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Inhibition of hepatic cholesterol biosynthesis by 3,5-dihydroxy-3,4,4-trimethylvaleric acid and its site of action
ARCHIVES
OF
BIOCHEMISTRY
inhibition
AND
146, 422-427 (1971)
BIOPHYSICS
of Hepatic
Cholesterol
Biosynthesis
3,4,4-Trimethylvaleric
Acid
FRANK The Department
of Biochemistry,
and
Its Site of Action
H. HULCHER
The Bowman Gray School of Medicine, Salem, North Carolina b71OS
Received
March
by 3,5-Dihydroxy-
8, 1971; accepted
July
Wake Forest University,
Winston-
6, 1971
A new analog of mevalonic acid, 3,5-dihydroxy-3,4,4-trimethylvaleric acid, was synthesized from 4-bromoacetoxy-3,3-dimethyl-2-butanone by an internal Reformatsky reaction. The compound strongly inhibits cholesterol biosynthesis by rat liver homogenates. The inhibitor in 4 X ~O+M @n-form) concentration decreased incorporation of mevalonate-2-i% into cholesterol by half. In the presence of this inhibitor, 5-phosphomevalonate accumulates from mevalonic acid and allylpyrophosphates could not be detected. Accumulation of the monophosphate suggests that a major site of inhibition is at the second phosphorylation step, in which B-phosphomevalonate is converted to 5-pyrophosphomevalonate by 5-phosphomevalonic kinase. A reaction mixture was developed for optimal accumulation of b-phosphomevalonate. The new inhibitor provides a competitive substrate that will be useful for examining the mechanism of mevalonic kinase and phosphomevalonic kinase.
L-Mevalonic acid is an important intermediate metabolite in the biosynthesis of cholesterol and coenzyme Q “in animal tissues. More recently, it was shown to be a precursor of N6-(A2-isopentenyl) adenosine (1, 2). The in the RNA of L. acidophihs promotion of cell division and differentiation by this compound (3) and the regulation of cholesterol biosynthesis in arteriosclerosis (4) has rekindled interest in isopentenyl biosynthesis and its control. Several studies with analogs of mevalonic acid have reported attempts to inhibit cholesterol biosynthesis. The reactions involved in attempted inhibitions are those given below. Mevalonic kinase is stereospecific for Lmevalonate (R-enantiomer) (5). (a) mevalonate
+ ATP
mevalonate -.
kinase
5-P04-mevalonate (b) 5-Pod-mevalonate
+ ADP
+ ATP
5-Pod-mevalonate
kinase
5-Pyro-P04-mevalonate
+ + ADP
(c) 5-Pyro-POa-mevalonate
+ ATP
5-Pyro-P04-mevalonate isopentenyl-Pyro-PO1
decarboxylase
>
+ CO* + ADP
Of several generically related compounds examined by Steward and Woolley (6) the most effective was 4-methylmevalonic acid. An intrinsic problem with this compound is that, if it is a competitive substrate for enzymes1-3 converting mevalonate to isopentenyl pyrophosphate, it could be decarboxylated and isomerized to the dimethallyl derivative. Thus, a pyrophosphate derivative of the inhibitor would be metabolized. In 3,5-dihydroxy-3,4,4-trimethylcontrast, valeric acid4 could not be converted to the 1 Mevalonate kinase is ATP: mevalonate-5phosphotransferase 2.7.1.36. 2 Phosphomevalonate kinase is ATP: 5-phosphomevalonate phosphotransferase 2.7.4.2. 3 Pyrophosphomevaonate decarboxylase is ATP. 5-pyrophosphomevalonate carboxy-lyase 4.1.1.33. acid. 4 3,5-Dihydroxy-3,4,4-trimethylvaleric The shorter, common name used for this compound in the text is dimethylmevalonic acid.
422
INHIBITOR
OF CHOLESTEROL
dimethallyl derivative because of the 2,2dimethyl substituents. Thus, it seemed likely to be a more potent inhibitor of cholesterol biosynthesis. This paper reports a preparation of a new inhibitor of cholesterol synthesis, dimethylmevalonic acid, its inhibition of cholesterol biosynthesis in rat liver, and t,he site of action. MAT.ERIALS
AND
METHODS
Synthesis of 3,5-dihydroxy-5,4,4-trimeth2/lvaleric acid (7). A mixture of 2.03 moles (218 ml) of methyl isopropyl ketone, 1.823 moles (134.3 ml) of 37.7% formalin, 100 ml of methanol, and 25 ml of 2 N NaOH was refluxed 30 min. The mixture was distilled, with use of a Dean and Stark moisture trap. After removal of a liquid boiling at 73”, the remaining water was removed by azeotropic distillation with benzene. Paraformaldehyde crystals precipitated by t,he benzene were removed by filtration and the filtrate was vacuum distilled. The product boiled at 8586” (16 mm Hg) and the yield was 48.2 g (23ye of theoretical) of 4-hydroxy3,3-dimethyl-2-butanone. A mixture ‘of 0.276 moles (33.2 ml) of the above product and 0.39 moles (54.2 g) of dry bromoacetic acid in 260 ml of benzene was refluxed through a Dean and Stark moisture trap to remove water from the reaction. The reaction mixture was cooled and added to 100 ml of 1% sodium bicarbonate. Bicarbonate was added with stirring until effervescence ceased. The organic layer was removed, the aqueous phase extracted three times with ether, and the extracts were combined with the organic layer. After drying, the organic solution was concentrated and the product distilled at 90” at a pressure of 0.3 mm Hg. Its density relative to water was 1.353 at 24.7”. The yield was 31.4 g (48yo of theoretical, of 4-bromoacetoxy-3,3dimethyl-2-butanone. Elemental analysis calculated: C, 40.52yo; H, 5.53yc; Br, 33.7y0. Found: C, 40.63%; H, 5.41%; Br, 32.36%. Finally, a lsmall portion of a solution of 9.92 ml ,(0.0565 mole) of 4-bromoacetoxy-3,3-dimethylSbutanone in anhydrous tetrahydrofuran was added to 4.0 g of metallic zinc globules covered with tetrahydrofuran. The zinc had previously been activated by successive washings with dilute HCl, water, dilute CuClt , dilute Hg(NOa)t , water, alcohol, and ether. The solvent was carefully evaporated until the reaction started. The remaining ketone solution was then added dropwise while refluxing until the zinc had dissolved. The reaction mixture was cooled, added to water, and adjusted with KOH to pH 7.2 to 7.8. Zinc hy-
