Bifunctional inhibitors of mevalonate kinase and mevalonate 5-diphosphate decarboxylase

Article abstract of DOI:10.1021/ol052573s

A bifunctional inhibitor of mevalonate kinase and mevalonate 5-diphosphate decarboxylase was synthesized. Both enzymes are in the cholesterol biosynthetic pathway and play an important role in regulating cholesterol biosynthesis. The molecule may become a

Full text of DOI:10.1021/ol052573s

ORGANIC
LETTERS
2006
Vol. 8, No. 6
1013-1016
Bifunctional Inhibitors of Mevalonate
Kinase and Mevalonate 5-Diphosphate
Decarboxylase
Yongge Qiu and Ding Li*
Department of Biology and Chemistry, City UniVersity of Hong Kong,
83 Tat Chee AVenue, Kowloon, Hong Kong SAR, P. R. China
bhdingli@cityu.edu.hk
Received October 24, 2005
ABSTRACT
A bifunctional inhibitor of mevalonate kinase and mevalonate 5-diphosphate decarboxylase was synthesized. Both enzymes are in the cholesterol
biosynthetic pathway and play an important role in regulating cholesterol biosynthesis. The molecule may become a useful lead compound
for further development for treating cardiovascular disease and cancer. This study provides a novel example of a single inhibitor blocking two
sequential steps simultaneously in the cholesterol biosynthetic pathway.
In animal cells, the cholesterol biosynthetic pathway contains
a unique series of three sequential ATP-dependent enzymes
that convert mevalonate to isopentenyl diphosphate (IPP):
mevalonate kinase (MVK), phosphomevalonate kinase (PMK),
and mevalonate 5-diphosphate decarboxylase (MDD) (Figure
1).1 These three enzymes catalyze consecutive steps down-
lowering drugs treating cardiovascular disease. HMG-CoA
reductase catalyzes the main rate-limiting step in the
synthesis of cholesterol and nonsteroid isoprenoid derivatives.
Inhibition of HMG-CoA reductase by lovastatin and related
compounds blocks cholesterol biosynthesis and increases
transcription of the LDL receptor gene.2 A variation on this
approach has been suggested for cancer chemotherapy.3
Extremely high levels of reductase inhibitors, given for a
short period could prevent farnesylation of Ras proteins,
inhibiting the growth of Ras-dependent tumor cells. Although
HMG-CoA reductase is a key regulatory site in the pathway,
enzymes and genes further along the pathway are also
important in regulation of pathway function. Several inves-
tigators have suggested the involvement of MVK and MDD
as important regulatory steps in the biosynthesis of choles-
terol.4
Figure 1. Mevalonate pathway in animal cells.
The X-ray structures of MVK and MDD from various
sources have been solved,5 and both enzymes belong to the
GHMP (Galactokinase, Homoserine kinase, Mevalonate
stream from the HMG-CoA reductase in the mevalonate
pathway, and are responsive to cholesterol intake in animals.
The pathway has been exploited in the design of cholesterol-
(2) (a) Brown, M. S.; Goldstein, J. L. Science 1986, 232, 34. (b) Ma, P.
T. S.; Gil, G.; Sudhof, T. C.; Bilheimer, D. W.; Goldstein, J. L.; Brown,
M. S. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8370.
(3) Schafer, W. R.; Kim, R.; Sterne, R.; Thorner, J.; Kim, S. H.; Rine,
J. Science 1989, 245, 379.
(1) (a) Goldstein, J. L.; Brown, M. S. Nature 1990, 343, 425. (b) Houten,
S. M.; Schneiders, M. S.; Wanders, R. J. A.; Waterham, H. R. J. Biol.
Chem. 2003, 278, 5736. (c) Kaur, M.; Kaul, D. FASEB J. 1997, 11, A1217.
10.1021/ol052573s CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/11/2006

