Acyl coenzyme A:cholesterol acyltransferase inhibition: potential atherosclerosis therapy or springboard for other discoveries?

Cholesterol is an essential building block without which humans and other animals could not exist. As with most necessities, under certain conditions, excess can sharply tip the scale and lead to an unfavourable outcome. Excess cholesterol is stored as cholesteryl ester through an esterification process regulated in part by acyl coenzyme A:cholesterol acyltransferase (ACAT). ACAT is found in many tissue types which require the storage of cholesterol. Most notably, for cardiovascular disease ACAT activity is significant in intestinal and hepatic tissue and arterial macrophages. Several ACAT inhibitors have been investigated for their potential to favourably alter serum lipoprotein levels by blocking intestinal absorption, hepatic inhibition and/or slowing the progression of atherosclerosis through a non-lipid arterial inhibition. Recent evaluations of ACAT and ACAT inhibitors have provided some insight into the therapeutic potential and risks of ACAT inhibition as a means of treating atherosclerosis.     

Acyl-coenzyme A:cholesterol acyltransferase inhibitors for controlling hypercholesterolemia and atherosclerosis

Acyl-coenzyme A:cholesterol acyltransferase (ACAT; Sterol O-acyltransferase/SOAT) is an intracellular enzyme that catalyzes the formation of cholesteryl esters from cholesterol and fatty acyl-coenzyme A. ACAT inhibitors reduce plasma cholesterol levels by suppressing absorption of dietary cholesterol and by suppressing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein in liver and chylomicron in intestine. Moreover, ACAT inhibitors prevent the conversion of macrophages into foam cells in the arterial walls. Thus, ACAT inhibitors are under investigation for controlling hypercholesterolemia and the development of atherosclerosis. Some potent ACAT inhibitors have been tested for their efficacy and safety in humans.     

Acyl coenzyme A:cholesterol acyltransferase inhibitors as hypolipidemic and antiatherosclerotic drugs

Acyl coenzyme A:cholesterol acyltransferase (ACAT) is the enzyme that catalyzes the conversion of intracellular cholesterol into cholesteryl esters. Two ACAT isoforms, termed ACAT1 and ACAT2, have been described. ACAT1 is ubiquitously found, with high expression levels in macrophages, adrenals, sebaceous glands and foam cells from human atherosclerotic lesions. In contrast, ACAT2 expression is restricted to the intestine and the liver of mice and non-human primates. The reaction catalyzed by ACAT is essential for intestinal cholesterol absorption, synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins, and intracellular storage of cholesterol. Therefore, ACAT inhibitors would theoretically reduce plasma cholesterol levels by blocking cholesterol absorption from the diet and by reducing hepatic VLDL synthesis. Moreover, ACAT inhibition could limit the accumulation of cholesteryl esters in the cytoplasm of macrophages, thus reducing the formation of foam cells. In view of these attractive possibilities, a great deal of molecules with ACAT inhibitory properties have been synthesized in the last 20 years. However, only a few of them have reached clinical studies, mainly due to unexpected side effects. On the other hand, most of the compounds assayed in humans have not shown substantial hypolipidemic efficacy. The present article focuses on the current knowledge of the pharmacology of ACAT inhibitors, and, specifically, on the different pharmacological approaches used to evaluate these compounds as hypolipidemic and antiatherosclerotic agents.     

Acyl coenzyme A:cholesterol acyltransferase inhibition: potential atherosclerosis therapy or springboard for other discoveries?

Cholesterol is an essential building block without which humans and other animals could not exist. As with most necessities, under certain conditions, excess can sharply tip the scale and lead to an unfavourable outcome. Excess cholesterol is stored as cholesteryl ester through an esterification process regulated in part by acyl coenzyme A:cholesterol acyltransferase (ACAT). ACAT is found in many tissue types which require the storage of cholesterol. Most notably, for cardiovascular disease ACAT activity is significant in intestinal and hepatic tissue and arterial macrophages. Several ACAT inhibitors have been investigated for their potential to favourably alter serum lipoprotein levels by blocking intestinal absorption, hepatic inhibition and/or slowing the progression of atherosclerosis through a non-lipid arterial inhibition. Recent evaluations of ACAT and ACAT inhibitors have provided some insight into the therapeutic potential and risks of ACAT inhibition as a means of treating atherosclerosis.     

