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Biochemical aspect of HMG CoA reductase inhibitors
BIOCHEMICAL ASPECT OF HMG COA REDUCTASE INHIBITORS AKIRA ENDO and KEIJI HASUMI Department of Agricultural and BiologicalChemistry, Tokyo Noko University, Fuchu, Tokyo,Japan INTRODUCTION The biosynthetic pathway for cholesterol involves more than 25 different enzymes. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase [mevalonate : NADP + oxidoreductase (CoA-acylating), EC 1.1.1.34], which catalyzes the conversion of HMG CoA to mevalonate, is the rate-limiting enzyme in this pathway (Fig. 1). In humans, approximately one-third of total body cholesterol is derived from the diet, and two-thirds is synthesized, mainly by the liver and intestine. The major cause of death in developed countries is coronary heart disease. A primary risk factor for this disease is known to be hypercholesterolemia. An attractive and potentially effective way to lower blood cholesterol levels would, therefore, be to control cholesterol synthesis by selectively inhibiting H M G CoA reductase. The highly functionalized fungal metabolite compactin (ML-236B) was first isolated by our group as a potent inhibitor of H M G CoA reductase (Fig. 1) (1). Subsequent to the studies disclosing the structure and biological activities of compactin, a series of compactin analogs have been obtained either from micro-organisms or by biological and chemical techniques. Several of those compounds are now on the market as hypocholesterolemic agents in the United States and in some European countries. In this review we will examine the isolation and synthesis of compactin analogs and their biochemical activities. ISOLATION AND SYNTHESIS OF HMG CoA REDUCTASE INHIBITORS
Compactin Analogs Isolated from Micro-organisms Compactin (ML-236B) is the first of a series of competitive inhibitors of H M G CoA reductase. This compound is produced by Penicillium citrinum (1), Pen. brevicompactum (2) and Pen. cyclopium (3). Along with compactin, ML-236A (Fig. 2), ML-236C, and dihydrocompactin, which are analogous to compactin, are produced by Pen. citrinum (1, 4). Monacolin K (mevinolin) (Fig. 2) is the main active compound produced by Monascus 53
54
A. ENDO and K. HASUMI HO~ooH
2NADPH÷2H*
HMG-CoA
HOC~)H
MevalOnicacid
FIG. 1. HMG CoA reductasereaction. ruber (5, 6) and Aspergillus terreus (7). In addition, monacolins J (Fig. 2), L, M and X and dihydromonacolin L were isolated from Mon. ruber (8-10) and dihydromevinolin from Asp. terreus (11). Microbial and Chemical Modification Compactin is converted to 31~-hydroxycompactin by Mucor hiemalis and Nocardia sp. (12, 13). Compactin is also hydroxylated to 3othydroxycompactin by Syncephalastrum nigricans (14) and to 6ot-hydroxy/so-compactin by Absidia coerulea (15). 8ot-Hydroxycompactin and 8othydroxymonacolin K are derived from compactin and monacolin K, respectively, by growing Schizophyllum commune (16). 5 '-Phosphocompactin acid and 5 '-phosphomonacolin K are produced by several fungal strains (17). The 2(S)-methylbutylyl ester of compactin and monacolin K can be removed, giving ML-236A and monacolin J, respectively, by chemical hydrolysis or by the action of a carboxylesterase of the fungus Emericella unguis (18, 19). A series of the side chain ester analogs of monacolin K (mevinolin) are derived from the silyl ether of monacolin J, which includes synvinolin (simvastatin) (Fig. 2) (20). H
0
o~
14t'k
o
R:H, Compactin (ML-236B) R=CH3.Monacolin K (mevinolin)
"CO z Na
~'- ° "
CS-514 (pravastat in)
0
: Ha
H3C,"W Synvinolin (simvastatin)
A
' '"~[~
:
H3
R,,~ R=H. ML-236A R=CH3.Monaco~inJ
FIG. 2. Majorcompactin-relatedcompounds.
HMG CoA REDUCTASE INHIBITORS
55
Chemical Synthesis A series of 7-(3,5-disubstituted [1,1'-biphenyl]-2-yl)-3,5-dihydroxy6-heptanoic acids were synthesized and assayed for inhibitory activity of HMG CoA reductase. Among these compounds, compounds 100(+) and 110(+) have 2.8 times the inhibitory activity of compactin (21). Of the HMG CoA reductase inhibitors described above, four compounds have been studied in the clinic: compactin, mevinolin, 3l~-hydroxycompactin acid monosodium salt (pravastatin, CS-514), and synvinolin. MECHANISM
OF HMG CoA REDUCTASE
INHIBITION
The lactone form of compactin analogs is inactive and, in vivo, hydrolyzed to the corresponding 3', 5'-dihydroxyacid, which is the principal active form of these compounds. Most of these compounds inhibit HMG CoA reductase of rat liver at - 1 nM (6, 22-24). The inhibition of HMG CoA reductase by compactin is reversible. As can be expected from the structure of the 3', 5'-dihydroxypentanoic acid portion, the inhibition is competitive with respect to HMG CoA and non-competitive with respect to NADPH. The K i values for the acid form of compactin, monacolin K (mevinolin) and CS-514 are 1.2, 0.49 and 2.3 nM, respectively, while under the same conditions, the K m value for HMG CoA is -10/xM (6, 22). Thus, the affinity of HMG CoA reductase for compactin is 10,000 times higher than its affinity for the natural substrate HMG CoA. The conformational change of the HMG CoA reductase molecule occurs by its binding to compactin, leading to alterations in antigenicity (25). STRUCTURE-ACTIVITY
RELATIONSHIP
The compactin molecule is composed of four moieties; (a) the lactone or the hydroxylic 3', 5'-dihydroxyacid, (b) the moiety bridging the lactone and the lipophilic groups, (c) the decarine ring, and (d) the side chain ester. The hydroxyl group at the 3'-position of the 3', 5'-dihydroxypentanoic acid portion is the trans-isomer and much more active than the cis-isomer (21, 26). The addition of a methyl group to the 3'-position to give the trans-form, a compound which more closely resembles the HMG portion of HMG CoA, does not stimulate activity appreciably (26). Conversion of either the 3'- or 5'-hydroxyl group to the corresponding methyl ester ablates activity (unpublished data), indicating a crucial role of these hydroxyl groups in activity. Replacement of the carboxyl group of compactin with a carboxamide group also abolishes activity (unpublished data). Increasing the length of the bridge to three carbons reduces activity (21). The addition of a methyl group to the 6-position of the decarine ring of compactin to give monacolin K (mevinolin) doubles activity (6). The introduction of a hydroxyl group to give CS-514 slightly reduces activity (24).