kinase, and Phosphomevalonate kinase) superfamily of small-
molecule kinases. MVK and MDD share the same fold,
catalyze phosphorylation of chemically similar substrates
(MDD decarboxylation involves phosphorylation of meva-
lonate diphosphate), and seem to have evolved from a
common ancestor.5c Kinases are a ubiquitous group of
enzymes central to many biochemical processes.6 The transfer
of the terminal phosphoryl group from one nucleotide to
another, or to a small molecule (by enzymes which we term
“small molecule kinases”), or to a protein substrate (by
protein kinase) is a fundamental process in many aspects of
metabolism, gene regulation, and signal transduction.6
lonate lactone is relatively expensive, which prevents large-
scale preparation of mevalonate 5-diphosphate. We devel-
oped a new synthetic method for the preparation of mevalonate
5-diphosphate as shown in Scheme 1, which facilitated our
Scheme 1. Organic Synthesis of Mevalonate Diphosphate
A variety of substrate analogues of MVK and MDD
targeting the mevalonate binding site have been studied,7 and
some have been found to be enzyme inhibitors. In addition,
MVK is subject to posttranscriptional regulation via competi-
tive inhibition at the ATP-binding site by prenyl phosphates
of varying length, including geranyl diphosphate (GPP),
farnesyl diphosphate (FPP), and geranylgeranyl diphosphate
(GGPP).8 Because of the potential importance of MVK and
MDD in the regulation of cholesterol biosynthesis, and their
similar structure and catalyzed reaction, we tried to develop
a common inhibitor effective for both enzymes. In the present
study, we report organic synthesis and biological character-
ization of a bifunctional molecule, which can inhibit both
MVK and MDD. This molecule may become a lead for
further development by the pharmaceutical industry for
treating cardiovascular disease and certain forms of cancer.
further study of MDD. 4-Hydroxy-2-butanone reacted with
toluenesulfonyl chloride in the presence of pyridine to give
4-tosyloxy-2-butanone (1), which was then reacted with
methyl 2-bromoacetate through a Reformatsky reaction
yielding methyl 3-hydroxy-3-methyl-5-tosyloxypentanoate
(2). Mevalonate 5-diphosphate (4) was obtained through
reaction of compound 2 with pyrophosphate followed by
hydrolysis with base.
Three bisubstrate analogues (compounds 38, 39, and 40)
were synthesized through three parallel syntheses as shown
in Scheme 2. Three corresponding analogues (compounds
The substrate of MDD, mevalonate 5-diphosphate, has
been obtained previously through hydrolysis of mevalonic
lactone followed by reaction with pyrophosphate.9 The
method required protection of the carboxylate group of
mevalonate through formation of methyl ester after hydroly-
sis of mevalonic lactone, and also needed deprotection of
the methyl group after reaction with pyrophosphate. Meva-
Scheme 2. Organic Synthesis of Bisubstrate Analogues
(4) (a) Iglesias, J.; Gonzalezpacanowska, D.; Marco, C.; Garciaperegrin,
E. Biochem. J. 1989, 260, 333. (b) Gonzalezpacanowska, D.; Marco, C.;
Garciamartinez, J.; Garciaperegrin, E. Biochim. Biophys. Acta 1985, 833,
449. (c) Mitchell, E. D.; Avigan, J. Circulation 1979, 60, 31. (d) Tanaka,
R. D.; Schafer, B. L.; Lee, L. Y.; Freudenberger, J. S.; Mosley, S. T. J.
Biol. Chem. 1990, 265, 2391. (e) Jabalquinto, A. M.; Cardemil, E. Arch.
Biochem. Biophys. 1981, 210, 132. (f) Gonzalezpacanowska, D.; Marco,
C.; Garciamartinez, J.; Linares, A.; Garciaperegrin, E. Nutr. Rep. Int. 1985,
31, 121. (g) Gonzalez-Pacanowska, D.; Marco, C.; Garcia-Martinez, J.;
Garcia-Peregrin, E. Biochim. Biophys. Acta 1986, 875, 605. (h) Tanaka, R.
D.; Lee, L. Y.; Schafer, B. L.; Kratunis, V. J.; Mohler, W. A.; Robinson,
G. W.; Mosley, S. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2872. (i)
Jabalquinto, A. M.; Cardemil, E. Lipids 1980, 15, 196.
(5) (a) Fu, Z. J.; Wang, M.; Potter, D.; Miziorko, H. M.; Kim, J. J. P. J.
Biol. Chem. 2002, 277, 18134. (b) Yang, D.; Shipman, L. W.; Roessner,
C. A.; Scott, A. I.; Sacchettini, J. C. J. Biol. Chem. 2002, 277, 9462. (c)
Bonanno, J. B.; Edo, C.; Eswar, N.; Pieper, U.; Romanowski, M. J.; Ilyin,
V.; Gerchman, S. E.; Kycia, H.; Studier, F. W.; Sali, A.; Burley, S. K.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12896.
(6) Matte, A.; Tari, L. W.; Delbaere, L. T. J. Structure 1998, 6, 413.
(7) (a) Nave, J. F.; d’Orchymont, H.; Ducep, J. B.; Piriou, F.; Jung, M.
J. Biochem. J. 1985, 227, 247. (b) Reardon, J. E.; Abeles, R. H. Biochemistry
1987, 26, 4717. (c) Wilde, J.; Eggerer, H. Eur. J. Biochem. 1994, 221,
463. (d) Wilde, J.; Eggerer, H. Liebigs Ann. Recl. 1997, 581. (e) Dhe-
Paganon, S.; Magrath, J.; Abeles, R. H. Biochemistry 1994, 33, 13355. (f)
Vlattas, I.; Dellureficio, J.; Ku, E.; Bohacek, R.; Zhang, X. Bioorg. Med.
Chem. Lett. 1996, 6, 2091.
(8) (a) Hinson, D. D.; Chambliss, K. L.; Toth, M. J.; Tanaka, R. D.;
Gibson, K. M. J. Lipid Res. 1997, 38, 2216. (b) Dorsey, J. K.; Porter, J. W.
J. Biol. Chem. 1968, 243, 4667.
32, 33, and 34) without a geranyl group were also synthesized
as shown in Scheme 2 for comparative study. The mono-
protected diols (8, 9, and 10) were oxidized with PCC,
(9) Reardon, J. E.; Abeles, R. H. Biochemistry 1987, 26, 4717.
1014
Org. Lett., Vol. 8, No. 6, 2006