BIOSYNTHESIS
423
droxide was removed by filtration and washed with methanol. The filtrate was concentrated to remove solvents. After readjustment of the pH to 7.8 and filtration to remove residual zinc hydroxide, the pH was increased to 9.0 with KOH, and methanol was added to dissolve the organic material. After the cooled solution was refluxed 30 min, it was extracted with et,her. The pH of the aqueous phase was adjusted to 3.0 with dilute H&J04 , and the solution was extracted three times with ether and once with chloroform. Basic and acidic extracts were separately concentrated to identical products. A typical yield was 3.0 g of 3,5-dihydroxy-3,4,4-trimethylvaleric acid, (yields varied from 42 to 60% of the theoretical). Successive extraction with water and then with ethyl ether removed nearly all contamination since the product was soluble in both solvents. The sodium salt crystallized from a concentrated water solution upon titration to pH 7.0. The N,N’-dibenzylethylene diammonium salt was prepared by the procedures of Hoffman et al. (8) and of Rilling and Block (9). The salt, in 38% of theoretical yield melted at 124.5”. The elemental analysis calculated: C, 60.7yc,;,; H, 8.85ye. The elemental analysis found was: C, 60.91~0; H, 8.87%. Physical data on the product. The infrared spectrum of dimethylmevalonic acid was obtained and compared with an authentic sample of mevalonic acid lactone (Sigma Chemical Co.). The two differ structurally only by the 4,4-dimethyl substitution. The spectra were nearly identical except for the region 1375-1475 cm-l. The synthetic compound gave bands at 1374 cm-’ (7.3 p) and 1383 cm-1 (7.25 JJ) indicating the 4,4-dimethyl groups, but mevalonic acid lactone did not show these bands. It gave bands at 1409 cm-l (6.81 p) for the CH,-C group. The structure of the product was confirmed by nuclear magnetic resonance and mass spectroscopy. The dimethylmevalonic acid was methylated and the nuclear magnetic resonance spectrum was taken. The results showed a strong peak at 0.93 ppm for the three methyl groups attached to carbon atoms. A peak at 1.63 ppm indicated the 3-methyl group attached to the tertiary-OH carbon. A peak at 3.38 ppm indicated the methyl ester protons. The integrated areas showed ratios at 0.93 ppm: 3.38 ppm of 9:3 indicating the nine protons on CHB-C methyl groups and three protons on the ester methyl group. The mass spectrum was obtained on the methyl ester (Fig. 1) which gave a molecular weight mass of 190; and the removal of one proton gave a more pronounced peak at 189 m/e. The base peak at m/e 43 is the same base peak obtained with mevaionic acid lactone (Fig. 1). The other features
424
HULCHER.
IO 20
30
40
50 60 70
80
MASS NUMBER
30 100 110 120 130
m/e
MASS NUMBER
m/e
FIG. 1. The mass spectra of (A) mevalonic acid lactone and the (B) methyl ester of dimethylmevalonic acid. The spectrum was produced by a Perkin-Elmer 270 mass spectrometer with an ionization voltage of 70 eV.
were similar to the fragmentation pattern of mevalonie acid showing the 70/71 m/e pair and 28 m/e (CO) masses. Abundant mass at 44 m/e occurred in both cases for carbon dioxide from decarboxvlation. The fragmentation of the methyl ester of dimethylmevalonic acid was rationalized as follows: the 190 m/e mass loses a proton to give a 189 m/e ion. This 189 m/e ion loses a molecule of vater to give the mass 171 m/e. This, in turn, %rould lose the methoxy group (31 m/e) to give a 1’40 m/e ion. Loss of three successive methyl ions (15 m/e) to produce 125 m/e, 110 m/e, and 95 m/e ions would account for the three CHa+ groups. The ion mass at 28 m/e arises from the carbonyl group. Both mevalonic acid lactone and the methyl ester of dimethylmevalonic acid showed similar characteristics stated above for S-valerola&one and methyl analogs (10, 11). The potassium salt was tritiated by the Wilzbath procedure (12) by New England Nuclear Corp. After repurification by ion-exchange chromatography (9) it was chromatographed on paper with the solvent system isobutyric acid:ammonia:water (66:1.5:15) and gave a single radioactive spot at Rp 0.80. This is the position expected, since mevalonate has an Rp of 0.66 and is more polar. Biochemicals. Disodium adenosined’-triphosphate, and p-diphosphopyridine nucleotide were purchased from Sigma Chemical Co. Mevalonic acid-2-r%, dibenzylethylene diammonium salt, or the lactone was purchased from Nuclear-Chicago Corp. The DBED salt was converted to the sodium salt after DBED was removed with ether (8).