56
A. ENDO and K. HASUMI
Activity is reduced one-third by the hydroxylation at the 8a-position (16), partial or complete hydrogenation of the decarine ring gives no detectable change in activity (unpublished data). Monacolin J and L (or ML-236A and ML-236C), which lack the side chain ester, are far less active than monacolin K (or compactin) (27). The introduction of an additional aliphatic group on the carbon to the carbonyl group of the side chain increases potency. Synvinolin, which has an additional methyl group on the carbon a, has about 2.5 times the inhibitory activity of mevinolin (20). Increasing the length of the side chain and the removal of the terminal methyl group lowers the inhibitory activity. Stereochemistry at the carbon a to the carbonyl is not crucial as the natural product (mevinolin) and its diastereomer are equally potent (20). INHIBITION
OF CHOLESTEROL
SYNTHESIS
In Cultured Cells
Compactin inhibits cholesterol biosynthesis in a variety of cultured animal and human cells at nM concentrations (20-30). In cultured human skin fibroblasts, inhibition of sterol synthesis from [14C]acetate by compactin is 50% at 1 riM, 80% at 10 nM, 90% at 100 riM, 95% at 1 /zM, and 100% at 10/zM, respectively (28). Under these conditions, sterol synthesis from [14C]mevalonate and fatty acid synthesis from [14C]acetate are not significantly affected. The inhibition of H M G CoA reductase by compactin analogs results in the depletion of mevalonate. In addition to cholesterol, several isoprenoids like ubiquinones and dolichols are also derived from mevalonate. In human skin fibroblasts grown in the presence of low density lipoprotein(LDL)cholesterol, compactin has no detectable effects on the synthesis of these two isoprenoids at 10 nM, a concentration that causes over 50% inhibition of sterol synthesis (unpublished data). When H M G CoA reductase is partially inhibited by compactin, cells must have some way of diverting the small amounts of synthesized mevalonate preferentially into these non-sterol isoprenoids. At higher concentrations where sterol synthesis is reduced by over 90%, compactin inhibits cell growth as well (28, 31, 32). This inhibition can be overcome and cells can grow normally if mevalonate is added to the culture medium, indicating that compactin is a specific inhibitor of H M G CoA reductase. In Animals and Humans
When administered orally to mice, compactin is readily absorbed into the liver, the major site of cholesterogenesis, as compared with other organs
HMG CoA REDUCTASE INHIBITORS
57
(33). When 50 mg/kg of compactin was given orally to rats, sterol synthesis in vivo in the liver was inhibited by 92% after 4 hr, while the inhibition in other organs (ileum, adrenal, kidney, lung, spleen and testis) was 50-80% (33). Inhibition of cholesterol synthesis after a single dose of mevinolin 46/zg/kg in rats was 50% (7). Cholesterol synthesis, as determined by labeled acetate incorporation in vitro, increased by 40% in isolated mononuclear leucocytes from 20 patients with familial hypercholesterolemia treated with mevinolin 20 and 40 mg twice daily (34). In a double-blind placebo-controlled single dose study, mevinolin (100 mg) produced a 78% reduction in plasma mevalonate concentrations between 2 and 6 hr after administration in healthy volunteers (35). Plasma mevalonate concentrations were directly correlated with the rate of whole body cholesterol synthesis. In a longer term study, mevinolin (25 and 50 mg twice daily for 4 weeks) reduced urinary output of mevalonate by 81% in healthy volunteers (35). Reduction of urinary mevalonate excretion by mevinolin was also observed in patients with familial hypercholesterolemia (FH) (36). Using the sterol balance method to estimate cholesterol synthesis, Grundy and Bilheimer (37) found mevinolin 20 mg twice daily decreased output of neutral steroids in patients with FH by 22 to 44%, although not below levels observed in healthy volunteers. INDUCTION
OF HMG CoA REDUCTASE
ACTIVITY
Compactin analogs block the production of mevalonate so that the cells, now dependent on exogenous mevalonate, are phenotypic auxotrophs. When the HMG CoA reductase activity of human skin fibroblasts is inhibited by compactin, where mevalonate production is completely inhibited, a striking increase in the cellular levels of HMG CoA reductase (3.5- to 15-fold) occurs (29). The induced HMG CoA reductase could not be fully suppressed by LDL; the suppression is 85% (30). In the presence of compactin the enzyme could be fully suppressed only when the cells are given a small amount of mevalonate in addition to LDL. The induction of HMG CoA reductase activity by compactin requires protein synthesis and thus is completely blocked by cycloheximide (29). The induction of reductase activity in CHO-K1 cells treated with mevinolin is due to decreases in the rate of enzyme degradation but not changes in enzyme synthesis (38). Increases in HMG CoA reductase activity by compactin analogs also occurs in the liver of rats (39) and in rat hepatocytes (40). A combination of mevinolin and cholestyramine in the diet of rats produces a greater increase in HMG CoA reductase activity than either drug alone. The increases in this enzyme are a result of increased synthesis and decreased degradation (41, 42). In these experiments, increases in HMG CoA reductase synthesis JAER 28---C*
58
A. ENDO and K. HASUMI
and decreases in degradation (increase in apparent half-life) are 12- and at least 4-fold, respectively, for rats fed both mevinolin and cholestyramine. The mechanism by which these drugs induce H M G CoA reductase activity appears to be due to activating the synthesis of the reductase protein mediated by increasing its mRNA (43-45). Mevinolin induces reductase activity in mice, even when the animals were fed on a cholesterol-enriched diet (46). This augmentation in reductase activity was suppressed 90% by mevalonate. Mevalonate inhibits the rate of synthesis and enhances the rate of degradation of H M G CoA reductase in rat hepatocytes (47). It is likely that synthesis and degradation of the reductase are regulated by either mevalonate or, more likely, a product of mevalonate metabolism. In an insect cell line (Drosophila Kc cells) that neither synthesizes nor requires cholesterol for growth, H M G CoA reductase is not modulated by cholesterol. However, compactin causes a 5- to 10-fold increase in reductase activity. Mevalonate blocks this elevation (48). The non-sterol regulatory product derived from mevalonate is distal to isopentenyl-l-phosphate in the insect cells (47). In Chinese hamster ovary cells, HMG CoA reductase and H M G CoA synthase (EC 4.1.3.5) are co-ordinately induced by mcvinolin (49). In rats treated with mevinolin, acetoacetyl CoA synthetase (EC, not available) is also increased by compactin (50).
