 μL of DMSO and 2 μL of saline containing 12% BSA, 10 mmol/L sodium oleate, and 41.7 μCi/mL [14C]oleic acid. Cellular lipids were extracted twice with 0.2 mL hexane/isopropanol (3:2) and radioactivities of cholesteryl [14C]oleate were determined as described in assay for ACAT activity.

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Cholesterol efflux from THP-1 macrophages [1]
Human monocytic THP-1 cells (ATCC TIB202; Dainippon Pharmaceutical Co. Ltd, Osaka, Japan) were maintained with 0.5 mL RPMI1640 containing 10% fetal bovine serum, 100 μg/mL streptomycin, and 50 μmol/L 2-mercaptoethanol (medium A). For experiments, THP-1 cells (5 × 105) were seeded onto 24-well plates and cultured for 3 days in medium A containing 0.1 μg/mL phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Cells were further incubated for 48 h with medium A containing 40 μg/mL AcLDL labeled with [3H]cholesterol. For analysis of HDL3-mediated cholesterol efflux, cells were washed with 0.5 mL of medium A and further incubated for 24 h in 0.5 mL of medium A containing HDL3 (200 μg/mL) in the presence or absence of various concentrations of K-604. For analysis of apolipoprtein A-I (apoA-I)-mediated cholesterol efflux, HDL3 was replaced with 10 μg/mL apo-AI under identical conditions. Cholesterol efflux was determined by counting [3H]cholesterol released from cells. Finally, cells were dissolved in 0.5 mL of 1.0 mmol/L HEPES containing 0.5% Triton X-100 for determination of cell-associated radioactivity.

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The mouse neuroblastoma cell line N2a was cultured in DMEM/Opti-MEM (50:50) with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2 in a humidified incubator. Cells were incubated for 24 h with the ACAT1-specific inhibitor K-604 or isotype-nonspecific ACAT inhibitor CI-1011 at concentration as indicated. K-604 and CI-1011 were dissolved in 100 % ethanol to make a 5 mM and 10 mM stock respectively, and stored at −20 °C till usage. Primary cortical neurons were isolated from mouse brains at postnatal day 0–3 using a procedure as described (Brewer, 1997; Bryleva et al., 2010). Cortical neurons were plated in 6 well plates at 350,000 cells/well and grown in 2 mL/well Neurobasal A with 1xB27, 0.5 mM L-glutamine, and 5 ng/mL fibroblast growth factor. Half of the media was replaced with fresh media once every 7 days. After 14–21 days in culture, cells were used for individual experiments as described in the legends of Figure 5 and Figure 6 [2].

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Analysis of human tau levels in N2a cells [2]
Plasmid DNA encoding WT-tau (pRK5-EGFP-Tau) and P301L-tau (pRK5-EGFPTau P301L) described previously were used. N2a cells were grown in 6 well plates to ~50% confluence and transfected with the plasmid as indicated by using Lipofectamine 3000. Twenty-four hours after transfection, cells were incubated for 24 h with or without K-604 at concentrations as indicated in fresh DMEM/Opti-MEM (50:50) containing 10% FBS, in the presence or absence of 5 mM 3-methyladenine (3MA). Cells were then lysed in RIPA buffer (50 mM Tris-HCl, pH 7.6, 1% Triton X, 1% SDS, 0.5% sodium deoxycholate) with protease and phosphatase inhibitor cocktails, and analyzed for tau levels by western blot.

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Analysis of cellular acidic compartments by flow cytometry [2]
The experiment was performed as described previously (Shibuya et al., 2014). Briefly, primary cortical neurons were pretreated for time as indicated with 0.5 µM K-604. The control cells received solvent vehicle only. Cells were then incubated with 50 nM LysoTracker for 30 min and washed twice with PBS. LysoTracker positive (cellular acidic) compartments were analyzed by using flow cytometry using a BD FACSCanto. Dead cells were excluded from analyses by propidium iodide staining. Data were analyzed by using FlowJo software.