Liver enzyme system. A rat liver homogenate was used for the biosynthesis of cholesterol from acetate according to Franz and Bucher (13). Freshly excised rat livers, usually two per experiment, were weighed and minced in 2.5 vol of icecold homogenizing medium per gram of tissue. The tissue was homogenized in a 50-ml Teflon homogenizer with a 0.5-mm od clearance for 1.5 min at 300 rpm in an ice bath. The cold homogenate was centrifuged at 500g for 10 min at O’, and the decanted preparation was kept at 0” and used immediately. The radioactive cholesterol was extracted and precipitated as the tomatinide by the procedure of Kabara et al. (14). The cholesterol tomatinide was dissolved in methanol and samples were placed on planchets together with egg lipids, which were added, to constant weight for correction of selfabsorption. The mixture was dried and counted in a Nuclear-Chicago gas-flow counter with automatic sample changer. Reaction midure. The reaction mixture for cholesterol biosynthesis contained 1.0 mg ATP added as 0.1 ml of solution at pH 7.0, 0.224 pmoles of nn-mevalonate-2-r%, 2 mg DPN+, and 2 ml of liver homogenate preparation. The latter was suspended in a homogenizing solution that was 0.044 M in potassium phosphate buffer (pH 7.4), 0.028 M in nicotinamide, and 0.007 M in MgClz prior to homogenization. The final volume was 2.5 ml. For detection of radioactive Fj-phosphomevalonate, &pyrophosphomevalonate, and mevalonic reaction mixtures were acid and its la&one, treated as described by DeWaard and Popjak
INHIBITOR
OF CHOLESTEROL TABLE
INCORPORATION
425
BIOSYNTHESIS I
OF MEVALONATE-2-‘% INTO CHOLESTEROL BY RAT IN THE PRESENCE OF DIMETHYLMEVALONATE Nmoles of medonate-2.1’C
Dimethylmevalonate bmoles)
0.0 0.1 1.0 5.0 10.0 20.0 30.0
LIVER
HOMOGENATE
converted into cholesterol per 10 min
Exp. 1
2
3
* Range, f
88.6 74.0 53.0 21.1 9.5 6.5 5.9
92.2 70.0 58.0 17.0 10.7 7.6 5.9
91.0 72.0 54.0 18.0 9.7 7.0 5.8
5.6 4.5 5.2 1.4 1.1 0.7 0.4
0 The reaction mixtures contained 1.0 mg of ATP (pH 7.0), 2.0 mg DPN, 0.224 pmoles of sodium mevalonate-2-14C, and 2.0 ml of rat liver homogenate in the homogenizing medium described in the Methods section. The reactions were incubated for 10 min at 37’. The experiments were done in triplicate flasks. The values were closely agreeing within experiments ranging from 6.2 to 12.5%.~ (15) and paper-chromatographed in the solvent system isobut:yric acid:ammonium hydroxide:water (66:3:30). Radioactivity was detected with an autoscanner or by cutting 0.5-cm strips and counting them. in a Nuclear-Chicago liquid-scintillation counter. RESULTS
Inhibition of cholesterol biosynthesis. The ability of dimethylmevalonic acid to inhibit incorporation of mevalonate-2-14C into cholesterol was examined in rat liver homogenate. The results obtained from different concentzations of racemic inhibitor are shown in Ta,ble I. The percentage of inhibition against inhibitor concentration is plotted in Fig. 2. The inhibitor gave halfmaximum inhibition of incorporation at an inhibitor:substrate ratio of 4.4 when the inhibitor concentration was 4 X lop4 M, (nL-forms) and considering only the active L-form of mevalonate. Nearly complete inhibition occurred with 10 pmoles of inhibitor per milliliter of reaction mixture. Reaction inhibited by dimethylmevalonate. Reactions were carried out as before, with and without inhibitor, and perchloric acid (to remove protein) samples were treated as described by DeWaard and Popjak (15) and paper-chromatographed in the system, previously described. Radioactivity was detected by an. autoscanner. Results with (top) and without inhibitor (bottom) are compared in Fig. 3. Radioactive areas are seen with RF values corresponding to those for
ok
’
4
’
6
’
IO
’
12
’
14
’
16
’
16
J
20
3,5-DIHYDROXY-3,4.4’-TRIMETHYLVALERATE ~rnoles D-, L-form
FIG. 2. Effect of dimethylmevalonic acid on the incorporation of mevalonate-2J4C into cholesterol in rat liver homogenate. The conditions of the experiment are described in Table I.
5-phosphomevalonate, 5-pyrophosphomevalonate (RF = O.lS), mevalonate, and mevalonolactone. In the control sample small radioactive areas were found for the mono- and pyrophosphomevalonate. The accumulation of 5-phosphomevalonat’e-2J4C in the presence of inhibitor is apparent (Top, Fig. 3). This evidence suggested that the major metabolic inhibition followed the synthesis of 5-phosphomevalonate.
426
HULCHER
Rt=0.61 mevalonate
5s
Rt =0.?2 mevolonoloctone
Rt=0.66
5-phosphomevalonate
mevalonote 5-phosphomevalonate Rt=0.35 Rt=0.16
1
I
FRONT
ORIGIN
FIG. 3. Identification of 5-phosphomevalonate from mevalonate-2J4C in liver homogenate containing dimethylmevalonic acid. Each drawing represents a radioactive scan of paper chromatograms of reaction mixtures chromatographed in isobutyric acid: ammonia: water (33:1.5:15). Top scan is from the reaction with inhibitor. The bottom scan represents the control reaction without inhibitor. The reaction mixture was as given in Fig. 2 except that the concentration of inhibitor was 20 pmoles of the racemic compound.