EFFECTS
ON CHOLESTEROL
AND LIPOPROTEINS
In Non-human Animals Compactin analogs fail to reduce plasma cholesterol levels in normal rats and mice (39). Compactin is, however, effective in rats treated with Triton WR-1339, a hyperlipidemic detergent (39, 51). In normal rats compactin causes a decrease in fecal excretion of bile acids and in the activity of hepatic cholesterol 7ot-hydroxylase, the rate-limiting enzyme in bile acid synthesis. In addition, compactin causes a marked increase in hepatic HMG CoA reductase activity (39). The unexpected changes in the activity of the two microsomal enzymes may in part explain the ineffectiveness of compactin. In normolipidemic dogs, compactin produces a rapid reduction of plasma cholesterol levels at a dose of 10 mg/kg (52). Triglyceride levels are not consistently changed. The LDL-cholesterol fraction which is responsible for atherosclerosis is preferentially lowered. Later, similar results were obtained with mevinolin and CS-514 (pravastatin) (7, 24, 53). Fecal excretion of bile acids is, unlike in the rats, not affected or slightly elevated by these inhibitors (52). In dogs and miniature pigs, a combination of colestipol (bile acid sequestrant) (700 mg/kg) in addition to mevinolin (10 mg/kg) produces
HMG CoA REDUCTASEINHIBITORS
59
greater reductions in LDL-cholesterol than occurs with either drug alone (54). In cynomolgus monkeys, compactin is effective in lowering plasma cholesterol levels at a dose of 20 mg/kg (55). Rabbits are sensitive to compactin analogs. When compactin is given to W H H L rabbits at a dose of 5 mg/kg, plasma cholesterol levels were reduced by 21% after 2 weeks (56). In normal rabbits, plasma cholesterol is lowered by mevinolin at 2-2.5 mg/kg (57). In Humans In healthy volunteers, compactin produces a rapid and profound cholesterol-lowering effect (58). Similar effects are obtained with mevinolin, which lowers serum total cholesterol by 23 to 27% at doses of 12.5 to 100 mg daily (59, 60). Yamamoto et al. (61) first studied the efficacy of compactin in patients with FH. In this study plasma cholesterol was reduced by 27% at 60 to 100 mg/day after 4 to 8 weeks treatment. In a detailed study with heterozygous FH, decreases in LDL-cholesterol levels were 29% at 30 to 60 mg daily (62). High density lipoprotein (HDL) levels were not reduced, but rather slightly elevated. Combined treatment with a bile acid-binding resin (cholestyramine) with compactin produces a much greater decrease in LDL-cholesterol levels than does either drug alone (63). When patients with heterozygous FH were treated with cholestyramine alone (12 g daily), LDL-cholesterol was lowered by 28%, and the addition of compactin (30 mg daily) produced a 53% reduction. Similar cholesterol-lowering effects in humans were obtained with mevinolin (64) and CS-514 (65). Mechanism for Lowering L D L-Cholesterol Compactin analogs lower plasma LDL-cholesterol levels both in animals and humans, while they do not show a significant effect on H D L and intermediate density lipoprotein (IDL). There are two possible mechanisms for lowering LDL-cholesterol levels by an HMG CoA reductase inhibitor, namely decreasing LDL synthesis and increasing its clearance from the circulation. In dogs, the lowering of LDL-cholesterol levels occurs in 2 ways by an inhibition of LDL synthesis, and through enhancing the receptor-mediated LDL catabolism in the liver (53). In humans, however, the relative importance of these two mechanisms in lowering plasma LDL-cholesterol varies in patients, depending on whether they have familial or non-familial hypercholesterolemia. Mevinolin treatment primarily enhances receptor-mediated LDL catabolism in patients with heterozygous FH (66), whereas Grundy and Vega found reduced LDL synthesis, rather than increased LDL catabolism, to be the predominant factor in patients with non-familial hypercholesterolemia (67).
60
A. ENDO and K. HASUMI SUMMARY
Subsequent to the discovery of compactin (ML-236B) as a specific inhibitor of HMG CoA reductase, a series of compactin analogs have been either isolated or synthesized. Several of these compounds, which include compactin, mevinolin (monacolin K) and CS-514, have been extensively studied. The inhibition of HMG CoA reductase by these compounds is reversible and competitive ( K i = - I nM). The 3', 5'-dihydroxypentanoic acid portion of the acid form of compactin analogs, which resembles the HMG portion of HMG CoA, plays a crucial role in inhibitory activity. These inhibitors block sterol synthesis both in cultured mammalian cells and in animals. Strong inhibition of sterol synthesis results in a marked increase in HMG CoA reductase activity both in vitro and in vivo. These compounds strongly lower plasma LDL-cholesterol levels in animals and humans. The lowering of LDL-cholesterol levels occurs by an inhibition of LDL synthesis and/or by an elevation of the receptor-mediated LDL catabolism in the liver.
