Metabolism is described as the series of chemical reactions required to sustain life. When it comes to drug metabolism, no one processes these chemicals better than the cytochrome P450 (CYP450) family of enzymes. Understanding key enzymes within this superfamily—including CYP2D6, CYP2C9 and CYP3A4—is essential to drug design. Were you expecting The Incredibles? Sorry, not that superfamily, but cytochrome are super nonetheless!
CYP450 is the name for a superfamily of enzymes in our liver. They are responsible for breaking down many foreign chemicals in our bodies, including drugs and toxins, through oxidation reactions. Out of over fifty CYP450 enzymes, three (CYP2D6, CYP2C9 and CYP3A4) are among the most important, oxidizing most of the medicines we take.
What is Drug Metabolism
Chemical Structure is Everything
In drug discovery, understanding how a potential drug is broken down in the body dramatically increases its chances of making it through development. If we can predict how a molecule behaves inside a patient’s body, we can modify its existing chemical structure.
For example, we want to increase the stability of a drug. If we think a certain part of the molecule will make it unstable, we can replace it with another to increase its overall half-life.
If we want the opposite, we can look for these stable groups and replace them with something less stable.
All About the Liver
The liver is the crucial organ for breaking down (metabolizing) drugs. Once we take a medicine, it goes into our bloodstream. Oral medications will diffuse through the stomach and intestines, while injectables directly enter the blood.
Blood moves around our body and eventually reaches our liver. In the liver, special carrier proteins move the drug compounds from the bloodstream into liver cells. These liver cells can then work their magic to break them down.
Generally, the liver cells make compounds more hydrophilic (water-soluble) to help them dissolve and be excreted as urine. And for that, they can count on the CYP450 superfamily of enzymes.
Introducing the CYP450 Family
How Does CYP450 Work?
Discovered in 1958, the cytochrome P450 (CYP450) family of enzymes was named as such because they absorb photons of light at 450 nm, hence their massively creative name.
CYP450 enzymes work by oxidation—jamming an oxygen atom onto electron-rich areas on molecules. It’s quite a brute-force approach to metabolism, but it works (most of the time). The drug molecule becomes more hydrophilic after oxidation, meaning it can dissolve in water and is removed through our urine.
The reason why CYP450 enzymes are so valuable is because of they’re not too picky with their substrates. We can rely on them to break down many drugs and toxic substances!
The wonderful non-specificity of CYP450 enzymes stems from its haem center. The haem group is an electron-deficient, highly reaction FeO3+ complex—iron in the +4 or +5 oxidation state! The highly reactive iron center activates oxygen so it can easily attach to drug molecules.
For those more chemically-inclined, this haem group is so reactive that it is a competent oxidant in alkene epoxidation and alkane hydroxylation through a 1-electron (radical) oxidation3!
CYP450 essentially rips hydrogens from carbons and forces an oxygen atom on them, which sounds simple, but is no mean feat. The three forms of CYP450 identified as the most important for human drug metabolism are CYP2D6, CYP2C9 and CYP3A4.
CYP2D6
The CYP2D6 enzyme is unique because it has an aspartic acid (COO–) group, allowing it to bind to nitrogen in drug molecules. One of these is propafenone, a drug used to control an irregular heartbeat (arrhythmia). It attaches to propafenone so firmly that other CYP450 enzymes can’t come close.
After binding to the nitrogen atom, it looks for electron-rich aromatic rings. The chemical structure of propafenone is below; can you guess which benzene ring it oxidizes?
The strength of the aspartic acid-nitrogen bond means that CYP2D6 can bind with a high affinity to certain compounds. Hence, despite its low concentration in the liver, it is vital in metabolizing nitrogen-containing drugs.
Below are two similar molecules, metoprolol and betaxolol, both beta-blockers for hypertension. However, metoprolol is broken down much quicker than betaxolol. Can you figure out why?
Remember, CYP2D6 binds to nitrogen, which both compounds have. Then, they look for areas rich with electrons, such as aromatic rings (especially at the para position) and terminal R-O-R’ groups (ether groups).
Because metoprolol has an ether group, CYP2D6 attacks the terminal carbon attached to its oxygen. In betaxolol, the cyclopropyl group doesn’t allow this due to its structure, preventing CYP450 enzymes from reacting at that end.