To test this idea, similar reaction mixtures were treated to extract any ally1 pyrophosphates, according to the techniques of Christophe and Popjak (16) and Goodman and Popjak (17). The final extracts were almost freeof radioactive material; evidently, no ally1 pyrophosphates were present in reactions having the inhibitor present. About 8 % of original counts in mevalonate2-14Cwere recovered as ally1 pyrophosphates without inhibitor. Optimal conditions for the enzymatic synthesis of Gphosphomevalonate by liver homogenate. To demonstrate the magnitude of the accumulation of 5-phosphomevalonate in liver preparations containing the inhibitor, optimal conditions were determined. The factors studied were concentration of ATP, mevalonate-2J4C, and dimethylmevalonic acid, the amount of liver homogenate, and the duration of incubation. The reaction was stopped by adding an equal volume of 10 % cold perchloric acid. Protein was removed by centrifugation, the perchloric acid neutralized with 30% KOH, and the KClO, removed by centrifugation. The lyophilized extracts were taken up in 2.0 ml of water and made to a final volume of 3.8 ml; 25-~1 aliquots were chromatographed as before. The product, 5-phosphomevalonate accu-
FIG. 4. Accumulation of 5-phosphomevalonate effected by inhibitor under optimal conditions. Counts per minute in l-cm sections of a paper chromatogram are plotted against centimeters of migration. The reaction mixture contained 2.0 ml of rat liver homogenate, 40 rmoles of ATP, 0.299 rmoles of mevalonate-2-W, and 15 pmoles of sodium dimethylmevalonate, and was incubated at 38’ for 20 min. The deproteinieed reaction mixtures were chromatographed in the same solvents as for Fig. 3, the paper cut into strips, and counted in a liquid-scintillation counter.
mulated from L-mevalonate-2J4C, had an RF of 0.32436. The results of strip paper scanning and of counting l-cm strips in a liquid-scintillation counter are given in
INHIBITOR
OF CHOLESTEROL
Fig. 4. The yield of 5-phosphomevalonate from n-mevalonate based on these data was from 82 to 100%; only the n-mevalonate-214C remains. The product was also isolated on, and eluted from Dowex-1 anion-exchange resin in the formate form (15). It was again chromatographed on paper with the systems isobutyric acid: ammonia: water (33 : 1.5 : 15) and t-butanol:formic acid:water (40: 10: 16). The RF values were the same as given by DeWaard and Popjak (15). The 5-phosphomevalonate-2J4C may be prepared in this way for use in studying the reaction catalyzed by phosphomevalonic kinase. DISCUSSION
As anticipated from previous work in which 4-methylmevalonic acid was an inhibitor of cholesterol biosynthesis (6), 4,4dimethylmevalonic acid is also an effective in vitro inhibitor. The results minimize the hypothesis that mevalonic kinase is the major site of inhibition; but suggest that the inhibitor may be a competitive substrate. Since 5phosphomevalonate accumulates in the presence of inhibitor in liver preparations, the activity of mevalonic kinase is not seriously impaired. Rather, the inhibitor or a phosphorylated derivative appears to inhibit phosphomevalonic kinase; yet, allowing the formation of 5-phosphomevalonate. This seems indicated because of the lack of accumulation of either 5pyrophosphomevalonate or ally1 pyrophosphates. Ally1 pyrophosphates could not be detected in inhibited systems and only small amounts were detected in uninhibited systems because of the rapid flow in the direction of cholesterol biosynthesis. In cholesterol biosynthesis when the synthesis of 5-pyrophosphomevalonate is blocked, likewise its decarboxylation product, isopentenyl-pyrophosphate, would not be produced. Stronger support was added to these findings by ‘establishing optimal conditions for producing 5phosphomevalonate using the inhibitor, a procedure which may be used to prepare the naturally occurring enantiomer ((5). It is not known if a single
BIOSYNTHESIS
427
enantiomer of the inhibitor is the active form. The new inhibitor may find usefulness to examine the reactions converting mevalonate to isopentenylpyrophosphate. ACKNOWLEDGMENTS This study was supported by the National Multiple Sclerosis Society Grant-222 and by the United Medical Research Foundation of North Carolina. The author gratefully acknowledges the help given by Dr. Phillip Hamrick in making the infrared analyses and by Dr. Edward 0. Oswald who performed the mass spectrometry and nuclear magnetic resonance analyses. REFERENCES 1. FITTLER, F., KLINE, L. K., AND HALL, Ii. H., Biochemistry 7, 94@944 (1968). 2. PETERKOFSKY, A., Biochemistry 7, 472 (1968). 3. HALL, R. H., AND SRIVAST~VA, B. I. S., Life Sci. 81, 4951 (1959). 4. Ho, K.-J., AND TAYLOR, C. B., Arch. Pathol. 90, 83-92 (1970). 5. CORNFORTH, J. W., CORNFORTH, R. H., .~ND POPJ~K, G., Tetrahedron 18, 1351 (1962). 6. STEWART, J. M., AND WOOLLEY, D. W., J. Amer. Chem. SGC. 81,495l (1959). 7. HULCHER, F. H., AND HOSICK, T. A., U. S. Patent 5, 119,842, Jan. 28, 1964. 8. HOFFMAN, C. H., WAGNER, A. F., WILSON, A. N., WALTON, E., SHUNK, C. H., WOLF, D. E., HOLLY, F. W., AND FOLKERS, K., J. Amer. Chem. Sot. 79,23X-2318 (1957). 9. RILLING, H. C., AND BLOCK, K., J. Biol. Chem. 284, 1424 (1959). 10. YAMAMURA, S., AND HIRATA, Y., Tetrahedron Lett. !I,79 (1964). 11. SAKAN, T., ISOE, S. B., HYEON, S. B., KATSUMUR, R., AND MAEDA, T., Tetrahedron Lett. 46,4097 (1965). 12. WILZBACH, K. E., J. Amer. Chem. Sot. 79, 1013 (1957). 13. FRANZ, I. D., AND BUCHER, N. L. R., J. Biol. Chem. 206, 471 (1954). 14. KABBRA, J. J., MCLAUGHLIN, J. T., AND RIEGEL, C. A., Anal. Chem. 33,305 (1961). 15. DEWAARD, A., AND POPJAK, G., Biochem. J. 73, 410 (1959). 16. CHRISTOPHE, J., AND POPJAK, G., J. Lipid Res. 2, 244-257 (1961). 17. GOODMAN, DEW. S., AND POPJAK, G., J. Lipid Res. 1, 286 (1960).