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10. A. ENDO, K. HASUMI, T. NAKAMURA, M. KUNISHIMA and M. MASUDA, Dihydromonacolin L and monacolin X, new metabolites that inhibit cholesterol biosynthesis, J. Antibiotics 38,321-327 (1985). 11. G. A. SCHONBERG, H. JOSHUA, M. B. LOPEZ, O. D. HENSENS, J. P. SPRINGER, J. CHEN, S. OSTROVE, C. H. HOFFMAN, A. W. ALBERTS and A. A. PATCHETT, Dihydromevinolin, a potent hypocholesterolemic metabolite produced by Aspergillus terreus, J. Antibiotics 34, 507-512 (1981). 12. N. SERIZAWA, K. NAKAGAWA, K. HAMANO, Y. TSUJITA, A. T E R A H A R A and H. KUWANO, Microbial hydroxylation of ML-236B (compactin) and monacolin K (MB-530B), J. Antibiotics 36, 604-607 (1983). 13. N. SERIZAWA, S. SERIZAWA, K. NAKAGAWA, K. FURUYA, T. OKAZAKI and A. T E R A H A R A , Microbial hydroxylation of ML-236B (compactin). Studies on organisms capable of 31~-hydroxylation of ML-236B, J. Antibiotics 36, 887-891 (1983). 14. N. SERIZAWA, K. NAKAGAWA, Y. TSUJITA, A. T E R A H A R A and H. KUWANO, 3et-Hydroxy-ML-236B (3~t-hydroxycompactin), microbial transformation product of ML-236B (compactin), J. Antibiotics 36, 608--610 (1983). 15. N. SERIZAWA, K. NAKAGAWA, Y. TSUJITA, A. T E R A H A R A , H. KUWANO and M. TANAKA, 6ct-Hydroxy-iso-ML-236B (6a-hydroxy-iso-compactin) and ML236A, microbial transformation products of ML-236B, J. Antibiotics 36, 918-920 (1983). 16. H. YAMASHITA, S. TSUBOKAWA and A. ENDO, Microbial hydroxylation of compactin (ML-236B) and monacolin K, J. Antibiotics 38, 6054509 (1985). 17. A. ENDO, H. YAMASHITA, H. NAOKI, T. IWASHITA and Y. MIZUKAWA, Microbial phosphorylation of compactin (ML-236B) and related compounds, J. Antibiotics 38, 328-332 (1985). 18. A . K . WILLARD and R. L. SMITH, Incorporation of 2(S)-methylbutanoic acid-1-14C into the structure of mevinolin, J. Labelled Comp. Radiopharm. 19, 337-344 (1981). 19. S. MURAKAWA, T. NAKAMURA, D. KOMAGATA, E. SUNAGAWA and A. ENDO, Purification and properties of carboxylesterase from Emericella unguis that catalyzes the conversion of ML-236B (compactin) to ML-236A, Agric. Biol. Chem. 51, 1879-1884 (1987). 20. W.F. HOFFMAN, A. W. ALBERTS, P. S. ANDERSON, J. S. CHEN, R. L. SMITH and A. K. WILLARD, 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 4. Side chain ester derivatives of mevinolin, J. Med. Chem. 29, 849-852 (1986). 21. G . E . STOKKER, A. W. ALBERTS, P. S. ANDERSON, E. J. CRAGOE, A. A. DEANA, J. L. GILFILLAN, J. HIRSHFIELD, W. J. HOLTZ, W. F. HOFFMAN, J. W. HUFF, T. J. LEE, F. C. NOVELLO, J. D. PRUGH, C. S. ROONY, R. L. SMITH and A. K. WILLARD, 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 3.7-(3,5-Disubstituted [1, l'-biphenyl]-2-yl)-3, 5-dihydroxy-6-hepatanoic acids and their lactone derivatives, J. Med. Chem. 29, 170-181 (1986). 22. A. ENDO, M. K U R O D A and K. TANZAWA, Competitive inhibition of 3-hydroxy3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B, fungal metabolites, having hypocholesterolemic activity, FEBS Left. 98,323-326 (1976). 23. K. TANZAWA and A. ENDO, Kinetic analysis of reaction catalyzed by rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase using two specific inhibitors, Eur. J. Biochem. 98, 195-210 (1979). 24. Y. TSUJITA, M. KURODA, Y, SHIMADA, K. TANZAWA, M. ARAI, I. KANEKO, M. TANAKA, H. MASUDA, C. TARUI, Y. WATANABE and S. FUJII, CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissueselective inhibition of sterol synthesis and hypolipidemic effect on various animal species, Biochim. Biophys. Acta 877, 50-60 (1986). 25. D. H. ROGERS and H. RUDNEY, Modification of 3-hydroxy-3-methylglutaryl coenzyme A reductase immunoinhibition curves by substrates and inhibitors. Evidence for conformational changes leading to alterations in antigenicity, J. Biol. Chem. 257, 10650-10658 (1982). 26. W. F. HOFFMAN, A. W. ALBERTS, E. J. CRAGOE, A. A. DEANA, B. E. EVANS, J. L. GILFILLAN, N. P. GOULD, J. W. HUFF, F. C. NOVELLO, J. D.
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27. 28. 29.
30. 31. 32.
33. 34. 35. 36. 37. 38. 39. 40. 41.
42. 43. 44.
A. ENDO and K. HASUMI PRUGH, K. E. RI'ITLE, R. L. SMITH, G. E. STOKKER and A. K. WILLARD, 3-Hydroxyl-3-methylglutaryl-coenzyme A reductase inhibitors. 2. Structural modification of 7-(substituted aryl)-3, 5-dihydroxy-6-heptanoic acids and their lactone derivatives, J. Med. Chem. 29, 159-169 (1986). A. ENDO, Compactin (ML-236B) and related compounds as potential cholesterollowering agents that inhibit HMG-CoA reductase, J. Med. Chem. 28, 401--405 (1985). 1. KANEKO, Y. H A Z A M A - S H I M A D A and A. ENDO, Inhibitory effects on lipid metabolism in cultured cells of ML-236B, a potent inhibitor of 3-hydroxy3-methylglutaryl-coenzyme A reductase, Eur. J. Biochem. 87, 313-321 (1978). M. S. BROWN, J. R. FAUST, J. L. GOLDSTEIN, I. KANEKO and A. ENDO, Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase, J. Biol. Chem. 253, 1121-1128 (1978). A. ENDO, Specific non-sterol inhibitor o f H M G CoA reductase, pp. 49-78 in Regulation o f H M G CoA Reductase (B. PREISS, ed.), Academic Press, New York (1985). J.L. GOLDSTEIN, J. A. S. HELGESON and M. S. BROWN, Inhibition of cholesterol synthesis with compactin renders growth of cultured ceils dependent on the low density lipoprotein receptor, J. Biol. Chem. 254, 5403-5409 (1979). J. RYAN, E. C. HARDEMAN, A. ENDO and R. D. SIMONI, Isolation and characterization of cells resistant to ML-236B (compactin) with increased levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, J. Biol. Chem. 256, 6762-6768 (1981). A. ENDO, Y. TSUJ1TA, M. K U R O D A and K. TANZAWA, Inhibition of cholesterol synthesis in vitro and in vivo by ML-236A and ML-236B, competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, Eur. J. Biochem. 77, 31-36 (1977). F . C . HAGEMENAS, S. LINDSAY and D. R. ILLINGWORTH, The influence of mevinolin on cholesterol homeostasis in mononuclear leucocytes from patients with familial hypercholesterolemia, Circulation 74 (Suppl. II), 797 (1986). T.S. PARKER, J. MacNAMARA, C. D. BROWN, R. COLB, E. H. AHRENS JR, A. W. ALBERTS, J. TOBERT, J. CHEN and P. J. DE SCHEPPER, Plasma mevalonate as a measure of cholesterol synthesis in man, J. Clin. Invest. 74, 795-804 (1984). A . S . PAPPU, S. P. BACON and D. R. ILLINGWORTH, The influence of lovastatin (mevinolin) on 24 hour urinary mevalonate in familial hypercholesterolemia, Clin. Res. 35, 625A (1987). S . M . G R U N D Y and D. W. BILHEIMER, Inhibition of 3-hydroxy-3-methylglutarylCoA reductase by mevinolin in familial hypercholesterolemia heterozygotes: effects on cholesterol balance, Proc. Natl. Acad. Sci. U.S.A. 81, 2538--2542 (1984). M. SINENSKY and J. ROGEL, Inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin, J. Biol. Chem. 258, 8547-8549 (1983). A. ENDO, Y. TSUJITA, M. K U R O D A and K. T A N Z A W A , Effects of ML-236B on cholesterol metabolism in mice and rats: lack of hypocholesterolemic activity in normal animals, Biochim. Biophys. Acta 575, 266--276 (1979). P. A. EDWARDS, D. LEMONGELLO, J. KANE, I. SHECHTER and A. M. FOGELMAN, Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity, J. Biol. Chem. 255, 3715-3725 (1980). R. D. TANAKA, P. A. EDWARDS, S. F. LAN, E. M. KNOPPEL and A. M. FOGELMAN, The effects of cholestyramine and mevinolin on the diurnal cycle of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, J. Lipid Res. 23, 1026--1031 (1982). P . A . EDWARDS, S. F. LAN and A. M. FOGELMAN, Alteration in the rates of synthesis and degradation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase produced by cholestyramine and mevinolin, J. Biol. Chem. 258, 10219--10222 (1983). C. F. CLARKE, P. A. EDWARDS, S. F. LAN, R. D. TANAKA and A. M. FOGELMAN, Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in rat liver, Proc. Natl. Acad. Sci. U.S.A. 80, 3305-3308 (1983). L. LISCUM, K. L. LUSKEY, D. J. CHIN, Y. K. HO, J. L. GOLDSTEIN and M. S. BROWN, Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and its
HMG CoA REDUCTASE INHIBITORS
45. 46. 47.
