Inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase and cholesterol biosynthesis by beta-lactone inhibitors and binding of these inhibitors to the enzyme.

The beta-lactones L-659,699 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2- oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic acid) and its radioactive derivative 3H-L-668,411 (the 2,3-ditritiated methyl ester of L-659,699) inhibited a partially purified preparation of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase with an IC50 of 0.1 microM. These compounds were also found to inhibit the incorporation of [14C]acetate into sterols in cultured Hep G2 cells with an IC50 of 3 microM. New kinetic evidence indicated that inhibition of the isolated enzyme was irreversible. In contrast, sterol biosynthesis in cultured Hep G2 cells was rapidly restored upon removal of the compound from the medium of inhibited cultures, suggesting reversibility of inhibition in the cells. Radioactivity was found to be associated with a single cytoplasmic protein by SDS/PAGE of the cytoplasm of Hep G2 cells after incubation of the cells with the inhibitor 3H-L-668,411. This protein was identified as cytoplasmic HMG-CoA synthase. Binding of the radioactive compound to the enzyme was decreased with time if the radioactive inhibitor was removed from the medium. Exposure of a gel containing the radioactive enzyme-inhibitor complex to neutral hydroxylamine also resulted in a loss of radioactivity from the gel. The purified rat liver enzyme reacted with the 3H-ligand to form a stable enzyme-inhibitor complex which could be isolated by h.p.l.c. Radioactivity was also subsequently lost from this complex when it was incubated with neutral hydroxylamine. Incorporation of [14C]acetate into cholesterol in mouse liver was inhibited in a reversible manner after oral administration of the beta-lactone inhibitor. These studies, as well as the kinetic evidence presented, suggest that the beta-lactone inhibitors acylate HMG-CoA synthase in a reaction which appears to be irreversible in vitro, but is easily reversed in cultured cells and in animals.

On the etiology of subcapsular lenticular opacities produced in dogs receiving HMG-CoA reductase inhibitors.

The administration of high dosages of various hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors has resulted in the development of subcapsular lenticular opacities in dogs. While dogs receiving cataractogenic doses of HMG-CoA reductase inhibitors experienced profound decreases in circulating serum cholesterol concentrations (40-60% reductions in total serum cholesterol), a causal relationship between serum cholesterol lowering and cataractogenesis was not established. A strong relationship was demonstrated, however, between the systemic exposure to inhibitor (plasma drug levels) and the cataractogenic potential of the various compounds studied. Analysis of lenses from dogs chronically dosed with various HMG-CoA reductase inhibitors revealed the presence of low drug levels in the lens (less than 500 ng equivalents g-1), but no correlation was observed between the amount of drug associated with the lens after chronic treatment and cataract development. In addition, no abnormalities in cholesterol content or sterol composition were observed in clear and/or cataract containing lenses from dogs chronically dosed with HMG-CoA reductase inhibitors. The kinetics of drug appearance in the aqueous and lens cortex was assessed after doses of various HMG-CoA reductase inhibitors, and suggested somewhat higher but not statistically significant peak concentrations of inhibitor were achieved by compounds which produced a higher incidence of cataracts. These data have suggested that high doses of HMG-CoA reductase inhibitors may increase lenticular exposure to drug via the aqueous humor by producing a substantial systemic exposure to drug substance. This may result in an increased concentration of inhibitor in the outer cortical region of the lens where cholesterol synthesis is critical, thereby resulting in the development of opacities. The production of lenticular changes by a HMG-CoA reductase inhibitor of diverse chemical structure establishes, with reasonable assurance, that these lens changes are mechanism based (i.e. a product of the biochemical mechanism of action of this class of compounds). An extrapolation of these findings to patients receiving therapeutic dosages enables a favorable risk evaluation since the doses to be employed clinically are much lower and result in a far lower systemic exposure to drug substance.

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631).

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.

Metabolism of lovastatin by rat and human liver microsomes in vitro.

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.