OF
BIOCHEMISTRY
inhibition
AND
146, 422-427 (1971)
BIOPHYSICS
of Hepatic
Cholesterol
Biosynthesis
3,4,4-Trimethylvaleric
Acid
FRANK The Department
of Biochemistry,
and
Its Site of Action
H. HULCHER
The Bowman Gray School of Medicine, Salem, North Carolina b71OS
Received
March
by 3,5-Dihydroxy-
8, 1971; accepted
July
Wake Forest University,
Winston-
6, 1971
A new analog of mevalonic acid, 3,5-dihydroxy-3,4,4-trimethylvaleric acid, was synthesized from 4-bromoacetoxy-3,3-dimethyl-2-butanone by an internal Reformatsky reaction. The compound strongly inhibits cholesterol biosynthesis by rat liver homogenates. The inhibitor in 4 X ~O+M @n-form) concentration decreased incorporation of mevalonate-2-i% into cholesterol by half. In the presence of this inhibitor, 5-phosphomevalonate accumulates from mevalonic acid and allylpyrophosphates could not be detected. Accumulation of the monophosphate suggests that a major site of inhibition is at the second phosphorylation step, in which B-phosphomevalonate is converted to 5-pyrophosphomevalonate by 5-phosphomevalonic kinase. A reaction mixture was developed for optimal accumulation of b-phosphomevalonate. The new inhibitor provides a competitive substrate that will be useful for examining the mechanism of mevalonic kinase and phosphomevalonic kinase.
L-Mevalonic acid is an important intermediate metabolite in the biosynthesis of cholesterol and coenzyme Q “in animal tissues. More recently, it was shown to be a precursor of N6-(A2-isopentenyl) adenosine (1, 2). The in the RNA of L. acidophihs promotion of cell division and differentiation by this compound (3) and the regulation of cholesterol biosynthesis in arteriosclerosis (4) has rekindled interest in isopentenyl biosynthesis and its control. Several studies with analogs of mevalonic acid have reported attempts to inhibit cholesterol biosynthesis. The reactions involved in attempted inhibitions are those given below. Mevalonic kinase is stereospecific for Lmevalonate (R-enantiomer) (5). (a) mevalonate
+ ATP
mevalonate -.
kinase
5-P04-mevalonate (b) 5-Pod-mevalonate
+ ADP
+ ATP
5-Pod-mevalonate
kinase
5-Pyro-P04-mevalonate
+ + ADP
(c) 5-Pyro-POa-mevalonate
+ ATP
5-Pyro-P04-mevalonate isopentenyl-Pyro-PO1
decarboxylase
>
+ CO* + ADP
Of several generically related compounds examined by Steward and Woolley (6) the most effective was 4-methylmevalonic acid. An intrinsic problem with this compound is that, if it is a competitive substrate for enzymes1-3 converting mevalonate to isopentenyl pyrophosphate, it could be decarboxylated and isomerized to the dimethallyl derivative. Thus, a pyrophosphate derivative of the inhibitor would be metabolized. In 3,5-dihydroxy-3,4,4-trimethylcontrast, valeric acid4 could not be converted to the 1 Mevalonate kinase is ATP: mevalonate-5phosphotransferase 2.7.1.36. 2 Phosphomevalonate kinase is ATP: 5-phosphomevalonate phosphotransferase 2.7.4.2. 3 Pyrophosphomevaonate decarboxylase is ATP. 5-pyrophosphomevalonate carboxy-lyase 4.1.1.33. acid. 4 3,5-Dihydroxy-3,4,4-trimethylvaleric The shorter, common name used for this compound in the text is dimethylmevalonic acid.
422
INHIBITOR
OF CHOLESTEROL
dimethallyl derivative because of the 2,2dimethyl substituents. Thus, it seemed likely to be a more potent inhibitor of cholesterol biosynthesis. This paper reports a preparation of a new inhibitor of cholesterol synthesis, dimethylmevalonic acid, its inhibition of cholesterol biosynthesis in rat liver, and t,he site of action. MAT.ERIALS
AND
METHODS
Synthesis of 3,5-dihydroxy-5,4,4-trimeth2/lvaleric acid (7). A mixture of 2.03 moles (218 ml) of methyl isopropyl ketone, 1.823 moles (134.3 ml) of 37.7% formalin, 100 ml of methanol, and 25 ml of 2 N NaOH was refluxed 30 min. The mixture was distilled, with use of a Dean and Stark moisture trap. After removal of a liquid boiling at 73”, the remaining water was removed by azeotropic distillation with benzene. Paraformaldehyde crystals precipitated by t,he benzene were removed by filtration and the filtrate was vacuum distilled. The product boiled at 8586” (16 mm Hg) and the yield was 48.2 g (23ye of theoretical) of 4-hydroxy3,3-dimethyl-2-butanone. A mixture ‘of 0.276 moles (33.2 ml) of the above product and 0.39 moles (54.2 g) of dry bromoacetic acid in 260 ml of benzene was refluxed through a Dean and Stark moisture trap to remove water from the reaction. The reaction mixture was cooled and added to 100 ml of 1% sodium bicarbonate. Bicarbonate was added with stirring until effervescence ceased. The organic layer was removed, the aqueous phase extracted three times with ether, and the extracts were combined with the organic layer. After drying, the organic solution was concentrated and the product distilled at 90” at a pressure of 0.3 mm Hg. Its density relative to water was 1.353 at 24.7”. The yield was 31.4 g (48yo of theoretical, of 4-bromoacetoxy-3,3dimethyl-2-butanone. Elemental analysis calculated: C, 40.52yo; H, 5.53yc; Br, 33.7y0. Found: C, 40.63%; H, 5.41%; Br, 32.36%. Finally, a lsmall portion of a solution of 9.92 ml ,(0.0565 mole) of 4-bromoacetoxy-3,3-dimethylSbutanone in anhydrous tetrahydrofuran was added to 4.0 g of metallic zinc globules covered with tetrahydrofuran. The zinc had previously been activated by successive washings with dilute HCl, water, dilute CuClt , dilute Hg(NOa)t , water, alcohol, and ether. The solvent was carefully evaporated until the reaction started. The remaining ketone solution was then added dropwise while refluxing until the zinc had dissolved. The reaction mixture was cooled, added to water, and adjusted with KOH to pH 7.2 to 7.8. Zinc hy-
