Cholesterol, the Mevalonate Pathway, and Inhibitors of HMG-CoA
Reductase
Cholesterol, the Mevalonate Pathway, and Inhibitors of HMG-CoA
Reductase
By Alice Yoo
Introduction
A topic of growing interest in the scientific community is the
pathway of cholesterol homeostasis. Although cholesterol is
necessary for functioning of the living cell, elevated levels of
blood cholesterol can result in the formation of atherosclerotic
plaques whose build-up can lead to heart attacks and strokes (Brown
and Goldstein, 1985). With the societal rise in
hypercholesterolemia, understanding the pathway of cholesterol
homeostasis is essential in treating and preventing the growth of
these populations.
MethodsCholesterol biosynthesis
Human and animal cells obtain useable cholesterol by two
mechanisms that occur in the liver. The first mechanism is de novo
cholesterol synthesis via the mevalonate pathway, in which
3-hydroxy-3-methylglutaryl coenzyme A reductase is involved in the
rate-determining step (see figure 1). At low cholesterol levels,
the liver and intestine synthesize sufficient amounts of
cholesterol to meet the bodys needs through the mevalonate pathway
(Endo 1992). In this pathway, acetyl CoA and acetoacetyl CoA are
converted to 3-hydroxy-3-methylglutaryl coenzyme A, which then is
converted to mevalonate. Mevalonate eventually forms cholesterol
after taking the form of numerous intermediates. The synthesized
cholesterol feeds into several pathways to form steroid hormones,
vitamin D, bile acids, and other lipoproteins.
Regulation of the mevalonate pathway occurs at the enzyme
3-hydroxy-3-methylglutaryl coenzyme A reductase (abbreviated HMG-R
hereafter). Negative feedback inhibition and cross-regulation are
regulatory mechanisms of this enzyme. In negative feedback
inhibition, cholesterol and isoprenoid intermediates of the
mevalonate pathway suppress HMG-R. According to Reynolds, et al.
1984, cholesterol suppresses HMG-R activity primarily by inhibiting
the rate of the reductase genes transcription. Through inhibition
of transcription, HMG-R ceases to be made for the pathway to
continue. Additionally in cross-regulation, the catalytic domain of
HMG-R is deemed inactive through phosphorylation by an
AMP-dependent kinase. Phosphorylation of the enzyme regulates
sterol synthesis since it alters the enzymes kinetic properties
resulting in cellular energy charge (Hampton, et al. 1996).
Cross-regulation also occurs at the bodys response to stresses
related to the invasion of pathogens. At the invasion of certain
bacterial toxins, cytokines are produced in response, which signals
increase in levels of HMG-R mRNA in the liver (Hampton, et al.
1996). At the resultant increase of HMG-R production, increases are
observed in enzyme activity.
An important aside must be made about the mevalonate pathway.
Mevalonate is not only essential in producing cholesterol, but also
serves as a precursor to numerous non-steroidal isoprenoid
compounds, such as dolichols, heme A, ubiquinone, and
isopentenyladenosine, that are essential for normal activity in the
cell (Bellosta, et al. 1998). According to Huang, et al. 2003,
mevalonate and mevalonate-derived isoprenoids, such as farnesyl
pyrophosphate and geranylgeranyl pyrophosphate are involved in
post-translational modification. This process occurs through
prenylation of several proteins in the signal transduction pathway,
such as the Rho GTPases and the Rac, Ras, Rab, and Rap family
proteins (Huang, et al. 2003). When the mevalonate pathway is
short-circuited by HMG-R inhibition, isoprenoids are not made,
resulting in the absence of protein modification. Without this
post-translational protein modification, proteins are not converted
to their more lipophilic states to permit interactions with cell
membranes. A second mode of obtaining useable cholesterol is by
receptor-mediated endocytosis of low-density lipoprotein
(abbreviated LDL). Since cholesterol is insoluble in water, it
cannot be directly transported in the blood; instead, it undergoes
endocytosis mediated by the LDL receptors in the adrenal gland and
liver (De Pinieux, et al. 1996). Cholesterol present in the body is
esterified with a long-chain fatty acid, essentially becoming
solubilized through sequestration by a surface monolayer of
phospholipids. It then becomes packaged into the core of a
low-density lipoprotein with partitioning by transient cell
membranes. However, this cholesteryl ester cannot pass through
membranes due to its hydrophobicity. Therefore, lipoprotein
receptors, located on the surfaces of cells, bind the lipoprotein
and carry it into the cell by receptor-mediated endocytosis. (See
figure 2).
