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Cholesterol is a biologically important molecule whose synthesis overview was featured in the steroids lecture, found using this link: Steroids. In this lecture, we will investigate cholesterol further, specifically its biological functions, its different packagings that help transport to and from the cell membrane, and methods of inhibition of biosynthesis of cholesterol that can be used to treat individuals with high levels of LDL packaging, also sometimes called "bad cholesterol." 

Cholesterol Functions

Function 1: Micelle Formation

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As shown above, only 1% of cholesterol is converted to steroidal hormones such as progesterone and estradiol.

Furthermore, about 50% of cholesterol is converted to cholate (bile salt) to aid in fat transport.  An initial oxidation occurs in the liver and is coupled with the reduction of iron oxides (FeIV to FeIII) in cytochrome enzymes. After a terminal methyl of the cholesterol is selectively oxidized to an alcohol, the resulting molecule is oxidized further to cholate (bile salt), a molecule with both nonpolar and polar regions. These regions assist the formation of micelles like the one pictured above. But, why do we need micelles and how do they form? 
Why cholate makes micelles: Acting as an adapter molecule, cholate helps the formation of micelles to aid in the transport of fats throughout the body. Usually, so-called "hydrophobic" interactions prevent water from solvating fats and allowing them to travel easily.

How micelles are made: The nonpolar end of cholate interacts with nonpolar ingested fat, while the polar carboxylate group dangling off of the D ring interacts with polar water molecules. In this way, the water molecules never have to see the fat, and a solvation shell is formed with a polar surface that allows solvation in water and transportation throughout the body.

Function 2: Cell Membrane Formation and Permeability

About another 50% of cholesterol made in our bodies is used for the structure and functioning of cell membranes. Because both the intracellular and extracellular environment is aqueous,  the cell membrane's task of maintaining the cell's water levels is difficult. It must keep water from escaping and entering the cell. This requires the cell membrane to have two features:

  1. Hydrophilic (polar) inside and outside edges.
  2. Hydrophobic (nonpolar) middle so that water from the inside and outside of the cell will not pass through the membrane.

These features create the phospholipid bilayer that we see in the cell membrane. The polar phosphate heads make up the inside and outside of the bilayer, while the middle is composed of the nonpolar tail regions of the phospholipids.  

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The phospholipid bilayer works remarkably well, but because of its structure, it is impervious to many things the cell wishes to take in or to expel. It is thought that the cholesterol molecules inserted into the cell membrane help regulate the permeability of the bilayer and thus facilitate the transport of molecules through the membrane. However, the use of cholesterol in the membrane may become a problem when either not enough of it is present or too much of it is present. When there is not enough cholesterol in the membrane, not enough important molecules may be able to enter the cell. On the other hand, when there is too much cholesterol in the membrane, important molecules inside the cell can exit. In these cases, the controlled permeability provided by cholesterol has lost its effectiveness.

Cholesterol Transport

How are cholesterol molecules transported to and from the cell membrane? Since cholesterol is very nearly insoluble in water, and our blood is water based, cholesterol will not solvate and travel in the blood unaided. Thus, cholesterol is transported by lipoprotein micelles that originate in the liver. These cholesterol packages have phospholipid monolayers embedded with proteins. The monolayer has an outward facing (polar/water-soluble) surface and inward facing (nonpolar/cholesterol-soluble) surface. The cholesterol is carried within these lipoproteins to and from cell membranes throughout the body.

There are two kinds of lipoproteins, specifically low-density lipoproteins (LDL) and high-density lipoproteins (HDL). LDL delivers cholesterol from the liver to the cell membrane and after it dumps the cholesterol into the cell membrane, this empty lipoprotein returns back to the liver to pick up more cholesterol molecules.  High-density lipoprotein (HDL) also originates from the liver, but it picks up cholesterol from the cell membrane and returns it to the liver.  HDL is named accordingly because its membrane contains more proteins than LDL's membrane.  Since high levels of LDL can lead to serious health problems, the packaging of cholesterol in LDL is often referred to as "bad cholesterol." The LDL pathway is labeled in red (stop!) ,while the HDL ("good cholesterol") pathway is labeled in green (go!). In the end, there are not two kinds of cholesterol but rather two different micelle packagings of cholesterol.