48. 49.
50.
51. 52. 53. 54. 55. 56.
57. 58.
59.
60.
63
mRNA in rat liver as studied with a monoclonal antibody and a cDNA probe, J. Biol. Chem. 258, 8450--8456 (1983). C.F. CLARKE, A. M. FOGELMAN and P. A. EDWARDS, Transcriptional regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver, J. Biol. Chem. 260, 14363-14367 (1985). T. KITA, M. S. BROWN and J. L. GOLDSTEIN, Feedback regulation of 3-hydroxy3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase, J. Clin. Invest. 66, 1094-1100 (1980). P. A. EDWARDS, S. F. LAN, R. D. TANAKA and A. M. FOGELMAN, Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes, J. Biol. Chem. 258, 7272-7275 (1983). K. BROWN, C. M. H A V E L and J. A. WATSON, Isoprene synthesis in isolated embryonic Drosophila cells. II. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, J. Biol. Chem. 258, 8512-8518 (1983). R. SCHNITZER-POLOKOFF, R. TOGERT, J. LOGEL and M. SINENSKY, Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts, Arch. Biochem. Biophys. 227, 71-80 (1983). J. D. BERGSTROM, G. A. WONG, P. A. EDWARDS and J. EDMOND, The regulation of acetoacetyl-CoA synthetase activity by modulators of cholesterol synthesis in vivo and the utilization of acetoacetate for cholesterogenesis, J. Biol. Chem. 259, 14548--14553 (1984). M. KURODA, K. T A N Z A W A , Y. TSUJITA and A. ENDO, Mechanism for elevation of hepatic cholesterol synthesis and serum cholesterol levels in Triton-induced hypercholesterolemia, Biochim. Biophys. Acta 489, 119-125 (1977). Y. TSUJITA, M. KURODA, K. T A N Z A W A , N. KITANO and A. ENDO, Hypolipidemic effects in dogs of ML-236B, a competitive inhibitor of 3-hydroxy3-methylglutaryl coenzyme A reductase, Atherosclerosis 32,307-313 (1979). P.T. KOVANEN, D. W. BILHEIMER, J. L. GOLDSTEIN, J. J. JARAMILLO and M. S. BROWN, Regulatory role for hepatic low density lipoprotein receptors in vivo in the dog, Proc. Natl. Acad. Sci. U.S.A. 78, 1194-1198 (1981). M. W. HUFF, D. E. TELFORD, K. WOODCROFT and W. L. P. STRONG, Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apolipoprotein B in miniature pigs, J. Lipid Res. 26, 1175-1186 (1985). M. KURODA, Y. TSUJITA, K. T A N Z A W A and A. ENDO, Hypolipidemic effects in monkeys of ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, Lipids 14, 585-589 (1979). Y. WATANABE, T. ITO, M. SAEKI, M. KURODA, K. T A N Z A W A , M. MOCHIZUKI, Y. TSUJITA and M. ARAI, Hypocholesterolemic effects of CS-500 (ML-236B) in WHHL-rabbit, a heritable animal for hyperlipidemia, Atherosclerosis 38, 27-31 (1981). P . A . KROON, K. M. HAND, J. W. HUFF and A. W. ALBERTS, The effects of mevinolin on serum cholesterol levels of rabbits with endogenous hypercholesterolemia, Atherosclerosis 44, 41--48 (1982). H. SHIGEMATSU, Y. HATA, M. YAMAMOTO, T. OIKAWA, Y. YAMAUCHI, N. NAKAYA and Y. GOTO, Treatment of hypercholesterolemia with an HMG CoA reductase inhibitor (CS-500). I. Phase I study in normal subjects, Geriatr. Med. (Japan) 17, 1564-1570 (1979). J. A. TOBERT, G. D. BELL, J. BIRTWELL, I. JAMES, W. R. KUKOVETZ, J. S. PRYOR, A. BUNTINX, I. B. HOLMES, Y. S, CHAO and J. A. BOLOGESE, Cholesterol-lowering effect of mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, in healthy volunteers, J. Clin. Invest. 69, 913-919 (1982). J . A . TOBERT, G. H I T Z E N B E R G E R , W. R. KUKOVETZ, I. B. HOLMES and K. H. JONES, Rapid and substantial lowering of human serum cholesterol by mevinolin (MK-803), an inhibitor of hydroxymethylglutaryl coenzyme A reductase, Atherosclerosis 41, 61-65 (1982).