BIOSYNTHESIS
423
droxide was removed by filtration and washed with methanol. The filtrate was concentrated to remove solvents. After readjustment of the pH to 7.8 and filtration to remove residual zinc hydroxide, the pH was increased to 9.0 with KOH, and methanol was added to dissolve the organic material. After the cooled solution was refluxed 30 min, it was extracted with et,her. The pH of the aqueous phase was adjusted to 3.0 with dilute H&J04 , and the solution was extracted three times with ether and once with chloroform. Basic and acidic extracts were separately concentrated to identical products. A typical yield was 3.0 g of 3,5-dihydroxy-3,4,4-trimethylvaleric acid, (yields varied from 42 to 60% of the theoretical). Successive extraction with water and then with ethyl ether removed nearly all contamination since the product was soluble in both solvents. The sodium salt crystallized from a concentrated water solution upon titration to pH 7.0. The N,N’-dibenzylethylene diammonium salt was prepared by the procedures of Hoffman et al. (8) and of Rilling and Block (9). The salt, in 38% of theoretical yield melted at 124.5”. The elemental analysis calculated: C, 60.7yc,;,; H, 8.85ye. The elemental analysis found was: C, 60.91~0; H, 8.87%. Physical data on the product. The infrared spectrum of dimethylmevalonic acid was obtained and compared with an authentic sample of mevalonic acid lactone (Sigma Chemical Co.). The two differ structurally only by the 4,4-dimethyl substitution. The spectra were nearly identical except for the region 1375-1475 cm-l. The synthetic compound gave bands at 1374 cm-’ (7.3 p) and 1383 cm-1 (7.25 JJ) indicating the 4,4-dimethyl groups, but mevalonic acid lactone did not show these bands. It gave bands at 1409 cm-l (6.81 p) for the CH,-C group. The structure of the product was confirmed by nuclear magnetic resonance and mass spectroscopy. The dimethylmevalonic acid was methylated and the nuclear magnetic resonance spectrum was taken. The results showed a strong peak at 0.93 ppm for the three methyl groups attached to carbon atoms. A peak at 1.63 ppm indicated the 3-methyl group attached to the tertiary-OH carbon. A peak at 3.38 ppm indicated the methyl ester protons. The integrated areas showed ratios at 0.93 ppm: 3.38 ppm of 9:3 indicating the nine protons on CHB-C methyl groups and three protons on the ester methyl group. The mass spectrum was obtained on the methyl ester (Fig. 1) which gave a molecular weight mass of 190; and the removal of one proton gave a more pronounced peak at 189 m/e. The base peak at m/e 43 is the same base peak obtained with mevaionic acid lactone (Fig. 1). The other features
424
HULCHER.
IO 20
30
40
50 60 70
80
MASS NUMBER
30 100 110 120 130
m/e
MASS NUMBER
m/e
FIG. 1. The mass spectra of (A) mevalonic acid lactone and the (B) methyl ester of dimethylmevalonic acid. The spectrum was produced by a Perkin-Elmer 270 mass spectrometer with an ionization voltage of 70 eV.
were similar to the fragmentation pattern of mevalonie acid showing the 70/71 m/e pair and 28 m/e (CO) masses. Abundant mass at 44 m/e occurred in both cases for carbon dioxide from decarboxvlation. The fragmentation of the methyl ester of dimethylmevalonic acid was rationalized as follows: the 190 m/e mass loses a proton to give a 189 m/e ion. This 189 m/e ion loses a molecule of vater to give the mass 171 m/e. This, in turn, %rould lose the methoxy group (31 m/e) to give a 1’40 m/e ion. Loss of three successive methyl ions (15 m/e) to produce 125 m/e, 110 m/e, and 95 m/e ions would account for the three CHa+ groups. The ion mass at 28 m/e arises from the carbonyl group. Both mevalonic acid lactone and the methyl ester of dimethylmevalonic acid showed similar characteristics stated above for S-valerola&one and methyl analogs (10, 11). The potassium salt was tritiated by the Wilzbath procedure (12) by New England Nuclear Corp. After repurification by ion-exchange chromatography (9) it was chromatographed on paper with the solvent system isobutyric acid:ammonia:water (66:1.5:15) and gave a single radioactive spot at Rp 0.80. This is the position expected, since mevalonate has an Rp of 0.66 and is more polar. Biochemicals. Disodium adenosined’-triphosphate, and p-diphosphopyridine nucleotide were purchased from Sigma Chemical Co. Mevalonic acid-2-r%, dibenzylethylene diammonium salt, or the lactone was purchased from Nuclear-Chicago Corp. The DBED salt was converted to the sodium salt after DBED was removed with ether (8).