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Your body needs cholesterol to regulate cell membrane permeability, and therefore needs both LDL and HDL, but too much LDL can be a bad thing. Your liver naturally produces about 1 gram of cholesterol per day, which is enough for normal biological function, but we can ingest much more than that. The increased levels of cholesterol lead to an increase of LDL production in the liver. The LDL deposits this excess cholesterol into the cell membranes, performing its normal function even if cholesterol levels are already too high.  High LDL levels can lead to health problems, such as a stroke:

  • Cell membranes of blood vessels have abnormally high cholesterol concentration-> reduces polarity of the cell membranes, causing a loss of adhesion->Failure of cells to adhere to one another causes them to separate and let other stuff get between cell masses.
  • White blood cells can now permeate the vein/artery walls--> white blood cells are designed to recognize and consume pathogens, foreign bodies, bacteria etc. that should not be present in the blood stream. Unaware that they are no longer in the blood stream, the white cells begin consuming everything around them, thinking it is a threat. The white cells undergo what is called an "engorging process," in which they consume so much that they burst. This leads to an immune system response, releasing more white blood cells to clean up the white blood cell pieces. Those white blood cells subsequently burst, and the cycle continues.
  • This iterative process leads to plaque formation, which can eventually constrict blood flow. ->If plaque is deposited in the brain, the subsequent constriction of blood flow can lead to a stroke.

Nearly all high LDL levels can be corrected by reducing cholesterol consumption, but the inconvenience of changing dietary habits and unwillingness to do so lead chemists to produce a cholesterol biosynthesis inhibitor.

Cholesterol Biosynthesis

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Cholesterol biosynthesis proceeds as drawn above, a biological pathway beginning with Acetyl CoA that undergoes a series of reactions with different enzymes, cofactors, and substrates to produce cholesterol. How should we shut down cholesterol biosynthesis? In order to inhibit the biosynthesis of cholesterol you could attempt to stop the mechanism at any one of these steps, however if you inhibit the wrong step, there could be many adverse effects on the body. When selecting an inhibitor, you want to use a molecule that will inhibit the "committed step," or the first step in which the product will go on to only make cholesterol.  In this case the committed step is the reduction of HMG-CoA to mevalonate (meaning once mevalonate is produced, the only biological product will be cholesterol).  If you were to inhibit a later step in the synthesis, it may be hard to get rid of these later products. For example,  if you prevented the formation of lanosterol from squalene oxide, you would cause a build up of squalene oxide in your cells, which may result in toxic shock from squalene oxide overdose. If you were to stop the synthesis too soon, sometime during its initial steps, you could inhibit the production of hundreds of other biologically useful molecules that have the same first steps as the cholesterol biosynthesis. However, HMG-CoA is used for other functions in the body, so if you stop the biosynthesis at the reduction of HMG-CoA, it will not proceed to form cholesterol, but may go on to do other useful chemistry in the body.

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The image above shows the mechanism of the committed step that we would like to inhibit. HMG-CoA is reduced by NADPH (looks like NADH) in HMG-CoA reductase. Electrons from the carbonyl bond are sent up onto the oxygen. One of the lone pairs of the now negatively charged oxygen will collapse down, reforming the double bond and eliminating the S-CoA substituent in the process. This conjugate addition and subsequent elimination normally goes on to produce mevalonate, and an inhibitor with structural similarities to HMG-CoA could work to prevent this step of cholesterol biosynthesis.

As can be seen in the above a lysine residue acts as an analog to the oxyanion hole seen in serine proteases. However, the enzyme also has characteristics of aspartyl proteases in that instead of forming the acyl-enzyme intermediate produced in serine proteases the enzyme forms a tetrahedral intermediate. These two elements are components of HMG-CoA reductase that can be targeted when creating inhibitors.