Brain and optic system pathology in hypocholesterolemic dogs treated with a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The cholesterol lowering compound lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34 HMG CoA reductase), was given in nine separate experiments to normocholesterolemic dogs at rates up to 180 times the maximum therapeutic dose in man (1 mg/kg/day). Mean serum total cholesterol concentrations were reduced as much as 88% below normal. Clinical evidence of neurotoxicity occurred in up to 37% of animals given 180 mg/kg/day lovastatin for 11 or more days, especially in one laboratory where the dosing regime resulted in higher concentrations of plasma drug levels. Dogs receiving 60 mg/kg/day or less never exhibited neurologic signs. The central nervous system (CNS) of affected dogs exhibited endothelial degeneration and hemorrhagic encephalopathy. Focal extravasation of horseradish peroxidase occurred frequently (6/8) in the retrolaminar optic nerve of asymptomatic or clinically affected dogs given 180 mg/kg/day lovastatin, with endothelial degeneration and discrete optic nerve degenerative lesions interpreted as ischemic. The association between the degree of hypocholesterolemia and occurrence of clinical signs was not exact. Total brain cholesterol was similar in treated and control dogs. Hypocholesterolemic dogs had proportionally lowered serum concentrations of alpha-tocopherol, but oral supplementation of this vitamin did not prevent the neurologic syndrome. Endothelial degeneration in the CNS and optic nerve may have reflected in vitro morphologic effects of HMG CoA reductase inhibitors due to extreme inhibition of nonsterol isoprene synthesis. Retinogeniculate axonal (Wallerian-like) degeneration occurred in greater than or equal to 12% of dogs given 60 mg/kg/day or more lovastatin, with central chromatolysis of occasional retinal ganglion cells. These neuroaxonal changes may have been secondary to vascular effects, but superimposed direct neurotoxic action at the high dosage levels of lovastatin could not be excluded. There was no evidence of drug induced adverse effects in the CNS of dogs given up to 30 mg/kg/day lovastatin for 2 years.

Separation and identification of triglycerides, cholesteryl esters, cholesterol, 7-dehydrocholesterol, dolichol, ubiquinone, alpha-tocopherol, and retinol by high performance liquid chromatography with a diode array detector.

A simple isocratic high performance liquid chromatography (HPLC) system is described that allows separation and identification of cholesteryl esters, triglycerides, ubiquinone, alpha-tocopherol, dolichol, cholesterol, 7-dehydrocholesterol, and retinol. This consisted of a normal phase cyanopropyl column with 0.1% isopropanol in heptane as the solvent. The effluent was monitored with an LKB model 2140 diode array detector which enabled the lipids to be identified by their characteristic absorption spectra. This system was applied to a sample of dog liver in which cholesteryl esters, retinyl esters, triglycerides, ubiquinone, dolichol, cholesterol, and retinol were identified. Retinyl esters and vitamin D esters were identified by their similarity in absorption spectra to retinol and vitamin D. A system to transfer and store the chromatograms on the VAX PDP-11 or an optical disc is also described.

Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699.

A beta-lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylating), EC, 4.1.3.5] from rat liver. The structure of this beta-lactone, termed L-659,699, is (E,E)-11-[3-(hydroxy-methyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4 - undecadienenoic acid. A partially purified preparation of cytoplasmic HMG-CoA synthase from rat liver was inhibited by L-659,699 with an IC50 of 0.12 microM. The enzyme HMG-CoA reductase, beta-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, and fatty acid synthase were not inhibited to any extent by this compound. In cultured Hep G2 cells, the compound inhibited the incorporation of [14C]acetate into sterols with an IC50 of 6 microM, while incorporation of [3H]mevalonate into sterols in these cells was not affected. The activity of HMG-CoA reductase in the cultured Hep G2 cells was induced in a dose-dependent manner by incubation with L-659,699. A 37-fold increase in reductase was observed after a 24-hr incubation with 62 microM L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed.

Mevinolinic acid biosynthesis by Aspergillus terreus and its relationship to fatty acid biosynthesis.

Mevinolinic acid, the open acid form of mevinolin, which is a metabolite of Aspergillus terreus, has been shown to be a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (Alberts et al., Proc. Natl. Acad. Sci. U.S.A. 77:3957-3961, 1980). The biosynthesis of mevinolinic acid was studied by examining the incorporation of [1-14C]acetate and [methyl-14C]methionine into the molecule. These isotopes were rapidly incorporated into mevinolinic acid, with [1-14C]acetate and [methyl-14C]methionine incorporation being linear for at least 10 and 30 min, respectively. A comparison of acetate incorporation into mevinolinic acid and fatty acids indicated that mevinolinic acid biosynthesis increased with a maximum between days 3 and 5 of growth; at this time cell growth had ceased and fatty acid biosynthesis was negligible. Hydrolysis of the mevinolinic acid and isolation of the products showed that [1-14C]acetate and [methyl-14C]methionine were incorporated into the 2-methylbutyric acid side chain as well as into the main (alcohol) portion of the molecule.

Studies on the in vivo synthesis of triacylglycerol in mouse liver.

The in vivo biosynthesis of hepatic glycerolipids was examined by studying the incorporation of [2-3H] glycerol into triacylglycerol in the mouse. The isotope was administered by rapid injection into the portal vein. The incorporation of glycerol was linear for about 1 min and maximal rates were seen in the presence of additional oleic acid. At 20 mumol of oleic acid bound to 1 mumol of bovine serum albumin, the highest level tested, glycerol incorporation was still increasing linearly, whereas a plateau was reached at 4 mumol of glycerol. The liver incorporated 200-300 nmol of [2-3H] glycerol into triacyglycerol per min when 20 mumol of albumin-bound oleic acid plus 4 mumol of glycerol were injected in a 0.2 ml volume. Studies on the uptake by the liver after intraportal injection revealed that at 0.5 min the liver retained 2 mumol of glycerol and 5 mumol of oleic acid, indicating that the uptake of substrate was not rate-limiting. The results suggest that the availability of substrate is a major factor in the regulation of triacyglycerol biosynthesis by the liver.