This receptor-mediated intake of cholesterol occurs at increased
demands for cholesterol in the liver (Goldstein and Brown 1984).
The LDL receptor is a glycoprotein on the cells surface that binds
to two proteins, apo B-100 and apo E. When transported into the
cell, the lipoprotein is delivered to lysosomes where the
cholesteryl ester is hydrolyzed and the freed cholesterol goes to
create plasma membranes, bile acids, steroids and steroid hormones.
Leftover cholesterol is also stored as cholesteryl ester droplets.
This process is summarized in figure 3. In instances of defects in
the LDL receptors, cholesterol builds up and leads to
hypercholesterolemia and atherosclerosis.
The proper functioning of LDL receptors is regulated by
cholesterol in the liver. The liver regulates cholesterol through
controlling the number of available LDL receptors to transport
cholesterol. In a study by Goldstein and Brown 1984, the number of
LDL receptors in relation to cholesterol levels was examined in
rabbits. In response to a high administration of cholesterol,
rabbits accumulated cholesterol in the liver, which then suppressed
the activity of HMG-R and effectually blocked cholesterol
biosynthesis. Cholesterol accumulation then caused suppression in
the production of LDL receptors, causing severe
hypercholesterolemia by the prolonged circulation of LDL in the
blood. However, when the same experiment was performed in rats,
suppression of LDL receptor production was not observed, permitting
effective clearance of LDL. Consequently, hypercholesterolemia did
not occur. Although the explanation for why the rats did not
suppress LDL receptor production was unknown, Goldstein and Brown
indicated that as long as LDL receptors in the liver remains at
high levels, hypercholesterolemia does not occur.
Role and structure of statins
The statin family competitively inhibits HMG-R. Statins have
rigid, hydrophobic groups covalently linked to HMG-like moieties.
Type 1 statinslovastatin, simvastatin, and pravastatinshare a
similar hydronaphthalene ring structure. Type 2 statinsfluvastatin,
cerivastatin, atorvastatin, and rosuvastatinare synthetic statins
that contain large groups attached to their HMG-like moieties.
Fluvastatin is a derivative of the mevalonolactone with a
fluorophenyl-substituted indole ring. The hydroxy acid side chain
allows fluvastatin to be more hydrophilic than the other statins.
The structures are summarized in figure 5. All statins are
administered to the body as the active -hydroxy acid, with the
exception of lovastatin and simvastatin, which are administered as
lactone prodrugs that must be hydrolyzed in the body to their
corresponding -hydroxy acids (Williams and Feely 2002).
Mechanism of HMG-CoA reductase inhibition
Statins curtail the biosynthesis of cholesterol through
inhibition of the rate-determining step in the biosynthesis of
isoprenoids and sterols. The reaction that is inhibited
follows:
(S)-HMG-CoA + 2 NADPH + 2 H+ ( (R)mevalonate + 2 NADP+ +
CoASH
in which NADPH is oxidized and CoA is reduced. As shown in
figure 5, statins bind to the active site of the enzyme, sterically
precluding HMG-CoA from binding. According to Williams, et al.
2002, statins have an affinity for HMG-R that is approximately
three orders of magnitude greater than that of HMG-CoA. This allows
for an effective inhibition of the mevalonate pathway in response
to high cellular levels of cholesterol.
In examining the binding mechanism, it is clear that the
HMG-like moieties attached to the statins occupy the enzyme active
sites of HMG-R. The orientation and bonding of these moieties are
very similar to those of the substrate (see figure 6A). The
HMG-binding pocket contains a cis loop in which polar interactions
are formed between the residues of the cis loop (Ser684, Asp690,
Lys691, Lys692) and the HMG-like moieties of the statins.
Additionally, Lys691 creates a hydrogen-bonding network with the
residues Glu559 and Asp767 and the O5-hydroxyl group of the
statins. Shape and charge complementarities are created between the
statins and the enzymes binding site as a result of the large
number of hydrogen bonds and ion pairs. It is presumed that similar
interactions take place between the normal reaction product
mevalonate and protein, although some of the stabilizing
interactions that take place between protein and substrate will be
missing to account for the feasible release of mevalonate. In
addition, van der Waals interactions exist between the statins and
the enzymes Leu562, Val683, Leu853, Ala856, and Leu857 residues of
the hydrophobic side chains (Istvan, et al. 2001).