64
A. ENDO and K. HASUMI
61. A. YAMAMOTO, H. SUDO and A. ENDO, Therapeutic effects of ML-236B in primary hypercholesterolemia, Atherosclerosis 35, 259-266 (1980). 62. H. MABUCHI, T. H A B A , R. TATAMI, S. MIYAMOTO, Y. SAKAI, T. WAKASUGI, A. WATANABE, J. KOIZUMI and R. TAKEDA, Effects of an inhibitor of 3-hydroxy3-methylglutaryl coenzyme A reductase on serum lipoproteins and ubiquinone-10 levels in patients with familial hypercholesterolemia, New Engl. J. Med. 305, 478--482 (1981). 63. H. MABUCHI, T. SAKAI, Y. SAKAI, A. YOSHIMURA, A. WATANABE, T. WAKASUGI, J. KOIZUMI and R. TAKEDA, Reduction of serum cholesterol in heterozygous patients with familial hypercholesterolemia, New Engl. J. Med. 308, 609--613 (1983). 64. H. M. HERWOOD and R. C. HEEL, Lovastatin; a preliminary review of its pharmacodynamic properties and therapeutic use in hyperlipidemia, Drugs, in press. 65. Epstatin sodium, Drugs of the Future 13, 437-442 (1987). 66. D. W. BILHEIMER, S. M. GRUNDY, M. S. BROWN and J. L. GOLDSTEIN, Mevinolin and colestipol stimulate receptor-mediated clearance of low density lipoprotein from plasma in familial hypercholesterolemia heterozygotes, Proc. Natl. Acad. Sci. U.S.A. 80, 4124-4128 (1983). 67. S.M. GRUNDY, G. L. VEGA and D. W. BILHEIMER, Influence of combined therapy with mevinolin and interruption of bile-acid reabsorption on low density iipoproteins in heterozygous familial hypercholesterolemia, Annal. Intern. Med. 103, 339-343 (1985).
Compactin Analogs Isolated from Micro-organisms Compactin (ML-236B) is the first of a series of competitive inhibitors of H M G CoA reductase. This compound is produced by Penicillium citrinum (1), Pen. brevicompactum (2) and Pen. cyclopium (3). Along with compactin, ML-236A (Fig. 2), ML-236C, and dihydrocompactin, which are analogous to compactin, are produced by Pen. citrinum (1, 4). Monacolin K (mevinolin) (Fig. 2) is the main active compound produced by Monascus 53
54
A. ENDO and K. HASUMI HO~ooH
2NADPH÷2H*
HMG-CoA
HOC~)H
MevalOnicacid
FIG. 1. HMG CoA reductasereaction. ruber (5, 6) and Aspergillus terreus (7). In addition, monacolins J (Fig. 2), L, M and X and dihydromonacolin L were isolated from Mon. ruber (8-10) and dihydromevinolin from Asp. terreus (11). Microbial and Chemical Modification Compactin is converted to 31~-hydroxycompactin by Mucor hiemalis and Nocardia sp. (12, 13). Compactin is also hydroxylated to 3othydroxycompactin by Syncephalastrum nigricans (14) and to 6ot-hydroxy/so-compactin by Absidia coerulea (15). 8ot-Hydroxycompactin and 8othydroxymonacolin K are derived from compactin and monacolin K, respectively, by growing Schizophyllum commune (16). 5 '-Phosphocompactin acid and 5 '-phosphomonacolin K are produced by several fungal strains (17). The 2(S)-methylbutylyl ester of compactin and monacolin K can be removed, giving ML-236A and monacolin J, respectively, by chemical hydrolysis or by the action of a carboxylesterase of the fungus Emericella unguis (18, 19). A series of the side chain ester analogs of monacolin K (mevinolin) are derived from the silyl ether of monacolin J, which includes synvinolin (simvastatin) (Fig. 2) (20). H
0
o~
14t'k
o
R:H, Compactin (ML-236B) R=CH3.Monacolin K (mevinolin)
"CO z Na
~'- ° "
CS-514 (pravastat in)
0
: Ha
H3C,"W Synvinolin (simvastatin)
A
' '"~[~
:
H3
R,,~ R=H. ML-236A R=CH3.Monaco~inJ
FIG. 2. Majorcompactin-relatedcompounds.
HMG CoA REDUCTASE INHIBITORS
55
Chemical Synthesis A series of 7-(3,5-disubstituted [1,1'-biphenyl]-2-yl)-3,5-dihydroxy6-heptanoic acids were synthesized and assayed for inhibitory activity of HMG CoA reductase. Among these compounds, compounds 100(+) and 110(+) have 2.8 times the inhibitory activity of compactin (21). Of the HMG CoA reductase inhibitors described above, four compounds have been studied in the clinic: compactin, mevinolin, 3l~-hydroxycompactin acid monosodium salt (pravastatin, CS-514), and synvinolin. MECHANISM
OF HMG CoA REDUCTASE
INHIBITION
The lactone form of compactin analogs is inactive and, in vivo, hydrolyzed to the corresponding 3', 5'-dihydroxyacid, which is the principal active form of these compounds. Most of these compounds inhibit HMG CoA reductase of rat liver at - 1 nM (6, 22-24). The inhibition of HMG CoA reductase by compactin is reversible. As can be expected from the structure of the 3', 5'-dihydroxypentanoic acid portion, the inhibition is competitive with respect to HMG CoA and non-competitive with respect to NADPH. The K i values for the acid form of compactin, monacolin K (mevinolin) and CS-514 are 1.2, 0.49 and 2.3 nM, respectively, while under the same conditions, the K m value for HMG CoA is -10/xM (6, 22). Thus, the affinity of HMG CoA reductase for compactin is 10,000 times higher than its affinity for the natural substrate HMG CoA. The conformational change of the HMG CoA reductase molecule occurs by its binding to compactin, leading to alterations in antigenicity (25). STRUCTURE-ACTIVITY
RELATIONSHIP
The compactin molecule is composed of four moieties; (a) the lactone or the hydroxylic 3', 5'-dihydroxyacid, (b) the moiety bridging the lactone and the lipophilic groups, (c) the decarine ring, and (d) the side chain ester. The hydroxyl group at the 3'-position of the 3', 5'-dihydroxypentanoic acid portion is the trans-isomer and much more active than the cis-isomer (21, 26). The addition of a methyl group to the 3'-position to give the trans-form, a compound which more closely resembles the HMG portion of HMG CoA, does not stimulate activity appreciably (26). Conversion of either the 3'- or 5'-hydroxyl group to the corresponding methyl ester ablates activity (unpublished data), indicating a crucial role of these hydroxyl groups in activity. Replacement of the carboxyl group of compactin with a carboxamide group also abolishes activity (unpublished data). Increasing the length of the bridge to three carbons reduces activity (21). The addition of a methyl group to the 6-position of the decarine ring of compactin to give monacolin K (mevinolin) doubles activity (6). The introduction of a hydroxyl group to give CS-514 slightly reduces activity (24).