格列本脲对泡沫细胞形成的影响

目的:研究格列本脲(glibenclamide)对THP-1单核巨噬细胞形成泡沫细胞的影响及机制。方法:体外培养人THP-1单核细胞系,由佛波酯(PMA)作用使其分化为巨噬细胞,后者再由乙酰化低密度脂蛋白(Ac-LDL)进行脂质负荷转变为泡沫细胞。不同质量浓度的格列本脲单独或与Ac-LDL共同作用于上述巨噬细胞,以放射性同位素标记底物法检测酰基辅酶A:胆固醇酰基转移酶(ACAT)活性的变化,油红O染色法检测泡沫细胞的形成,酶比色法检测细胞中的总胆固醇、游离胆固醇和胆固醇酯含量。结果:格列本脲使脂质负荷的巨噬细胞转变为泡沫细胞的数目减少,并明显抑制脂质负荷前后巨噬细胞中ACAT酶活性;随着格列本脲剂量增加,脂质负荷前后的巨噬细胞中胆固醇酯含量逐渐降低,呈剂量依赖性趋势。结论:格列本脲抑制泡沫细胞形成,其作用机制可能与其抑制ACAT酶活性和细胞中胆固醇酯含量有关。     

Section Review: Cardiovascular and Renal: ACAT inhibitors in development

Acyl-CoA:cholesterol acyltransferase (ACAT), which catalyses the intracellular formation of cholesteryl esters, plays an important role in the intestinal absorption of cholesterol, foam cell formation within the arterial wall and may be very low density lipoprotein production in the liver. Therefore, the inhibition of ACAT as treatment for hypercholesterolaemia and atherosclerosis is an attractive target. Indeed, many of the ACAT inhibitors have shown potent hypocholesterolaemic and anti-atherosclerotic effects in various experimental animal models. Despite initial failures to show efficacy in humans, several ACAT inhibitors are now under clinical evaluation. The results obtained from these clinical studies should help to determine the future of ACAT inhibitors as hypocholesterolaemic and anti-atherosclerotic agents.     

Update on the role of acyl-CoA:cholesterol acyltransferase inhibitors in atherosclerosis

Cardiovascular disease is one of the leading causes of mortality and morbidity in industrialised nations. Hypercholesterolaemia is one of a number of risk factors identified that influences the development and progression of atherosclerosis. While drugs such as HMGCoA reductase inhibitors or statins have been shown to significantly reduce cholesterol levels and the risk for cardiovascular disease, there is still a pressing need to identify other compounds that might further reduce the risk. One such class of drugs, currently in preclinical and clinical studies, is acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors. Two isoforms of ACAT have been identified; ACAT₁ has a more ubiquitous distribution in steroidogenic tissue, pancreas, intestine and macrophages and ACAT₂ is predominantly expressed in hepatocytes and intestines. In human tissues, ACAT₂ expression is high in foetal hepatocytes but declines in adult hepatocytes. Its expression remains unchanged in foetal and adult human intestines. ACAT enzymes participate in the assembly of chylomicrons and very low density lipoproteins (triglyceride rich lipoproteins) and also in the formation of cholesteryl ester storage droplets within cells residing in the vessel wall. The initial results from preclinical and clinical studies suggest that ACAT inhibitors may have a beneficial effect in altering lipid profiles and in retarding the progression of atherosclerotic disease.     

