Metabolism is described as the series of chemical reactions required to sustain life, and when it comes to drug metabolism no one does it better than the cytochrome P450 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 super family, but still super nonetheless!
Metabolism in Drug Design
In drug discovery, understanding the way a drug candidate is metabolized greatly increases its chances of making it through development, as it allows us to predict how a drug might behave once it is inside a patient’s body. To put it simply, if we suspect it will be broken down too quickly to due a certain functional group, we can replace it with another to increase its overall half life, and vice versa.
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 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 picky with their food, meaning they can metabolize exogenous (foreign) compounds, including 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 allows CYP2D6 to bond with high affinity to compounds, such that despite it being present only in low concentrations in the liver it is extremely important in drug metabolism.
Once bound, CYP2D6 performs oxidation on areas of high electron density, such as 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 time 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 located a set distance away from the site of oxidation, allowing them to oxidize a wide range of compounds. Tolbutamide was found to be metabolized quickly by CYP2C9 at the para methyl group on the aromatic ring, and was exploited by replacing the methyl group with a chlorine atom (chlorpromamide). The chlorine atom makes the entire compound more resistant to oxidation, resulting in chlorpromamide having a much longer half life.
Replacing electron rich hydrogen groups with halogens is a common technique used by medicinal chemists to prolong the duration of action of drugs. This is due to the much stronger C-X bond compared with C-H making it more difficult to metabolize, and also the steric effects of having a large, bulky halogen group instead of a tiny little 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. The expulsion of water provides the driving force to ‘activate’ the iron-oxygen center and allows oxidation to take place.
In particular, CYP3A4 favors N-demthylation 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, and 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 targeted alteration 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.
- 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.