56
A. ENDO and K. HASUMI
Activity is reduced one-third by the hydroxylation at the 8a-position (16), partial or complete hydrogenation of the decarine ring gives no detectable change in activity (unpublished data). Monacolin J and L (or ML-236A and ML-236C), which lack the side chain ester, are far less active than monacolin K (or compactin) (27). The introduction of an additional aliphatic group on the carbon to the carbonyl group of the side chain increases potency. Synvinolin, which has an additional methyl group on the carbon a, has about 2.5 times the inhibitory activity of mevinolin (20). Increasing the length of the side chain and the removal of the terminal methyl group lowers the inhibitory activity. Stereochemistry at the carbon a to the carbonyl is not crucial as the natural product (mevinolin) and its diastereomer are equally potent (20). INHIBITION
OF CHOLESTEROL
SYNTHESIS
In Cultured Cells
Compactin inhibits cholesterol biosynthesis in a variety of cultured animal and human cells at nM concentrations (20-30). In cultured human skin fibroblasts, inhibition of sterol synthesis from [14C]acetate by compactin is 50% at 1 riM, 80% at 10 nM, 90% at 100 riM, 95% at 1 /zM, and 100% at 10/zM, respectively (28). Under these conditions, sterol synthesis from [14C]mevalonate and fatty acid synthesis from [14C]acetate are not significantly affected. The inhibition of H M G CoA reductase by compactin analogs results in the depletion of mevalonate. In addition to cholesterol, several isoprenoids like ubiquinones and dolichols are also derived from mevalonate. In human skin fibroblasts grown in the presence of low density lipoprotein(LDL)cholesterol, compactin has no detectable effects on the synthesis of these two isoprenoids at 10 nM, a concentration that causes over 50% inhibition of sterol synthesis (unpublished data). When H M G CoA reductase is partially inhibited by compactin, cells must have some way of diverting the small amounts of synthesized mevalonate preferentially into these non-sterol isoprenoids. At higher concentrations where sterol synthesis is reduced by over 90%, compactin inhibits cell growth as well (28, 31, 32). This inhibition can be overcome and cells can grow normally if mevalonate is added to the culture medium, indicating that compactin is a specific inhibitor of H M G CoA reductase. In Animals and Humans
When administered orally to mice, compactin is readily absorbed into the liver, the major site of cholesterogenesis, as compared with other organs
HMG CoA REDUCTASE INHIBITORS
57
(33). When 50 mg/kg of compactin was given orally to rats, sterol synthesis in vivo in the liver was inhibited by 92% after 4 hr, while the inhibition in other organs (ileum, adrenal, kidney, lung, spleen and testis) was 50-80% (33). Inhibition of cholesterol synthesis after a single dose of mevinolin 46/zg/kg in rats was 50% (7). Cholesterol synthesis, as determined by labeled acetate incorporation in vitro, increased by 40% in isolated mononuclear leucocytes from 20 patients with familial hypercholesterolemia treated with mevinolin 20 and 40 mg twice daily (34). In a double-blind placebo-controlled single dose study, mevinolin (100 mg) produced a 78% reduction in plasma mevalonate concentrations between 2 and 6 hr after administration in healthy volunteers (35). Plasma mevalonate concentrations were directly correlated with the rate of whole body cholesterol synthesis. In a longer term study, mevinolin (25 and 50 mg twice daily for 4 weeks) reduced urinary output of mevalonate by 81% in healthy volunteers (35). Reduction of urinary mevalonate excretion by mevinolin was also observed in patients with familial hypercholesterolemia (FH) (36). Using the sterol balance method to estimate cholesterol synthesis, Grundy and Bilheimer (37) found mevinolin 20 mg twice daily decreased output of neutral steroids in patients with FH by 22 to 44%, although not below levels observed in healthy volunteers. INDUCTION
OF HMG CoA REDUCTASE
ACTIVITY
Compactin analogs block the production of mevalonate so that the cells, now dependent on exogenous mevalonate, are phenotypic auxotrophs. When the HMG CoA reductase activity of human skin fibroblasts is inhibited by compactin, where mevalonate production is completely inhibited, a striking increase in the cellular levels of HMG CoA reductase (3.5- to 15-fold) occurs (29). The induced HMG CoA reductase could not be fully suppressed by LDL; the suppression is 85% (30). In the presence of compactin the enzyme could be fully suppressed only when the cells are given a small amount of mevalonate in addition to LDL. The induction of HMG CoA reductase activity by compactin requires protein synthesis and thus is completely blocked by cycloheximide (29). The induction of reductase activity in CHO-K1 cells treated with mevinolin is due to decreases in the rate of enzyme degradation but not changes in enzyme synthesis (38). Increases in HMG CoA reductase activity by compactin analogs also occurs in the liver of rats (39) and in rat hepatocytes (40). A combination of mevinolin and cholestyramine in the diet of rats produces a greater increase in HMG CoA reductase activity than either drug alone. The increases in this enzyme are a result of increased synthesis and decreased degradation (41, 42). In these experiments, increases in HMG CoA reductase synthesis JAER 28---C*
58
A. ENDO and K. HASUMI
and decreases in degradation (increase in apparent half-life) are 12- and at least 4-fold, respectively, for rats fed both mevinolin and cholestyramine. The mechanism by which these drugs induce H M G CoA reductase activity appears to be due to activating the synthesis of the reductase protein mediated by increasing its mRNA (43-45). Mevinolin induces reductase activity in mice, even when the animals were fed on a cholesterol-enriched diet (46). This augmentation in reductase activity was suppressed 90% by mevalonate. Mevalonate inhibits the rate of synthesis and enhances the rate of degradation of H M G CoA reductase in rat hepatocytes (47). It is likely that synthesis and degradation of the reductase are regulated by either mevalonate or, more likely, a product of mevalonate metabolism. In an insect cell line (Drosophila Kc cells) that neither synthesizes nor requires cholesterol for growth, H M G CoA reductase is not modulated by cholesterol. However, compactin causes a 5- to 10-fold increase in reductase activity. Mevalonate blocks this elevation (48). The non-sterol regulatory product derived from mevalonate is distal to isopentenyl-l-phosphate in the insect cells (47). In Chinese hamster ovary cells, HMG CoA reductase and H M G CoA synthase (EC 4.1.3.5) are co-ordinately induced by mcvinolin (49). In rats treated with mevinolin, acetoacetyl CoA synthetase (EC, not available) is also increased by compactin (50).
