Cytochrome P450 Enzymes: Meet the Superfamily
Metabolism is described as the series of chemical reactions required to sustain life; when it comes to drug metabolism, no one does it better than the cytochrome P450 (CYP450) family of enzymes. Understanding of key enzymes within this superfamily – such as CYP2D6, CYP2C9 and CYP3A4 – is an important part of drug design. Were you expecting The Incredibles? Sorry, not that superfamily, but still super nonetheless!
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Metabolism in Drug Design
In drug discovery, understanding the metabolism pathway of a drug candidate greatly increases its chances of making it through development. It allows us to predict how a drug might behave once it is inside a patient’s body, as well as make modifications to its existing structure. To put it simply, if we suspect that a certain functional group will make it unstable, we can replace it with another to increase its overall half-life and vice versa if it is too stable.
The liver plays a key role in the metabolism and clearance of drugs. Passive diffusion, as well as various carrier proteins, are responsible for moving the drug compounds from the bloodstream into hepatic cells, which can then work their magic. Generally, the role of the liver is to make the compounds more hydrophilic (or water soluble) such that it can be dissolved and excreted as urine (which is mostly water).
Cytochrome P450 (CYP450)
Discovered in 1958, these enzymes exhibit a unique and intense absorption band at 450nm, giving them their name. Drug metabolism is commonly broken down into phase I and phase II. They are first oxidized (fitted with an –OH group) and then conjugated (existing -OH group replaced with a big functional group).
The CYP450 family carries out phase I oxygen atom insertions by targeting a drug’s H atoms or electrons, oxidizing the compound and making it more hydrophilic. CYP450s are important in drug metabolism because enzymes in this family are usually not too picky with their substrates. This means they can metabolize exogenous (foreign) compounds, including many drugs!
The wonderful non-specificity of CYP450 enzymes stems from its haem center, namely the highly electron deficient, highly valent FeO3+ complex – that’s iron in the +4 or +5 oxidation state! The high valence of the iron center ‘activates’ oxygen, such that it is a competent oxidant in alkene epoxidation and alkane hydroxylation via a 1-electron (radical) oxidation3.
This means it essentially rips hydrogens from carbons and forces an oxygen atom in between them, which is no mean feat. The three major forms of CYP450 identified to be the most active in drug metabolism in humans are CYP2D6, CYP2C9 and CYP3A4.
The CYP2D6 enzyme bears an aspartic acid (COO–) residue that allows it to bind to compounds containing basic nitrogen atoms, such as propafenone – a drug used to control irregular heartbeat (arrhythmia). The strength of the aspartic acid-nitrogen bond means that CYP2D6 can bind with high affinity to certain compounds. Hence, it is extremely important in drug metabolism despite its relatively low concentrations in the liver.
Once bound, CYP2D6 performs oxidation on areas of high electron density, such as electron-rich aromatic rings (especially at the para position) and terminal R-O-R’ substituents. Both metoprolol and betaxolol are beta blockers, but metoprolol has a much shorter half-life compared to betaxolol and has to be dosed multiple times a day.
Substrates of CYP2C9 are unique in that they have hydrogen bond donating groups a set distance away from the site of oxidation, allowing them to oxidize a wide range of compounds.
Tolbutamide (a drug candidate) was found to be metabolized too quickly by CYP2C9 at the para-methyl group on the aromatic ring. By replacing the methyl group with a chlorine atom (
Replacing electron rich hydrogen groups with halogens is a common technique medicinal chemists use in order to prolong the duration of action of drugs. The stronger C-X bond compared with C-H makes it harder to break, combined with steric effects of having a large, bulky halogen group instead of a tiny proton!
The jack of all trades, CYP3A4 is able to metabolize probably the largest range of drugs in the CYP450 superfamily. Its enzyme-substrate bonding forces are relatively weak, meaning the active site can adapt to different orientations depending on the substrate.
Rather, substrates are targeted based on their lipophilicity, as they are thought to be able to drive water out of the active site of CYP3A4. This expulsion of water provides the driving force needed to ‘activate’ the iron-oxygen center and allows oxidation to take place.
In particular, CYP3A4 favors N-demethylation and carbon oxidation at allylic/benzylic positions as hydrogen atoms in those locations tend to be more reactive. As with other chemical reactions, the statistical probability has to be taken into account (i.e. multiple sites of oxidation) – as seen in terfenadine, an antihistamine.
Trying to circumvent drug metabolism by CYP3A4 poses a big challenge in drug design due to its affinity for many substrates as well as tolerance to changes in the substrate structure, courtesy of its binding mechanism. However, the two strategies that are first considered are:
- Removal or replacement of the culprit group(s) that are the major site of oxidation
- Reduction of lipophilicity of the entire compound (by oxygen insertion, reduction of carbon chain length, etc.)
There are, of course, many other enzymes involved in drug substrate metabolism. CYP450 activity is just one of the things (albeit an important thing) that has to be understood when designing a new ‘hit’ compound. It allows us to model and predict pharmacokinetic profiles of a drug even before they are synthesized, saving both time and money. It also enables modification of an existing drug to improve its properties, such as increasing/decreasing its rate of clearance or designing a prodrug for more efficient membrane transfer.
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