Liver enzyme system. A rat liver homogenate was used for the biosynthesis of cholesterol from acetate according to Franz and Bucher (13). Freshly excised rat livers, usually two per experiment, were weighed and minced in 2.5 vol of icecold homogenizing medium per gram of tissue. The tissue was homogenized in a 50-ml Teflon homogenizer with a 0.5-mm od clearance for 1.5 min at 300 rpm in an ice bath. The cold homogenate was centrifuged at 500g for 10 min at O’, and the decanted preparation was kept at 0” and used immediately. The radioactive cholesterol was extracted and precipitated as the tomatinide by the procedure of Kabara et al. (14). The cholesterol tomatinide was dissolved in methanol and samples were placed on planchets together with egg lipids, which were added, to constant weight for correction of selfabsorption. The mixture was dried and counted in a Nuclear-Chicago gas-flow counter with automatic sample changer. Reaction midure. The reaction mixture for cholesterol biosynthesis contained 1.0 mg ATP added as 0.1 ml of solution at pH 7.0, 0.224 pmoles of nn-mevalonate-2-r%, 2 mg DPN+, and 2 ml of liver homogenate preparation. The latter was suspended in a homogenizing solution that was 0.044 M in potassium phosphate buffer (pH 7.4), 0.028 M in nicotinamide, and 0.007 M in MgClz prior to homogenization. The final volume was 2.5 ml. For detection of radioactive Fj-phosphomevalonate, &pyrophosphomevalonate, and mevalonic reaction mixtures were acid and its la&one, treated as described by DeWaard and Popjak
INHIBITOR
OF CHOLESTEROL TABLE
INCORPORATION
425
BIOSYNTHESIS I
OF MEVALONATE-2-‘% INTO CHOLESTEROL BY RAT IN THE PRESENCE OF DIMETHYLMEVALONATE Nmoles of medonate-2.1’C
Dimethylmevalonate bmoles)
0.0 0.1 1.0 5.0 10.0 20.0 30.0
LIVER
HOMOGENATE
converted into cholesterol per 10 min
Exp. 1
2
3
* Range, f
88.6 74.0 53.0 21.1 9.5 6.5 5.9
92.2 70.0 58.0 17.0 10.7 7.6 5.9
91.0 72.0 54.0 18.0 9.7 7.0 5.8
5.6 4.5 5.2 1.4 1.1 0.7 0.4
0 The reaction mixtures contained 1.0 mg of ATP (pH 7.0), 2.0 mg DPN, 0.224 pmoles of sodium mevalonate-2-14C, and 2.0 ml of rat liver homogenate in the homogenizing medium described in the Methods section. The reactions were incubated for 10 min at 37’. The experiments were done in triplicate flasks. The values were closely agreeing within experiments ranging from 6.2 to 12.5%.~ (15) and paper-chromatographed in the solvent system isobut:yric acid:ammonium hydroxide:water (66:3:30). Radioactivity was detected with an autoscanner or by cutting 0.5-cm strips and counting them. in a Nuclear-Chicago liquid-scintillation counter. RESULTS
Inhibition of cholesterol biosynthesis. The ability of dimethylmevalonic acid to inhibit incorporation of mevalonate-2-14C into cholesterol was examined in rat liver homogenate. The results obtained from different concentzations of racemic inhibitor are shown in Ta,ble I. The percentage of inhibition against inhibitor concentration is plotted in Fig. 2. The inhibitor gave halfmaximum inhibition of incorporation at an inhibitor:substrate ratio of 4.4 when the inhibitor concentration was 4 X lop4 M, (nL-forms) and considering only the active L-form of mevalonate. Nearly complete inhibition occurred with 10 pmoles of inhibitor per milliliter of reaction mixture. Reaction inhibited by dimethylmevalonate. Reactions were carried out as before, with and without inhibitor, and perchloric acid (to remove protein) samples were treated as described by DeWaard and Popjak (15) and paper-chromatographed in the system, previously described. Radioactivity was detected by an. autoscanner. Results with (top) and without inhibitor (bottom) are compared in Fig. 3. Radioactive areas are seen with RF values corresponding to those for
ok
’
4
’
6
’
IO
’
12
’
14
’
16
’
16
J
20
3,5-DIHYDROXY-3,4.4’-TRIMETHYLVALERATE ~rnoles D-, L-form
FIG. 2. Effect of dimethylmevalonic acid on the incorporation of mevalonate-2J4C into cholesterol in rat liver homogenate. The conditions of the experiment are described in Table I.
5-phosphomevalonate, 5-pyrophosphomevalonate (RF = O.lS), mevalonate, and mevalonolactone. In the control sample small radioactive areas were found for the mono- and pyrophosphomevalonate. The accumulation of 5-phosphomevalonat’e-2J4C in the presence of inhibitor is apparent (Top, Fig. 3). This evidence suggested that the major metabolic inhibition followed the synthesis of 5-phosphomevalonate.
426
HULCHER
Rt=0.61 mevalonate
5s
Rt =0.?2 mevolonoloctone
Rt=0.66
5-phosphomevalonate
mevalonote 5-phosphomevalonate Rt=0.35 Rt=0.16
1
I
FRONT
ORIGIN
FIG. 3. Identification of 5-phosphomevalonate from mevalonate-2J4C in liver homogenate containing dimethylmevalonic acid. Each drawing represents a radioactive scan of paper chromatograms of reaction mixtures chromatographed in isobutyric acid: ammonia: water (33:1.5:15). Top scan is from the reaction with inhibitor. The bottom scan represents the control reaction without inhibitor. The reaction mixture was as given in Fig. 2 except that the concentration of inhibitor was 20 pmoles of the racemic compound.