Selective ACAT inhibitors as promising antihyperlipidemic,antiathero-sclerotic and anti-Alzheimer drugs

Inhibition of ACAT, the enzyme which catalyses the intracellular formation of cholesteryl esters, is a very attractive target for the treatment of hypercholesterolaemia and atherosclerosis. However, in the past years many ACAT inhibitors gave disappointing results in clinical trials showing very low efficacy. In addition, their development was affected by the adrenotoxicity observed in many compounds. The discovery of two isoforms of the enzyme, namely ACAT1 and ACAT2, with different substrate specificity and different potential function, offers a precious information for planning selective inhibitors with reduced secondary effects. Today some potent, bioavailable and non adrenotoxic ACAT inhibitors are under clinical evaluation. Amongst others, a very promising compound is Avasimibe, presently in phase III clinical trials as anti-hyperlipidemic and anti-atherosclerotic agent. Finally, ACAT inhibitors have recently been proposed for the treatment of Alzheimer's disease.     

Acute P-407 Administration to Mice Causes Hypercholesterolemia by Inducing Cholesterolgenesis and Down-Regulating Low-Density Lipoprotein Receptor Expression

Purpose Poloxamer 407 (P-407) is a chemical that induces a dose-controlled dyslipidemia in mice. Our aim was to determine the acute effects of P‐407 treatment on the mechanisms that influence hepatic cholesterol homeostasis. Methods We measured lipid levels in plasma and liver samples from control and P-407-treated mice (24 h post-i.p. injection of 0.5 g kg⁻¹ of P-407 or saline for the control mice). We measured acyl-coenzyme A:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities in liver microsomes. The protein expression of ACAT2, scavenger receptor class B, type I (SR-BI), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G8 (ABCG8), low-density lipoprotein receptor (LDLr), and actin was measured by immunoblot. Results We found an increase in plasma cholesterol and triglyceride levels as well as increased hepatic cholesteryl esters (CE) in P-407-treated mice. The hepatic ACAT microsomal activity and ACAT2 protein expression were not altered by P-407. The protein expression of the LDLr was decreased in the livers of P-407-treated mice. This decrease was specific, because the expression of the SR-BI was unchanged. The P-407-induced hypercholesterolemia was accounted for by increased activity and protein expression of HMG-CoA reductase. ATP-binding cassette transporters A1 and G8 protein expression were not significantly different in P-407-treated mice compared to controls. Conclusions The increased hepatic CE levels, following P-407 treatment, was neither related to an up-regulation of ACAT2 nor enhanced SR-BI expression. Hypercholesterolemia was associated with an up-regulation of both the protein expression and activity of HMG-CoA reductase and decreased LDLr expression.     

Importance of acyl-coenzyme A:cholesterol acyltransferase 1/2 dual inhibition for anti-atherosclerotic potency of pactimibe

Pactimibe sulfate, [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate, a novel Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was investigated in vitro and in vivo to characterize its potential. Pactimibe exhibited dual inhibition for ACAT1 and ACAT2 (concentrations inhibiting 50% [IC₅₀s] at micromolar levels) more potently than avasimibe. Kinetic analysis revealed pactimibe is a noncompetitive inhibitor of oleoyl-CoA (K_i value: 5.6 μM). Furthermore, pactimibe markedly inhibited cholesteryl ester formation (IC₅₀: 6.7 μM) in human monocyte-derived macrophages, and inhibited copper-induced oxidation of low density lipoprotein more potently than probucol. Pactimibe exerted potent lipid-lowering and anti-atherosclerotic effects in atherogenic diet-fed hamsters. At doses of 3 and 10 mg/kg for 90 days, pactimibe decreased serum total cholesterol by 70% and 72%, and aortic fatty streak area by 79% and 95%, respectively. Despite similar cholesterol lowering, fatty streak area reduction was greater by 10 mg/kg. These results suggest that ACAT1/2 dual inhibitor pactimibe has anti-atherosclerotic potential beyond its plasma cholesterol-lowering activity.     

