Statins: Powerful Statins: Powerful Inhibitors Inhibitors

of of Cholesterol Cholesterol BiosynthesisBiosynthesis

Cholesterol: What is it?Cholesterol: What is it?11

Cholesterol is a fatty steroid made primarily in the liver of most animals and humans. It is an integral component in the synthesis of hormones, can also be found in cell walls of animals and humans.

Isolated cholesterol is a white, flaky solid that is insoluble in aqueous environments.

H O

Cholesterol

Two types of transportation Two types of transportation for cholesterolfor cholesterol

In order to transport the steroid through blood, cholesterol is attached to a set of proteins called lipoproteins. There are two types of lipoproteins: high density and low density lipoproteins.

HDL: High-density lipoproteins – collect cholesterol particles as they travel through blood vessels and deposits them in the liver where they are transferred to bile acids and disposed off.

LDL: Low-density lipoproteins –deposits on the walls of blood vessels, and over time, builds up into cholesterol plaque and blocks blood vessels, especially arteries that feed blood to the heart.

1. The liver manufactures, secretes and removes LDL cholesterol from the body. To remove LDL cholesterol from the blood, there are special LDL receptors on the surface of liver cells.

2. LDL receptors remove LDL cholesterol particles from the blood and transport them inside the liver. A high number of active LDL receptors on the liver surfaces is necessary for the rapid removal of LDL cholesterol from the blood and low blood LDL cholesterol levels.

A deficiency of LDL receptors is associated with high LDL cholesterol blood levels.

Diets that are high in cholesterol diminish the activity of LDL receptors!!!!

Biological Role:Biological Role:11

It is an important component of cell linings It helps in the digestion of lipids It is a key component in the building of hormones

Hypercholestraemia: High blood cholesterol

Usually a result of high LDL/low HDL cholesterol levels Leads to

narrowing of artery walls (atherosclerosis) decreased blood and oxygen supply to heart heart attack death

Coronary heart disease1: Leading cause of death in western countries.

Initial treatment of hypercholesteraemia was directed toward limiting LDL-cholesterol levels through:

Low-cholesterol dietand regular exercise.Exercise burns fat so it is not coverted to cholesterol which the Body will have to dispose off.

This approach was not very successful because high blood cholesterol is also hereditary (Familial Hypercholestraemia (FH))1 and a chronic condition. People with FH have defective or nonexistent LDL receptors and need rigorous, long-term treatment.

Scientific Approach: Know and understand how the body makes

cholesterol

Find a way to effectively control cholesterol levels with minimum adverse effects

The Mevanolate PathwayThe Mevanolate Pathway22

The biosynthesis of cholesterol and isoprenoids (a group of compounds responsible for cell fluidity and cell proliferation)

+ HSCoA

H2CC

CH3HO

CH2

C O O

C SCoA

O

H2CC

CH3HO

CH2

C O O

H2C OH

2NADP+

2NADPH

HMG-CoA

mevalonate

HMG-CoA Reductase

5-pyrophosphomevalonate

isopentenyl pyrophosphate

geranyl pyrophosphate

farnesyl pyrophosphate

squalene 2,3-oxidosqualene

HO HO

lanosterol cholesterol

19 steps

In 1976……..In 1976…….. ML-236A, ML-236B, ML-236C: metabolites isolated

from a fungus (Penicillium citrinum) were found to reduce serum cholesterol levels in rats.

This work was done by Akira Endo, Masao Kuroda and Yoshio Tsujita at the Fermentation Research Laboratories, Tokyo, Japan.3

Preliminary experiments showed that these fungal metabolites had no effect on mevanolate or other steps in the biosynthetic pathway.

This led to the speculation that their action was somewhere between the mevanolate and the HMG-CoA

β

Target: HMG-CoA Target: HMG-CoA Reductase (HMGR)Reductase (HMGR)

The enzyme that catalyzes the conversion of HMG-CoA to mevanolate.

This reaction is the rate-determining step in the synthetic pathway.

+ HSCoA

H2CC

CH3HO

CH2

C O O

C SCoA

O

H2CC

CH3HO

CH2

C O O

H2C OH

2NADP+

2NADPH

HMG-CoA

mevalonate

HMG-CoA Reductase

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)

RESULTSRESULTS Rats received oral dose of test

compounds (5 mg/kg suspended in 0.5 mL of saline)

Control group received 0.5 mL of saline

Of the 3 substances tested, ML-236B had the highest level of hypocholesterolemic activity.