Animal experiments [1]
Male F1B hamsters (n = 30) at 8 weeks old were used for animal experiments. The animal room was controlled at 23 ± 3 °C and relative humidity of 50 ± 20%. Animals were fed a CE-2 chow diet, followed by supplementation with CE-2 containing 0.3% cholesterol and 10% coconut oil for 10 weeks. During fat loading, K-604 was administered orally at 1, 3, 10, or 30 mg/kg/day (n = 6 for each dose group). The control group (n = 6) received an aqueous solution of 0.5% methylcellulose instead of K-604. Tap water was given ad libitum. Body weight was measured at the time of drug administration. Blood was sampled for determination of plasma cholesterol levels using a commercially available kit.

[1]. A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels. Atherosclerosis. 2007 Apr;191(2):290-7.

[2]. Acyl-coenzyme A:cholesterol acyltransferase 1 blockage enhances autophagy in the neurons of triple transgenic Alzheimer's disease mouse and reduces human P301L-tau content at the presymptomatic stage. Neurobiol Aging. 2015 Jul;36(7):2248-2259.

Background: Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1), a major ACAT isozyme in macrophages, plays an essential role in foam cell formation in atherosclerotic lesions. However, whether pharmacological inhibition of macrophage ACAT-1 causes exacerbation or suppression of atherosclerosis is controversial.
Methods and results: We developed and characterized a novel ACAT inhibitor, K-604. The IC(50) values of K-604 for human ACAT-1 and ACAT-2 were 0.45 and 102.85 micromol/L, respectively, indicating that K-604 is 229-fold more selective for ACAT-1. Kinetic analysis indicated that the inhibition was competitive with respect to oleoyl-coenzyme A with a K(i) value of 0.378 micromol/L. Exposure of human monocyte-derived macrophages to K-604 inhibited cholesterol esterification with IC(50) of 68.0 nmol/L. Furthermore, cholesterol efflux from THP-1 macrophages to HDL(3) or apolipoprotein A-I was enhanced by K-604. Interestingly, administration of K-604 to F1B hamsters on a high-fat diet at a dose of >or=1mg/kg suppressed fatty streak lesions without affecting plasma cholesterol levels.
Conclusions: K-604, a potent and selective inhibitor of ACAT-1, suppressed the development of atherosclerosis in an animal model without affecting plasma cholesterol levels, providing direct evidence that pharmacological inhibition of ACAT-1 in the arterial walls leads to suppression of atherosclerosis. [1]

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In summary, we developed and characterized a novel ACAT inhibitor, K-604, which is highly selective for ACAT-1, and observed that this compound suppressed the formation of the macrophage enriched-fatty streak in fat-fed hamsters without affecting plasma cholesterol levels. Our results indicate that pharmacological inhibition of macrophage ACAT-1 in vascular walls leads to suppression of experimental atherosclerosis. The therapeutic potential of K-604 for the progression of atherosclerosis in human should be examined in the future clinical studies. [1]

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The “ACAT inhibitor” CI-1011 fed to animals or fed to humens decreased the plasma concentrations of total triglyceride content (Llaverías et al., 2003), presumably because CI 1011 also inhibits the enzyme activity of diacyl glycerol acyltransferase 1 (DGAT1), a different MBOAT member. At present K-604 is the only small molecule ACAT1 inhibitor available. In cell free biochemical assays, K604 at 0.5 µM inhibited ACAT1 enzymatic activity by 70% without significantly inhibiting the ACAT2 enzyme activity (Ikenoya et al., 2007). In cultured neurons and cultured microglial cells, our results show that the effects of K604 at 0.5 µM corroborate the effects of Acat1 KO. However, these results do not exclude the possibility that at higher concentration, K-604 may inhibit other member(s) of the MBOAT family. A second concern for using the currently available ACAT inhibitors to treat neurodegenerative diseases is that many ACAT inhibitors are very hydrophobic compounds and possess membrane active property (Homan and Hamelehle, 2001). The membrane active compounds can be sequestered within the lipid bilayer and reach high local concentration to affect membrane properties non-specifically. The short term and long term effects of these compounds in the CNS are unknown. To treat AD and other related neurodegenerative diseases, we encourage the development of new small molecule ACAT1-specific inhibitors that are blood brain barrier permeable while devoid of various off-target side effect(s) described above.[2]