Inhibitors of acyl-coenzyme a: cholesterol acyltransferase

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and fatty acyl-coenzyme A. Animal experiments showed that ACAT inhibitors reduce plasma cholesterol levels by suppressing absorption of dietary cholesterol and by suppressing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein in liver and chylomicron in intestine. Moreover, ACAT inhibitors were shown to prevent formation of macrophage-derived foam cells in the arterial walls. However, a recent double-blind, placebo-controlled, randomized trial of a potent ACAT inhibitor, avasimibe, failed to show significant beneficial effects on coronary atherosclerosis assessed by intravascular ultrasound. For clinical application of ACAT inhibitors, development of more potent compounds and improvements of the methods to evaluate their clinical efficacy are strongly needed.     

Bavachin and isobavachalcone, acyl-coenzyme A: Cholesterol acyltransferase inhibitors from Psoralea corylifolia

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification and plays important roles in intestinal absorption of cholesterol, hepatic production of lipoproteins and accumulation of cholesteryl ester within macrophages and smooth muscle cells. Ethanol extract of Psoralea corylifolia showed a significant inhibition of ACAT enzyme. Via bioactivity-guided fractionation of the ethanol extract of Psoralea corylifolia, two prenylated flavonoids were isolated. Their structures were determined as bavachin (1) and isobavachalcone (2) by spectroscopic analysis (¹H-, ¹³C-NMR, 2DNMR, and ESI-MS). The IC₅₀ values were 86.0 (1) and 48.0 (2) μM in the ACAT assay system using rat liver microsome. Compound 2 also decreased cholesteryl ester formations in HepG2 cells. In addition, this compound showed a noncompetitive type of inhibition of ACAT.     

Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and secretions of apolipoprotein B-containing lipoprotein and bile acids in HepG2

We studied the effect of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl) ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on intracellular cholesterol esterification and the secretion of apolipoprotein B100 (apoB)-containing lipoprotein and bile acids in the human hepatoma cell line HepG2. NTE-122 markably inhibited [3H]oleate incorporation into cholesteryl esters in HepG2 cells incubated with 5 microg/ml 25-hydroxycholesterol as a stimulus for ACAT (IC50=6.0 nM). On the other hand, NTE-122 did not affect [3H]oleate incorporation into triglycerides and phospholipids and [14C]acetate incorporation into cholesterol. The stimulation of ACAT by 25-hydroxycholesterol caused significant increases in the secretion of radiolabeled cholesteryl esters, radiolabeled triglycerides and apoB mass. NTE-122 pronouncedly inhibited the secretion of radiolabeled cholesteryl esters in proportion to the inhibition of cellular cholesterol esterification, and it significantly reduced the secretion of radiolabeled triglycerides and apoB mass in HepG2 cells incubated with 25-hydroxycholesterol. Furthermore, NTE-122 increased the secretion of bile acids synthesized from [14C]-cholesterol. These results suggest that NTE-122 is capable of exhibiting anti-hyperlipidemic effects by reducing both the cholesterol content and the amount of secreted very low-density lipoprotein and enhancing the excretion of bile acid from the liver.     

Effect of pitavastatin on macrophage cholesterol metabolism

OBJECTIVES: Pitavastatin is the first totally synthetic HMG-Co A reductase inhibitor in Japan that significantly reduces LDL cholesterol while raising HDL cholesterol. Clinical trial showed that pitavastatin has potent effects for LDL cholesterol lowering and is expected effectively to prevent atherosclerosis. To clarify the mechanism of reduction of atherosclerosis by pitavastatin, we examined the effect of pitavastatin on foam cell formation of RAW264.7 macrophages. METHODS & RESULTS: Macrophages were cultured with pitavastatin for 24 h and exposed to oxidized LDL with pitavastatin for 3 days. Pitavastatin decreased the cellular cholesteryl ester content in a dose-dependent manner, and this effect was not via inhibition of HMG-CoA reductase because the 3-30 nM pitavastatin did not inhibit [14C]cholesterol synthesis from [14C]acetic acid and the effect was not influenced by addition of mevalonic acid. Pitavastatin increased neutral cholesterol esterase (NCEase) activity and did not affect ACAT activity, and decreased the expression of CD36 and ABCA1 mRNA. The mechanism of the increase of NCEase activity was that pitavastatin directly modified the substrate state, which was cholesterol oleate emulsified with lecithin. CONCLUSION: Clinical blood concentrations of pitavastatin prevent foam cell formation of RAW macrophages by oxidized LDL, and this was not via inhibition of HMG-CoA reductase, and modify substrate condition.     

