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.
Methods
Cholesterol 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.

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.

Regulation of the mevalonate pathway occurs at the enzyme 3-hydroxy-3-methylglutaryl

coenzyme A reductase (abbreviated HMG-R hereafter). Negative feedback inhibition and crossregulation 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 HMGR 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 lowdensity 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 receptormediated endocytosis. (See figure 2).

Figure 2. The packaged cholesterol as a cholesteryl ester sequestered by a lipoprotein.

Adapted from Brown and Goldstein 1985.

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.

Figure 3. Path of endocytosis occurring for packaged cholesterol via LDL receptor in
mammalian cells. Adapted from Brown and Goldstein 1985.

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 pravastatin
share 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 fluorophenylsubstituted 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).

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.

Figure 5. The statin family of compounds that inhibit HMG-R. Adapted from Ucar, et al. 2000.

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.

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.

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, Lys 691
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.

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.

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 LDLcholesterol levels by 15 to 30 percent of total cholesterol and 20 to 40 percent of LDLcholesterol 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 ratedetermining 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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