The result is that metoprolol has a much shorter half-life compared to betaxolol, and patients on metoprolol have to take multiple doses every day.
CYP2C9
CYP2C9 is unique within the CYP450 family because it looks explicitly for unprotected hydrogen atoms. Many drugs have similar ‘hanging’ methyl groups, making CYP2C9 able to metabolize a wide range of compounds.
For example, we can look at two similar drug molecules, tolbutamide and chlorpropamide. One has a methyl group at the end, while the other has a chlorine group attached at the same position.
Tolbutamide (a drug candidate) was actually developed first, but was found to be metabolized too quickly by CYP2C9 because of the methyl group at the benzene ring. The researchers replaced the methyl group with a chlorine atom, which made the molecule (
Replacing electron-rich hydrogen groups with halogens is a common technique medicinal chemists use to prolong the action of a drug in the body. The stronger C-X bond compared with C-H makes it harder to break down. Furthermore, the chlorine atom is huge compared to the methyl group, blocking CYP450 enzymes from attaching to the molecule.
CYP3A4
The jack of all trades, CYP3A4 can metabolize the widest variety of drugs out of all the enzymes in the CYP450 superfamily. It forms relatively weak bonds, which means it can adapt to different orientations that fit whatever molecule is thrown at it.
Instead of looking for specific atoms, CYP3A4 targets lipophilic areas (parts of the molecule that hates water). This is because CYP3A4 holds water molecules in its active site. By binding to a drug, the water molecules can escape, which drives the reaction through entropy. After binding, the energy released is used to activate the haem center and oxidation occurs.
An example of its non-specificity, CYP3A4 can metabolize terfenadine, an antihistamine. Looking at the chemical structure of terfenadine, there isn’t a particularly reactive group. However, there is a group of three methyl groups on the right side of the molecule.
Even though none of the three methyl groups are reactive (they aren’t electron-rich, since they are bound to another carbon), CYP3A4 can still attach at this location! This is because of the number of methyl group—three groups means it is three times as likely to bind. Sometimes, statistical probability is all it takes.
CYP450 in Drug Design
Stop Metabolizing My Drugs!
Due to how efficient the CYP450 superfamily of enzymes are, medicinal chemists face considerable challenges when designing drugs. Often, we want to increase the duration a drug is active for, but trying to circumvent drug metabolism by nonspecific enzymes like CYP3A4 is difficult.
Even if a drug escapes metabolism by one enzyme, there are another fifty in the family that can do a job. In general, however, there are two strategies we can use if we want to protect our drug from CYP450 metabolism:
- Removing or replacing the group(s) that are significant sites of oxidation (electron-rich areas)
- Reducing the lipophilicity (increasing the water affinity) of the entire compound. This includes adding oxygen atoms to the structure and reducing the length of hydrocarbon chains
Other Considerations
Of course, many other enzymes are involved in drug substrate metabolism. CYP450 activity is just one of the factors (albeit an important factor) that has to be understood when designing a potential drug candidate. However, knowing how a drug is broken down allows us to model and predict pharmacokinetic profiles like ADME before drug development, saving time and money.
We can also use this information to improve existing drugs (in what is known as me-too drug design). Using a known drug as a template, we can change its properties such, as increasing or decreasing its rate of metabolism to alter the duration of action.
Reference
- Smith, D. A., & Van de Waterbeemd, H. (2012). Pharmacokinetics and metabolism in drug design. John Wiley & Sons.
- Gupta, G. K., & Kumar, V. (2016). Chemical Drug Design. Walter de Gruyter GmbH & Co KG.
- Hohenberger, J., Ray, K., & Meyer, K. (2012). The biology and chemistry of high-valent iron-oxo and iron-nitrido complexes. Nature communications, 3, 720.
- Danielson, P. B. (2002). The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Current drug metabolism, 3(6), 561-597.
- Hernandes, M. Z., Cavalcanti, S. M. T., Moreira, D. R. M., de Azevedo, J., Filgueira, W., & Leite, A. C. L. (2010). Halogen atoms in the modern medicinal chemistry: hints for the drug design. Current drug targets, 11(3), 303-314.
About the Author
Sean is a consultant for clients in the pharmaceutical industry and is an associate lecturer at La Trobe University, where unfortunate undergrads are subject to his ramblings on chemistry and pharmacology.