followed by a regioselective aldol condensation10 giving
organic compounds with â-ketol structure (14, 15, and 16).
The protection with dihydropyran followed by a Reformatsky
reaction generated compounds 20, 21, and 22. The depro-
tection accompanied by self-formation of lactone (23, 24,
and 25), followed by tosylation, gave key intermediate
compounds 26, 27, and 28. Compounds 32, 33, and 34 were
synthesized through the reaction of compounds 26, 27, and
28 with pyrophosphate followed by hydrolysis of lactone.
The bisubstrate analogues (compounds 38, 39, and 40) were
also obtained from intermediate compounds 26, 27, and 28
through a one-pot reaction with pyrophosphate and geranyl
bromide, followed by hydrolysis of lactone. This reaction
followed a previously reported method for syntheses of
isoprenoid conjugates of nucleoside 5′-diphosphates11 with
significant improvement, which made the desired product
in a one-pot reaction from geranyl bromide, pyrophosphate,
and tosylates of mevalonate derivatives instead of the
stepwise reactions used previously.
used. It is also possible that enzymes from two sources have
slightly different structures, and rat MVK is less susceptible
to feedback inhibition. P′-Geranyl 3,5,8-trihydroxy-3-
methyloctanate 8-diphosphate (39) and P′-geranyl 3,5,9-
trihydroxy-3-methylnonanate 9-diphosphate (40) showed a
slightly better inhibitory effect while P′-geranyl 3,5,7-
trihydroxy-3-methylheptanate 7-diphosphate (38) showed a
slightly less inhibitory effect than GPP on MVK. This result
showed that the bisubstrate analogue, compound 39, is a
much better inhibitor than the analogue without the geranyl
group (33). Compounds 38, 39, and 40 all exhibit competitive
inhibition with respect to both ATP and mevalonate, while
compounds 32, 33, and 34 show competitive inhibition on
mevalonate only.
The inhibitory effect of the above synthetic substrate
analogues on the activities of MDD was also investigated
and the results are shown in Table 1. Mevalonate-5-
diphosphate derivatives (32, 33, and 34) and GPP showed
weak inhibitory activity against rat MDD, while bisubstrate
analogues 38, 39, and 40 showed a much stronger inhibitory
effect than GPP. The result showed that the bisubstrate
analogue, compound 39, is a much better inhibitor than the
analogue without a geranyl group (33). Compounds 38, 39,
and 40 all exhibit competitive inhibition with respect to both
ATP and mevalonate, while compounds 32, 33, and 34 show
competitive inhibition on mevalonate-5-diphosphate only.
Two other organic molecules, compounds 42 and 43, were
also synthesized as reference molecules, using the same
reactions for comparison, as shown in Scheme 3. Octanoyl-
The inhibitory effect of the above synthetic substrate
analogues on the activities of MVK was investigated by using
Dixon plot,12 and the results are summarized in Table 1.
Table 1. IC50 Values of Synthetic Compounds for Rat MVK
and MDD
IC50 for rat
IC50 for rat
compd
MVK (µM)
MDD (µM)
4
32
33
34
GPP
38
39
39a
39b
40
180
275
300
225
5.0
8.0
3.8
2.5
7.5
3.9
450
5.5
215
185
200
65
2.6
2.0
1.9
2.0
6.5
600
600
Scheme 3. Organic Synthesis of Control Compounds
42
43
Mevalonate-5-diphosphate (4) and its derivatives (32, 33, and
34) showed weak inhibitory activity against rat MVK. A IC50
value of 5.0 µM was obtained for geranyl diphosphate on
MVK, which is comparable with that previously reported
on rat liver MVK.13 A considerably lower Ki value of 104-
116 nM has been reported for geranyl diphosphate on human
MVK.14 It should be mentioned that measured IC50 and Ki
values are dependent on assay conditions, and should be used
for comparison only when the same assay conditions are
1-diphosphate (42) shows much weaker competitive inhibi-
tion on both MVK and MDD than compounds 38, 39, and
40 (Table 1). P′-Geranyl octanate 8-diphosphate (43) exhibits
rather weak competitive inhibition on MDD, while compa-
rable inhibition with that of GPP on MVK. This control
experiment indicates simple lipophilic chains bearing a
pyrophosphate moiety are not good inhibitors.
Since compound 39 includes two stereo centers, further
effort was made to separate diastereomers. Lipase was used
to resolve the racemic mixtures without success. The cyclic
intramolecular esters, which are resistant to hydrolysis by
lipase, are quite stable. The mixture of four diastereomers
was separated by silica gel column into two spots 39a and
39b on TLC. Spot 39a includes two diastereomers, (3S),(5R)-
P′-geranyl 3,5,8-trihydroxy-3-methyloctanate 8-diphosphate
and (3R),(5S)-P′-geranyl 3,5,8-trihydroxy-3-methyloctanate
(10) Kourouli, T.; Kefalas, P.; Ragoussis, N.; Ragoussis, V. J. Org. Chem.
2002, 67, 4615.
(11) Ryu, Y. H.; Scott, A. I. Org. Lett. 2003, 5, 4713.
(12) (a) Dixon, M. Biochem. J. 1953, 55, 170. (b) Burlingham, B. T.;
Widlanski, T. S. J. Chem. Educ. 2003, 80, 214.
(13) (a) Dorsey, J. K.; Porter, J. W. J. Biol. Chem. 1968, 243, 4667. (b)
Tanaka, R. D.; Schafer, B. L.; Lee, L. Y.; Freudenberger, J. S.; Mosley, S.
T. J. Biol. Chem. 1990, 265, 2391.
(14) Hinson, D. D.; Chambliss, K. L.; Toth, M. J.; Tanaka, R. D.; Gibson,
K. M. J. Lipid Res. 1997, 38, 2216.
Org. Lett., Vol. 8, No. 6, 2006
1015