Therapeutic potential of ACAT inhibitors as lipid lowering and anti-atherosclerotic agents

Hypercholesterolemia is one of the few independent risk factors definitively linked to increased morbidity and mortality due to myocardial infarction. One possible therapy of current interest is the prevention of the absorption of dietary cholesterol by inhibiting the enzyme, acyl-CoA: cholesterol acyltransferase (ACAT), which catalyses the intracellular formation of cholesterol esters. Evidence is now accumulating that suggests that ACAT inhibition may not only lower plasma cholesterol levels, but may also have a direct effect at the artery wall, where ACAT has been shown to be responsible for the accumulation of cholesterol esters in arterial lesions. Drago Sliskovic and Andrew White discuss the importance of ACAT in the lipid transport system and the consequences of its inhibition in a variety of tissues, with emphasis on both lipid-lowering and anti-atherosclerotic effects.     

The liver metabolite S-422 of the hypolipidaemic drug benfluorex decreases cholesterol esterification in fibroblasts and monocyte-like cells

Summary The effects of S-422 (1-(3-trifluoromethylphenyl)-2-[N-(2-hydroxyethyl) amino] propane), an hepatic metabolite of the hypolipidaemic drug Benfluorex, on lipid metabolism have been investigated in two experimental models: in human fetal lung fibroblasts, for study of the apo B/E receptor-mediated regulation of cholesterol metabolism, and in murine J 774 monocyte-like cells, for study of the scavenger receptor-mediated induction of cholesteryl ester accumulation.In human fibroblasts S-422 increased low density lipoprotein (LDL) catabolism by about 20%, whereas it decreased oleic acid incorporation into triacylglycerols and cholesteryl esters by 25 and 35%, respectively. In J 774 cells, S-422 decreased acetylated LDL degradation and cholesteryl ester formation by about 35%. In both cell types, ACAT activity was significantly reduced by the drug, either after a 24 h pretreatment of the cultured cells, or after an in vitro 30 min preincubation of cell homogenates.The results suggest that S-422, and thus Benfluorex, might prevent the development of atherosclerotic plaques.Abbreviations LDL Low Density Lipoprotein - AcLDL Acetylated Low Density Lipoprotein - ACAT Acylcoenzyme A: Cholesterol-O-Acyl Transferase     

Acyl-CoA:cholesterol O-acyl transferase (ACAT) inhibitors. 1. 2-(Alkylthio)-4,5-diphenyl-1H-imidazoles as potent inhibitors of ACAT.

A potent, bioavailable ACAT inhibitor may have beneficial effects in the treatment of atherosclerosis by (i) reducing the absorption of dietary cholesterol, (ii) reducing the secretion of very low density lipoproteins into plasma from the liver, and (iii) preventing the transformation of arterial macrophages into foam cells. We have found that a mevalonate derivative 2, which contains a 4,5-diphenyl-1H-imidazol-2-yl moiety, inhibits rat hepatic microsomal ACAT in vitro and produces a significant hypocholesterolemic effect in the cholesterol-fed rat. Structure-activity relationships for analogues of 2 demonstrate that the 4,5-diphenyl-1H-imidazole moiety is a pharmacophore for inhibition of rat microsomal ACAT.     