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Patients with Alzheimer's disease (AD) display amyloidopathy and tauopathy. In mouse models of AD, pharmacological inhibition using small molecule enzyme inhibitors or genetic inactivation of acyl-coenzyme A (Acyl-CoA):cholesterol acyltransferase 1 (ACAT1) diminished amyloidopathy and restored cognitive deficits. In microglia, ACAT1 blockage increases autophagosome formation and stimulates amyloid β peptide1-42 degradation. Here, we hypothesize that in neurons ACAT1 blockage augments autophagy and increases autophagy-mediated degradation of P301L-tau protein. We tested this possibility in murine neuroblastoma cells ectopically expressing human tau and in primary neurons isolated from triple transgenic AD mice that express mutant forms of amyloid precursor protein, presenilin-1, and human tau. The results show that ACAT1 blockage increases autophagosome formation and decreases P301L-tau protein content without affecting endogenous mouse tau protein content. In vivo, lacking Acat1 decreases P301L-tau protein content in the brains of young triple transgenic AD mice but not in those of old mice, where extensive hyperphosphorylations and aggregation of P301L-tau take place. These results suggest that, in addition to ameliorating amyloidopathy in both young and old AD mice, ACAT1 blockage may benefit AD by reducing tauopathy at early stage.[2]

C23H31CLN6OS3

539.179839372635

574.117

C, 47.99; H, 5.60; Cl, 12.32; N, 14.60; O, 2.78; S, 16.71

217094-32-1

217094-32-1 (HCl);561023-90-3;

22272715

White to light yellow solid powder

4

8

9

35

623

0

N(C1=C(N=C(C)C=C1SC)SC)C(=O)CN1CCN(CCSC2NC3C=CC=CC=3N=2)CC1.Cl

DEKWEGUBUYKTAV-UHFFFAOYSA-N

InChI=1S/C23H30N6OS3.2ClH/c1-16-14-19(31-2)21(22(24-16)32-3)27-20(30)15-29-10-8-28(9-11-29)12-13-33-23-25-17-6-4-5-7-18(17)26-23;;/h4-7,14H,8-13,15H2,1-3H3,(H,25,26)(H,27,30);2*1H

2-[4-[2-(1H-benzimidazol-2-ylsulfanyl)ethyl]piperazin-1-yl]-N-[6-methyl-2,4-bis(methylsulfanyl)pyridin-3-yl]acetamide;dihydrochloride

K-604 HCl; K-604 Hydrochloride; K-604 dihydrochloride; 217094-32-1; 2-(4-(2-((1H-Benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide dihydrochloride; 2-[4-[2-(1H-benzimidazol-2-ylsulfanyl)ethyl]piperazin-1-yl]-N-[6-methyl-2,4-bis(methylsulfanyl)pyridin-3-yl]acetamide;dihydrochloride; 2-[4-[2-(BENZIMIDAZOL-2-YLTHIO)ETHYL]PIPERAZIN-1YL]-N-[2,4-BIS(METHYLTHIO)-6-METHYL-3-PYRIDYL]ACETAMIDE DIHYDROCHLORIDE; 2-{4-[2-(1H-1,3-benzodiazol-2-ylsulfanyl)ethyl]piperazin-1-yl}-N-[6-methyl-2,4-bis(methylsulfanyl)pyridin-3-yl]acetamide dihydrochloride; SCHEMBL6283825; SIA09432; K-604; K604; K 604;

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)

H2O : ~100 mg/mL (~173.72 mM)
DMSO : ~62.5 mg/mL (~108.57 mM)

Solubility in Formulation 1: ≥ 2.25 mg/mL (3.91 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 22.5 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: ≥ 2.25 mg/mL (3.91 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 22.5 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: ≥ 2.25 mg/mL (3.91 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 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (173.72 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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