8-diphosphate. Spot 39b includes two other diastereomers,
(3S),(5S)-P′-geranyl 3,5,8-trihydroxy-3-methyloctanate 8-
diphosphate, and (3R),(5R)-P′-geranyl 3,5,8-trihydroxy-3-
methyloctanate 8-diphosphate. It appears that 39a and 39b
have similar competitive inhibition on both MVK and MDD
(Table 1). 39a is slightly better than 39b on inhibition of
both MVK and MDD.
been previously reported for inhibition of other enzymes,16
they were designed and synthesized with a different strategy.
Two enzymes are usually significantly different, and two
known inhibitors are linked together through a covalent
chemical bond. Our strategy takes advantage of similar
active-site structures of two consecutive enzymes involved
in the biosynthesis of cholesterol, which provides a new
rational approach for drug design. More and more enzymes
are grouped into superfamilies, and members of the same
superfamily normally have similar peptide folding; therefore,
our approach may have general utility in drug design.
Compound 39 may be a useful lead compound for further
pharmaceutical development. Compound 39 is composed of
a hydrophilic part (mevalonate) and a hydrophobic part
(geranyl group), which is structurally similar to fatty acids
and may facilitate penetration of lipid membranes to reach
the target enzymes therefore increasing its effectiveness.
Compound 39 showed the strongest inhibition for both
MVK and MDD, indicating that a 3-carbon bridge is the
most suitable chain length between the mevalonate part and
geranyl diphosphate for both enzymes. It may reflect the
natural distance between ATP and mevalonate in the active
sites of MVK and MDD. It should be mentioned that drug
combination has been used in the clinical treatment of various
diseases,15 and sequential blocking of two enzymes in a
metabolic or biosynthetic pathway is one strategy for
enhancing treatment. Because it is difficult to inhibit an
enzyme completely (particularly with a reversible inhibitor),
inhibition of two consecutive enzymes in a pathway by using
a combined drug treatment can block the pathway much more
effectively than a single inhibitory drug therapy. Reversible
enzyme inhibition, however, is hyperbolic in nature and
requires a large excess of the inhibitory drugs, which may
have toxic side effects. Normally, two separate drugs are
given to patients when using a drug combination treatment.
In contrast, our bisubstrate analogue compound 39 provides
a novel example of a single inhibitor carrying out the
sequential blocking of MVK and MDD in the biosynthesis
of cholesterol. Although some bifunctional inhibitors have
Acknowledgment. The work described in this paper was
substantially supported by a grant from the City University
of Hong Kong (Project No. 7001793).
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for all new compounds, and
assay methods for MVK and MDD. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL052573S
(15) Silverman, R. B. The organic chemistry of drug design and drug
action, 2nd ed.; Elsevier Academic Press: Boston, MA, 2004.
(16) Jaulent, A. M.; Leatherbarrow, R. J. Protein Eng. Des. Sel. 2004,
17, 681.
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