Influence of 5-tridecylpyrazole-3-carboxylic acid, a new hypolipidaemic agent, on cholesteryl ester formation in rabbit intestinal mucosa

The comparative effects of 5-tridecylpyrazole-3-carboxylic acid (TDPC), beta-sitosterol and melinamide on the esterification of cholesterol (CH) have been investigated in rabbit intestinal microsomes and cytosol in-vitro. The three agents did not show an effect on cholesteryl ester formation by cholesterol esterase (CEase). TDPC and beta-sitosterol did not affect cholesteryl oleate formation from oleoyl CoA by microsomal acyl CoA:cholesterol acyltransferase (ACAT), whereas melinamide significantly inhibited cholesteryl oleate formation. TDPC significantly inhibited the incorporation of oleic acid into cholesteryl oleate, which is associated with acyl CoA synthetase (ACS) plus ACAT in mucosal microsomes, at a concentration of 20-100 microM. On the other hand, 5-tridecylpyrazole-3-carbinol (TDPC-OH) a congener of TDPC, and beta-sitosterol did not show any effect. From these results, it is demonstrated that carboxylic moiety of TDPC is necessary to inhibit ACS in-vitro. According to the kinetic analytical results, it is suggested that TDPC acts as a competitive inhibitor of ACS. These results suggest that the inhibitory effect of TDPC on cholesteryl ester formation may be mediated by an inhibition of ACS activity. It is apparent from the data presented that there are substantial differences between TDPC, beta-sitosterol and melinamide with respect to their action on cholesteryl ester formation in rabbit intestinal mucosa.     

Inhibition of acyl-CoA: cholesterol acyltransferase activity by cyclodepsipeptide antibiotics.

The effect was studied of the fungal cyclodepsipeptide antibiotics beauvericin and seven distinct enniatins on acyl-CoA: cholesterol acyltransferase (ACAT) activity. In an enzyme assay using rat liver microsomes, all the compounds were found to inhibit ACAT activity. The drug concentration that caused 50% inhibition (IC50 value) of the enzyme activity was determined to be 3.0 microM for beauvericin, indicating that the compound is one of the most potent ACAT inhibitors of microbial origin. Enniatins exhibited much higher IC50 values of 22 to 110 microM. More hydrophobic enniatins showed more potent inhibitory activity. Furthermore, the ACAT inhibitory activity was evaluated as inhibition of cholesteryl ester formation in a cell assay using J774 macrophages. Calculation of the ratio, CD50 value (the drug concentration causing 50% cell damage)/IC50 value of cholesteryl ester formation, indicated that beauvericin shows the highest specificity. These data indicate that beauvericin is one of the most potent and specific ACAT inhibitors of microbial origin.     

Direct effect of an acyl-CoA:cholesterol acyltransferase inhibitor, F-1394, on atherosclerosis in apolipoprotein E and low density lipoprotein receptor double knockout mice

The acyl-CoA:cholesterol acyltransferase (ACAT) enzyme is thought to be responsible for foam cell formation and the subsequent progression of atherosclerosis. The apolipoprotein E and low density lipoprotein receptor double knockout (apoE/LDLr-DKO) mouse is an animal model that develops severe hyperlipidaemia and atherosclerosis. Here we have examined the effect of oral administration of an ACAT inhibitor, F-1394, on atherosclerosis in apoE/LDLr-DKO mice fed a regular chow diet. In en face analysis, a dose of 10, 30, or 100 mg kg(-1) day(-1) F-1394 for 10 weeks reduced the extent of lesions visible in the aorta by 24, 28 and 38%, respectively, as detected by staining with oil red O, without affecting serum cholesterol level in these mice. At the highest dose 100 mg kg(-1) day(-1) of F-1394, the reduction was statistically significant. For quantitative analysis of the cellular and non-cellular components comprising the lesions at the aortic sinus, the effects of an oral dose of 100 mg kg(-1) day(-1) F-1394 for 15 weeks were studied. There was a significant reduction (31.9%) in the oil-red O-stained area in cross-sections of the aortic sinus. In addition, the neointimal area, as well as levels of ACAT-1 protein tended to be decreased (15.2 and 25.8%, respectively, not significant). However, the areas containing macrophages, smooth muscle cells, and collagen were not affected by F-1394. In vitro, F-1394 attenuated foam cell formation in mouse peritoneal macrophages. These results indicate that ACAT may be primarily responsible for lipid accumulation in atherosclerotic lesions, and that its inhibition diminishes the lipid deposition via a direct effect on macrophages in the arterial wall.