Amounts required for 50% inhibition

ML-236A 0.18 µg/mLML-236B 0.01 µg/mLML-236C 0.08 µg/mL

StatinsStatins ML-236B was later called compactin(6-demethylmevinolin

or mevastatin). A related fungal metabolite called lovastatin (mevinolin) was also found to be another good inhibitor of HMG-CoA reductase. Lovastatin was isolated from Aspergillus terreus.

compactinlovastatin

Today, there are two classes of statins:Natural Statins: Lovastatin(mevacor), Compactin, Pravastatin (pravachol), Simvastatin (Zocor).Synthetic Statins: Atorvastatin (Lipitor),Fluvastatin (Lescol).

Statins are competitive inhibitors of HMG-CoA reductase. They are bulky andliterally get “stuck” in the active site.This prevents the enzyme from binding with its substrate, HMG-CoA.

Ester side-chain

Making the synthetic Making the synthetic statinsstatins

Lovastatin and compactin can be made in the lab in multistep syntheses.

This allowed scientists to study the structural-activity relationship of statins. The lactone was found to be the business end of the drugs.4

Modification of Modification of LovastatinLovastatin Since statins are competitive inhibitors, an increase in the

amount of HMG-CoA will reduce the effectiveness of the drugs.

New drug design approaches are geared towards making lovastatin analogs that will have longer interaction with the enzyme –increase duration of drug occupancy of active site.

Structural modification: i. making ether side-chain analogs(Lee, et. al. 1982) ii. homologation of the lactone ring

iii. converting lovastatin to mevanolate analog

(changing stereochemistry at the hydroxy- bearing carbon in the lactone)

i. making ether side-chain analogs5

ii. homologation of the lactone ring6

Purpose is to develop a lactone homolog that is compatible with the complex and sensitive structural features of lovastatin.

As in the case of making the ether analogs, the hydroxy-bearing carbon had to be protected

The hydroxy-bearing carbon in HMG-CoA and mevanolate have a methyl group. This substituent is lacking in lovastatin

Purpose is to investigate the biological consequence of this methyl group

16 and 11 are epimers: diastereomers that differ in configuration at only one stereogenic center.

iii. converting lovastatin to mevanolate analog (placing a methyl group at the hydroxy-bearing carbon in the lactone)6

ResultsResults Mevanolate and lactone modifications: no biological

test and results have been report. Results from ether analogs (Lee, et. al. in 1991)5

i. The ethers were tested against their ester analogsii. Compactin was used as standard and assigned a relative potency of 100

In vitro HMG-CoA reductase inhibitory activityshowed that absence of the carbonyl has detrimental effect on the inhibitory strength.

General conclusion: side-chain ether analogs areweaker inhibitors of HMGR than their Corresponding ether analog.

The role of the ester group in the synthetic pathway is still under investigation.

ConclusionsConclusions Coronary heart disease, a condition caused by

hypercholestraemia is a major leading cause of death in most western countries.

The discovery of natural statins (lovastatin and compactin) lead to innovative approaches to treatment of high cholesterol.

These natural statins have also served as templates for making synthetic statins, most of which are on the market today.

With understanding of the SAR of statins and their interactions with HMGR (bonding nature, etc), we can improve the effectiveness of these drugs and limit side-effects.

ReferencesReferences1. Lee, D. Cholesterol and the heart.

http://www.medicinenet.com/cholesterol/ (Sept 2004).2. Diwan, J. J. Cholesterol Synthesis.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/cholesterol.htm (Sept 2004).

3. Endo, A.; Kuroda, M.; Tsujita, Y. J. Antibio. (Tokyo) 1976, 29, 1346-1348.

4. Istvan, E. S. American Heart Journal 2002, 144, S27-32.5. Lee, T. J.; Holtz, W. J.; Smith, R. L.; Alberts, A. W.; Gilfillan,

J. L. J Med Chem 1991, 34, (8), 2474-7.6. Lee, T. J. H., W. J.; Smith, R. L. Journal of Organic Chemistry

1982, 47, (24), 4750.