Even though statins are a diverse family of compounds, they are
able to effectively inhibit HMG-R because their conformational
adaptability to allow their hydrophobic groups to maximize contacts
with the hydrophobic pocket of the enzyme (Istvan, et al. 2001).
This maximization of contacts between the hydrophobic groups and
the binding pocket is differentiated through the different types of
statins. Type 1 statins possess a decalin group, while the type 2
statins possess a methylethyl group. Additionally, the butyryl
group binding of the type 1 statins is paralleled to the
flurophenyl group in the type 2 statins. Although differences
exist, they still adhere to an optimization of binding between the
HMG-moieties of the statins and the binding pocket of the
enzyme.
Comparison between the six different structures indicates how
the differences among the statins in their binding to the enzyme
are subtle (see figure 7). Of note, however, are the
dissimilarities of rosuvastatin with the other statins.
Rosuvastatin has the greatest number of bonding interactions with
HMG-R and a unique polar interaction between the Arg568 side chain
of the inhibitor and a sulfone group of the protein. Unique to
rosuvastatin and atorvastatin is the hydrogen bonding between
Ser565 and a carbonyl oxygen atom or a sulfone oxygen atom.
In studies with mevastatin, the precursor to the statins, it was
shown that mevastatin had an inhibitor constant Ki of around 10-9 M
and was involved in a ring-opening of its acid form in inhibiting
HMG-CoA reductase. In quantitative structure-analysis relationship
studies, it was shown that derivatives of this statin that lack
amethylbutryl ester and a decalin ring were ineffective in their
inhibitory action. This indicated how these moieties serve as
pharmacophores in their role as inhibitors of the mevalonate
pathway (Endo 1988).
Overall effect of statins
Assessing the overall effect of statins, these inhibitors can
reduce total and LDL-cholesterol levels by 15 to 30 percent of
total cholesterol and 20 to 40 percent of LDL-cholesterol levels
based on dosage and type of statin used (Endo 1992). This response
can lead to a reduction in risks of coronary and atherosclerotic
complications.
Side effects
The main adverse side effects of statins are elevated levels of
serum creatine kinase, myalgia, rhabdomyolysis, and inflammatory
myopathies. Additionally, gastrointestinal effects such as
diarrhea, pain, constipation, and flatulence have been reported,
along with rashes, dizziness, pruritus, and headache (Williams and
Feely 2002). These side effects are muscular, yet the pathology of
the statin-induced muscle side effects is still unclear (De Pinieux
1996).
Treatment of Atherosclerosis
In addition to treating hypercholesterolemia, statins also
demonstrate significant gains in improving conditions of
atherosclerosis. Atherosclerosis, defined as the hardening of the
arterial blood vessel, is caused by the accumulation of ruptured
plaques within the arteries. The protective fibrous cap of a plaque
in a coronary artery can rupture in the instance of atherosclerotic
complications and can lead to instances of myocardial infarction
and unstable angina. Such plaque instability can be manifested as
ulceration of the fibrous cap, plaque rupture, and intraplaque
hemorrhage and is a result of high lipid concentrations with excess
content of macrophages in the fibrous cap (Bellosta, et al. 1998).
Excess macrophages secrete proteolytic enzymes that degrade
collagen, a major component that provides tensile strength for the
fibrous caps. Through phagocytosis or secretion of metalloproteases
(MMPs), macrophages weaken the cap and render it susceptible to
rupture (Bellosta, et al. 1998). Nevertheless, statins inhibit the
production of such metalloproteases, therefore conferring stability
to plaques and effectually lowering the risks for myocardial
infarctions and angina.
