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.

References

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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.