EFFECTS
ON CHOLESTEROL
AND LIPOPROTEINS
In Non-human Animals Compactin analogs fail to reduce plasma cholesterol levels in normal rats and mice (39). Compactin is, however, effective in rats treated with Triton WR-1339, a hyperlipidemic detergent (39, 51). In normal rats compactin causes a decrease in fecal excretion of bile acids and in the activity of hepatic cholesterol 7ot-hydroxylase, the rate-limiting enzyme in bile acid synthesis. In addition, compactin causes a marked increase in hepatic HMG CoA reductase activity (39). The unexpected changes in the activity of the two microsomal enzymes may in part explain the ineffectiveness of compactin. In normolipidemic dogs, compactin produces a rapid reduction of plasma cholesterol levels at a dose of 10 mg/kg (52). Triglyceride levels are not consistently changed. The LDL-cholesterol fraction which is responsible for atherosclerosis is preferentially lowered. Later, similar results were obtained with mevinolin and CS-514 (pravastatin) (7, 24, 53). Fecal excretion of bile acids is, unlike in the rats, not affected or slightly elevated by these inhibitors (52). In dogs and miniature pigs, a combination of colestipol (bile acid sequestrant) (700 mg/kg) in addition to mevinolin (10 mg/kg) produces
HMG CoA REDUCTASEINHIBITORS
59
greater reductions in LDL-cholesterol than occurs with either drug alone (54). In cynomolgus monkeys, compactin is effective in lowering plasma cholesterol levels at a dose of 20 mg/kg (55). Rabbits are sensitive to compactin analogs. When compactin is given to W H H L rabbits at a dose of 5 mg/kg, plasma cholesterol levels were reduced by 21% after 2 weeks (56). In normal rabbits, plasma cholesterol is lowered by mevinolin at 2-2.5 mg/kg (57). In Humans In healthy volunteers, compactin produces a rapid and profound cholesterol-lowering effect (58). Similar effects are obtained with mevinolin, which lowers serum total cholesterol by 23 to 27% at doses of 12.5 to 100 mg daily (59, 60). Yamamoto et al. (61) first studied the efficacy of compactin in patients with FH. In this study plasma cholesterol was reduced by 27% at 60 to 100 mg/day after 4 to 8 weeks treatment. In a detailed study with heterozygous FH, decreases in LDL-cholesterol levels were 29% at 30 to 60 mg daily (62). High density lipoprotein (HDL) levels were not reduced, but rather slightly elevated. Combined treatment with a bile acid-binding resin (cholestyramine) with compactin produces a much greater decrease in LDL-cholesterol levels than does either drug alone (63). When patients with heterozygous FH were treated with cholestyramine alone (12 g daily), LDL-cholesterol was lowered by 28%, and the addition of compactin (30 mg daily) produced a 53% reduction. Similar cholesterol-lowering effects in humans were obtained with mevinolin (64) and CS-514 (65). Mechanism for Lowering L D L-Cholesterol Compactin analogs lower plasma LDL-cholesterol levels both in animals and humans, while they do not show a significant effect on H D L and intermediate density lipoprotein (IDL). There are two possible mechanisms for lowering LDL-cholesterol levels by an HMG CoA reductase inhibitor, namely decreasing LDL synthesis and increasing its clearance from the circulation. In dogs, the lowering of LDL-cholesterol levels occurs in 2 ways by an inhibition of LDL synthesis, and through enhancing the receptor-mediated LDL catabolism in the liver (53). In humans, however, the relative importance of these two mechanisms in lowering plasma LDL-cholesterol varies in patients, depending on whether they have familial or non-familial hypercholesterolemia. Mevinolin treatment primarily enhances receptor-mediated LDL catabolism in patients with heterozygous FH (66), whereas Grundy and Vega found reduced LDL synthesis, rather than increased LDL catabolism, to be the predominant factor in patients with non-familial hypercholesterolemia (67).
60
A. ENDO and K. HASUMI SUMMARY
Subsequent to the discovery of compactin (ML-236B) as a specific inhibitor of HMG CoA reductase, a series of compactin analogs have been either isolated or synthesized. Several of these compounds, which include compactin, mevinolin (monacolin K) and CS-514, have been extensively studied. The inhibition of HMG CoA reductase by these compounds is reversible and competitive ( K i = - I nM). The 3', 5'-dihydroxypentanoic acid portion of the acid form of compactin analogs, which resembles the HMG portion of HMG CoA, plays a crucial role in inhibitory activity. These inhibitors block sterol synthesis both in cultured mammalian cells and in animals. Strong inhibition of sterol synthesis results in a marked increase in HMG CoA reductase activity both in vitro and in vivo. These compounds strongly lower plasma LDL-cholesterol levels in animals and humans. The lowering of LDL-cholesterol levels occurs by an inhibition of LDL synthesis and/or by an elevation of the receptor-mediated LDL catabolism in the liver.
REFERENCES 1. A. ENDO, M. K U R O D A and Y. TSUJITA, ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum, J. Antibiotics 29, 1346-1348 (1976). 2. A. G. BROWN, T. C. SMALE, T. J. KING, R. HASENKAMP and R. H. THOMPSON, Crystal and molecular structure of compactin, a new antifungal metabolite from Penicillium brevicompactum, J. Chem. Soc., Perkin Trans. I 1976, 1165-1170 (1976). 3. S. L. DOSS, C. K. CHU, M. K. MESBAK, H. G. CUTLER, P. D. COLE, R. F. A R R E N D A L E and J. P. SPRINGER, Isolation of compactin (a hypocholesterolemic metabolite) from a new source: Penicillium cyclopium, J. Natur. Prod. 49, 357-358 (1986). 4. Y. K. T. LAM, V. P. GULLO, R. T. GOEGELMAN, D. JORN, L. HUANG, C. DE RISO, R. L. M O N A G H A M and I. PUTTER, Dihydrocompactin, a new potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase from Penicillium citrinum, J. Antibiotics 34, 614-616 (1981). 5. A. ENDO, Monacolin K, a new hypocholesterolemic agent produced by a Monascus species, J. Antibiotics 32, 852-854 (1979). 6. A. ENDO, Monacolin K, a new hypocholesterolemic agent that specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase, J. Antibiotics 33, 334-336 (1980). 7. A . W . ALBERTS, J. CHEN, G. KURON, V. HUNT, C. HOFFMAN, J. ROTHROCK, M. LOPEZ, H. JOSHUA, E. HARRIS, A. PATCHETF, S. MONAGHAN, S. CURRIE, E. STAPLEY, G. ALBERS-SCHONBERG, O. HENSENS, J. HIRSHFIELD, K. HOOGSTEEN, J. LIESCH and J. SPRINGER, Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent, Proc. Natl. Acad. Sci. U.S.A. 77, 3957-3961 (1980). 8. A. ENDO, K. HASUMI and S. NEGISHI, Monacolins J and L, new inhibitors of cholesterol biosynthesis produced by Monascus tuber, J. Antibiotics 38, 420-422 (1985). 9. A. ENDO, D. K O M A G A T A and H. SHIMADA, Monacolin M, a new inhibitor of cholesterol biosynthesis, J. Antibiotics 39, 1670-1673 (1986).
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