Additionally, thickening of the arterial blood vessel and
formation of lesions in the cell wall caused by the deposition of
lipids lead to atherosclerosis. Through the migration of smooth
muscle cells, the arterial blood vessel maintains a thickness to
exacerbate atherosclerosis. Smooth muscle cell migration occurs
through the mevalonate pathway, in which isoprenoid intermediates
produced in the pathway prenylate the necessary proteins to
initiate growth factor signal transduction. Therefore, statins role
of short-circuiting the mevalonate pathway disallows the migration
of smooth muscle cells. Simvastatin, fluvastatin, and cerivastatin
inhibit the migration and proliferation of arterial smooth muscle
cells in a dose-dependent manner (Bellosta, et al. 1998). Since the
isoprenoid intermediates are not being made in the mevalonate
pathway, apoptosis is induced in smooth muscle cells, which may
explain why migration of these cells is inhibited. As a result of
the curtailing of smooth muscle cell migration and proliferation,
the thickness of blood vessel walls is reduced in patients with
carotid atherosclerosis. Nevertheless, when mevalonate and the
isoprenoids all-trans farnesol and all-trans geranylgeraniol are
added, the inhibitory effect of statins is prevented in a
dose-dependent manner, suggesting regulation of myocyte regulation
by isoprenoid metabolites of the mevalonate pathway.
Discussion
The studies outlined here implicate an understanding of
cholesterol to be pertinent to treatment and precautionary measures
for hypercholesterolemia and atherosclerosis. Two methods of
cholesterol synthesis are available to the human cells. The
mevalonate pathway allows for de novo synthesis of cholesterol
through converting acetyl CoA into mevalonate and other isoprenoid
intermediates. Cholesterol is also created through the use of
receptor-mediated endocytosis in the liver via LDL-receptors. The
mevalonate pathway, in which a rate-determining step is the
reaction involving HMG-R, is inhibited by statins in order to block
de novo synthesis of cholesterol.
Future outlook
A future study that may enhance the understanding of cholesterol
in the human body is to examine whether some individuals in the
population have genetic defects that regulate the expression of LDL
receptors and HMG-R. Examining this genetic variability may shed
light on the speculation that some individuals in the population
seem to have a greater sensitivity to high levels of dietary
cholesterol, either allowing for a response that promotes
hypercholesterolemia (as in the study with rabbits) or a response
that promotes clearance of cholesterol and prevents
hypercholesterolemia (as in the study with rats).
Additionally, one must question the genetic and dietary
variability for the high levels of plasma LDL that are present in
many Western industrialized societies. Although it has been seen
that people who consume diets low in animal fats have plasma
LDL-cholesterol levels that remain low, the rise in plasma
LDL-cholesterol levels resulting from an increase in dietary animal
fats is not uniform among individuals. An investigation of this
variability must take into account the response to diet as well as
the genetic components that may predispose an individual to have
high levels of plasma LDL.
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Figure 2. The packaged cholesterol as a cholesteryl ester
sequestered by a lipoprotein. Adapted from Brown and Goldstein
1985.
Figure 1. The mevalonate pathway in animal cells. Negative
feedback inhibition occurs via cholesterol in the steps producing
HMG-CoA and mevalonate. Adapted from Goldstein and Brown 1990.
Figure 3. Path of endocytosis occurring for packaged cholesterol
via LDL receptor in mammalian cells. Adapted from Brown and
Goldstein 1985.
Figure 5. The statin family of compounds that inhibit HMG-R.
Adapted from Ucar, et al. 2000.
Figure 6. Statins exploit the conformational flexibility of
HMG-R to create a hydrophobic binding pocket at the active site. A.
Active site of human HMG-R in complex with HMG, CoA, and NADP. One
monomer is yellow, the other is blue. The ball-and-stick
representation shows the side chains of residues that come into
contact with the statin. HMG an CoA are in magenta and NADP is in
green. B. Binding of rosuvastatin to HMG-R. Rosuvastatin is purple.
Adapted from Istvan, et al. 2001.
Figure 7. The binding of HMG-R with the indicated statins.
Interactions between HMG-like moieties of statins and protein are
of ionic or polar nature and are indicated by the dotted lines.
Hydrophobic groups of statins are in the shallow groove between
helices La1 and La10. Interactions between Arg590 and a
fluorophenyl group occur in type 2 statins (C,D,E,F). A hydrogen
bond exists between Ser565 and a carbonyl oxygen atom (E) or a
sulfone oxygen atom (F). Adapted from Istvan, et al. 2001.
Figure 4. HMG-CoA and Mevastatin in its acid form. The HMG-like
moiety can be seen on the appendage of the double-ring structure of
the statin. Adapted from Endo 1992.