A sensitive assay for phosphatidate phosphohydrolase in mouse liver microsomes.

A technique is described for the assay of phosphatidate phosphohydrolase using 1,2-[9,10-3H]dioleoyl-sn-glycero-3-phosphate as a substrate. This substrate was prepared enzymatically using mouse liver microsomes washed with 0.5 M NaCl, which synthesize minimal amounts of neutral lipids at high enzyme concentrations. Measurement of the product, 1,2-[9,10-3H]dioleoylglycerol, was 10-fold more sensitive than the usual colorimetric assay for inorganic phosphate release. In addition, the assay provides information about the relative contribution of other activities which limit the availability of diacylglycerols for further esterification to triacylglycerols and/or phospholipids.

The effct of cerulenin on sterol biosynthesis in Saccharomyces cerevisiae.

Cerulenin specifically inhibited fatty acid biosynthesis in Saccharomyces cerevisiae without having an effect on sterol formation. Ergosterol was not required for cell growth in the presence of cerulenin (1 microgram/ml). The addition of fatty acids to the growth medium reduced the amount of ergosterol formed by 45%; further addition of cerulenin to the media had no effect on the amount of ergosterol synthesized by the cells. The incorporation of 3H from 3H2O into ergosterol was not affected by cerulenin whereas incorporation into fatty acids was inhibited by 90%.

Effect of halofenate and clofibrate on lipid synthesis in rat adipocytes.

The free acids of the plasma lipid-lowering agents, halofenate and clofibrate inhibited the incorporation of radioactive glucose and pyruvate into fatty acids of isolated adipocytes prepared from rat epididymal fat pads. The concentration which inhibited fatty acid synthesis was dependent on the bovine serum albumin concentration in the incubation. The 50 per cent inhibitory concentration of the free acid of halofenate in 1 per cent, 2 percent and 4 per cent albumin was 0.9 mM, 2.3 MM and 4.4 mM, respectively. The potency of clofibrate was also lowered by increasing the albumin concentration. These compounds inhibited the uptake of both [14C]glucose and [14C]pyruvate to the same degree as the incorporation of these substrates into fatty acids. However, the drugs either had no effect on , or stimulated the uptake of palmitate by the cells. Leucine accumulation by the adipocytes was unaffected by halofenate (free acid) and inhibited by clofibrate (free acid). A comparison of these agents with (minus)-hydroxycitrate, kynurenate and cerulenin (inhibitors of ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase, respectively) on the oxidation of pyruvate suggested that they inhibited pyruvate metabolism at or near the enzyme, pyruvate dehydrogenase.

Effect of halofenate and clofibrate on growth and lipid synthesis in Saccharomyces cerevisiae.

Halofenate-free acid (HFA) inhibited the growth of Saccharomyces cerevisiae by 50% at a concentration of 0.34 mm. This inhibitory effect was prevented by addition of either oleate or acetate, but not by pyruvate. When cell growth was supported by oleate, HFA inhibited the incorporation of radioactive carbon from glucose-U-(14)C or pyruvate-2-(14)C into fatty acids and sterols. The incorporation of radioactive carbon into fatty acids and sterols from acetate-2-(14)C was unaffected by the compound. When cell growth was supported by either oleate or acetate, HFA inhibited the conversion of pyruvate-1-(14)C to (14)CO(2). These results suggest that HFA inhibits the conversion of pyruvate to acetate in yeast. Partially purified yeast pyruvate dehydrogenase was inhibited 50% by 5.5 mm HFA; however, the concentration required for 50% inhibition was considerably reduced when the enzyme was preincubated with the compound at room temperature. In a similar manner, the hypolipidemic agent clofibrate-free acid inhibited the growth of yeast by 50% at 3.0 mm. This inhibition was also prevented by acetate and not by pyruvate. In addition, clofibrate-free acid inhibited partially purified pyruvate dehydrogenase by 50% at a concentration of 37.0 mm.

Enzyme specificity as a factor in regulation of fatty acid chain length in Escherichia coli.

Various acyl-acyl carrier protein intermediates in saturated and unsaturated fatty acid biosynthesis were tested as substrates for beta-ketoacyl-acyl carrier protein synthetase. With both classes of substrates the condensing enzyme in fatty biosynthesis demonstrates specificities which indicate that it might be an important factor in determining fatty acid chain length in Escherichia coli.