HMG-CoA Reductase Inhibition

Based on what we know about inhibitors of enzymes we can predict what the HMG-CoA enzyme would look like. Since the intermediate of the reductase is a tetrahedral intermediate very similar to that seen in aspartyl proteases we can employ a similar strategy to "fool" the enzyme. Secondary or tertiary alcohols have proved useful as aspartyl protease inhibitors. We can thus propose a potential inhibitor which simply replaces the carbonyl in the substrate with an alcohol group. This would create a molecule that would fit perfectly into the enzyme's specificity pocket but be non-processable as the alcohol will interact with the lysine residue but not be able to "collapse down" like the carbonyl.

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Circled in green is the alcohol that has replaced the carbonyl of HMG-CoA. There are, however, problems with the above structure for use as a drug. The carboxyl group circled in red is highly polar. Due to this group this molecule will be unable to pass through the gastrointestinal tract and from there be circulated throughout the body. For this reason chemists had to come up with a creative strategy to mask this group during uptake and then unmask the group so the inhibitor can act on the reductase and reduce cholesterol biosynthesis.

In 1978, a Japanese chemist discovered a molecule called mevastatin, a compound produced by a soil-dwelling fungus called penicillium citrinum. Mevastatin, a lactone and cyclic ester, was the first of a group of inhibitors known as the "statins" to be discovered.  When mevastatin is reacted with esterase (functions like a protease), it is hydrolyzed, breaking open its ring and forming a reversible, transition state analog inhibitor to HMG-CoA reductase.

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Mevastatin is drawn alongside the molecule it is mimicking in order to see the similarities in the two structures. The functionally critical elements of the structures are circled. (mevastatin on left) The large rings attached to the lactone functionality are also important in recognition by the enzyme. Looking back to the mechanism of HMG-CoA reductase we can see that NADPH is a critical cofactor in the reduction reaction. The two rings serve to mimic the structure of the NADPH cofactor, thus allowing for the inhibitor be better fitted to the enzyme's active site.

Mevastatin is not biologically active until the lactone ring is broken open by the esterase. This new structure looks very much like the HMG-CoA intermediate, shown to the right of the hydrolyzed mevastatin, that is involved in the uninhibited mechanism. The carboxylic acid circled at the upper left end of both molecules serves as the recognition element. This carboxylic acid probably hydrogen bonds to the enzyme, temporarily holding it in the active site. The circled hydroxyl group is in the right place for enzymatic activity as well; however, if you were to deprotonate this oxygen and collapse the electrons down (as you do in the uninhibited reaction), there is no leaving group to kick off. The elimination step fails in this inhibited reaction. In the normal HMG-CoA reductase mechanism, the lone pair on the oxygen collapses down to kick off the S-CoA group. But, this never occurs with the mevastatin. The remaining structural elements of the inhibitor are similar in size to the normal intermediate structure to the right, making it that much more likely to be admitted into the enzyme it inhibits.

Reversible inhibitors are very appealing to pharmaceutical companies and are useful for blocking cholesterol biosynthesis temporarily. Because reversible inhibitors only remain in the active site of the enzyme for a finite period of time, one must continuously take the drug to maintain effective inhibiting concentrations in the body, which leads to our next topic involving different types of statins.

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The Half Maximal Inhibition Concentration, or IC50 , is the concentration of inhibitor in a drug required for 50% inhibition of a biological process/part of a process. (in our case, the inhibition of the HMG-CoA Enzymes)

Characteristics and Facts of Statins

  • Mevastatin: made by fungus, highly reactive, (IC50=1nM), too many side effects to be used as a drug
  • Lovastatin (aka Mevacor): made by red yeast rice, considerably less powerful then mevastatin (IC50=50nM), fewer side effects, approved in 1987 by FDA
  • Simvastatin (aka Zocor): developed after lovastatin was off patent from fermentation of a fungus, more effective than lovastatin (IC50=11nM), much cheaper to produce, half life of 2 hours, approved in 1992
  • Atorvastatin (aka Lipitor): was developed after simvastatin was off patent.  The drug's developers recognized that in simvastatin, the molecule was hydrolyzed before it was activated.  So for a supposedly "new" drug idea, they simply came up with an already hydrolyzed, open-ring form of simvastatin with an insignificant bond rotation and a different bottom half of the inhibitor. The result had IC50= 8nM and a half life of 24 hours, which was much better than the half life of Zocor. The drug Lipitor was approved in 1997.
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