To test this idea, similar reaction mixtures were treated to extract any ally1 pyrophosphates, according to the techniques of Christophe and Popjak (16) and Goodman and Popjak (17). The final extracts were almost freeof radioactive material; evidently, no ally1 pyrophosphates were present in reactions having the inhibitor present. About 8 % of original counts in mevalonate2-14Cwere recovered as ally1 pyrophosphates without inhibitor. Optimal conditions for the enzymatic synthesis of Gphosphomevalonate by liver homogenate. To demonstrate the magnitude of the accumulation of 5-phosphomevalonate in liver preparations containing the inhibitor, optimal conditions were determined. The factors studied were concentration of ATP, mevalonate-2J4C, and dimethylmevalonic acid, the amount of liver homogenate, and the duration of incubation. The reaction was stopped by adding an equal volume of 10 % cold perchloric acid. Protein was removed by centrifugation, the perchloric acid neutralized with 30% KOH, and the KClO, removed by centrifugation. The lyophilized extracts were taken up in 2.0 ml of water and made to a final volume of 3.8 ml; 25-~1 aliquots were chromatographed as before. The product, 5-phosphomevalonate accu-
FIG. 4. Accumulation of 5-phosphomevalonate effected by inhibitor under optimal conditions. Counts per minute in l-cm sections of a paper chromatogram are plotted against centimeters of migration. The reaction mixture contained 2.0 ml of rat liver homogenate, 40 rmoles of ATP, 0.299 rmoles of mevalonate-2-W, and 15 pmoles of sodium dimethylmevalonate, and was incubated at 38’ for 20 min. The deproteinieed reaction mixtures were chromatographed in the same solvents as for Fig. 3, the paper cut into strips, and counted in a liquid-scintillation counter.
mulated from L-mevalonate-2J4C, had an RF of 0.32436. The results of strip paper scanning and of counting l-cm strips in a liquid-scintillation counter are given in
INHIBITOR
OF CHOLESTEROL
Fig. 4. The yield of 5-phosphomevalonate from n-mevalonate based on these data was from 82 to 100%; only the n-mevalonate-214C remains. The product was also isolated on, and eluted from Dowex-1 anion-exchange resin in the formate form (15). It was again chromatographed on paper with the systems isobutyric acid: ammonia: water (33 : 1.5 : 15) and t-butanol:formic acid:water (40: 10: 16). The RF values were the same as given by DeWaard and Popjak (15). The 5-phosphomevalonate-2J4C may be prepared in this way for use in studying the reaction catalyzed by phosphomevalonic kinase. DISCUSSION
As anticipated from previous work in which 4-methylmevalonic acid was an inhibitor of cholesterol biosynthesis (6), 4,4dimethylmevalonic acid is also an effective in vitro inhibitor. The results minimize the hypothesis that mevalonic kinase is the major site of inhibition; but suggest that the inhibitor may be a competitive substrate. Since 5phosphomevalonate accumulates in the presence of inhibitor in liver preparations, the activity of mevalonic kinase is not seriously impaired. Rather, the inhibitor or a phosphorylated derivative appears to inhibit phosphomevalonic kinase; yet, allowing the formation of 5-phosphomevalonate. This seems indicated because of the lack of accumulation of either 5pyrophosphomevalonate or ally1 pyrophosphates. Ally1 pyrophosphates could not be detected in inhibited systems and only small amounts were detected in uninhibited systems because of the rapid flow in the direction of cholesterol biosynthesis. In cholesterol biosynthesis when the synthesis of 5-pyrophosphomevalonate is blocked, likewise its decarboxylation product, isopentenyl-pyrophosphate, would not be produced. Stronger support was added to these findings by ‘establishing optimal conditions for producing 5phosphomevalonate using the inhibitor, a procedure which may be used to prepare the naturally occurring enantiomer ((5). It is not known if a single
BIOSYNTHESIS
427
enantiomer of the inhibitor is the active form. The new inhibitor may find usefulness to examine the reactions converting mevalonate to isopentenylpyrophosphate. ACKNOWLEDGMENTS This study was supported by the National Multiple Sclerosis Society Grant-222 and by the United Medical Research Foundation of North Carolina. The author gratefully acknowledges the help given by Dr. Phillip Hamrick in making the infrared analyses and by Dr. Edward 0. Oswald who performed the mass spectrometry and nuclear magnetic resonance analyses. REFERENCES 1. FITTLER, F., KLINE, L. K., AND HALL, Ii. H., Biochemistry 7, 94@944 (1968). 2. PETERKOFSKY, A., Biochemistry 7, 472 (1968). 3. HALL, R. H., AND SRIVAST~VA, B. I. S., Life Sci. 81, 4951 (1959). 4. Ho, K.-J., AND TAYLOR, C. B., Arch. Pathol. 90, 83-92 (1970). 5. CORNFORTH, J. W., CORNFORTH, R. H., .~ND POPJ~K, G., Tetrahedron 18, 1351 (1962). 6. STEWART, J. M., AND WOOLLEY, D. W., J. Amer. Chem. SGC. 81,495l (1959). 7. HULCHER, F. H., AND HOSICK, T. A., U. S. Patent 5, 119,842, Jan. 28, 1964. 8. HOFFMAN, C. H., WAGNER, A. F., WILSON, A. N., WALTON, E., SHUNK, C. H., WOLF, D. E., HOLLY, F. W., AND FOLKERS, K., J. Amer. Chem. Sot. 79,23X-2318 (1957). 9. RILLING, H. C., AND BLOCK, K., J. Biol. Chem. 284, 1424 (1959). 10. YAMAMURA, S., AND HIRATA, Y., Tetrahedron Lett. !I,79 (1964). 11. SAKAN, T., ISOE, S. B., HYEON, S. B., KATSUMUR, R., AND MAEDA, T., Tetrahedron Lett. 46,4097 (1965). 12. WILZBACH, K. E., J. Amer. Chem. Sot. 79, 1013 (1957). 13. FRANZ, I. D., AND BUCHER, N. L. R., J. Biol. Chem. 206, 471 (1954). 14. KABBRA, J. J., MCLAUGHLIN, J. T., AND RIEGEL, C. A., Anal. Chem. 33,305 (1961). 15. DEWAARD, A., AND POPJAK, G., Biochem. J. 73, 410 (1959). 16. CHRISTOPHE, J., AND POPJAK, G., J. Lipid Res. 2, 244-257 (1961). 17. GOODMAN, DEW. S., AND POPJAK, G., J. Lipid Res. 1, 286 (1960).