Drug Metabolism: Small but significant

August 9, 2004

..

http://www.nature.com/cgi-taf/gateway.taf?g=5 & file=/drugdisc/res_high/articles/nrd1506.html

Drug metabolism:Small but significant

from Nature Reviews Drug Discovery

Joanna Owens

The crystal structure of arguably the most important enzyme in drug metabolism — cytochrome P450 3A4 (CYP3A4) — has been published, and with it comes the surprise discovery that its active site is much smaller than previously thought. Although the overall structure of the enzyme is characteristic of the P450 family, this paper describes several unique features that provide new insights into the ability of CYP3A4 to metabolize a wide variety of diverse substrates.

CYP3A4 metabolizes 50% of all marketed drugs — more than any other P450 isoform — and the diversity of its substrates indicate that it is capable of metabolizing several compounds simultaneously.

In the absence of a crystal structure, understanding the complexity of CYP3A4 substrate binding and conformational change has relied on the use of homology models. However, these have limited use because the sequence similarity between P450s is low — just 24% between CYP3A4 and the recently published CYP2C9.

Writing in Science, Harren Jhoti and colleagues describe three structures of CYP3A4: unliganded, bound to a substrate (progesterone) and bound to an inhibitor (metyrapone). They report that there are some unique features of CYP3A4 that could help in the design of drugs with improved metabolic and toxicity profiles.

One particular feature unique to CYP3A4 is the observed 'Phe-cluster' of seven phenylalanine residues that form a hydrophobic core pointing towards the active site. Some of the residues that form this cluster have been shown by mutational analysis to have a role in CYP3A4 activity. The presence of this cluster means that the accessible volume of the active site is much smaller than would be expected considering the large molecular size of some CYP3A4 substrates. The authors propose that conformational change of the Phe-cluster could reposition phenylalanine residues, resulting in an extended active site that is capable of binding more than one substrate. In addition, the active site in CYP3A4 has greater access to the haem moiety compared with CYP2C9. This proximity could enable two substrate molecules to have access to reactive oxygen, potentially providing the means for CYP3A4 to metabolize more than one substrate simultaneously.

The authors investigated whether conformational change was a prerequisite for substrate or inhibitor binding. They were surprised to find that there was no conformational change in metyrapone-bound CYP3A4, but postulated that this might be because metyrapone is a small molecule that the CYP3A4 active site could accommodate easily. To test this hypothesis, they studied the conformation of the protein when bound to the CYP3A4 substrate progesterone. Again, no conformational change was observed, but, more significantly, progesterone was shown to bind peripherally to the active site close to the Phe-cluster, in a novel binding pocket of CYP3A4, which might be involved in recognition of substrate and effector molecules and cooperativity. Furthermore, the authors suggest that movement of the Phe-cluster could facilitate the transfer of substrates from the peripheral site to the active site by forming a substrate-access channel. Further insights into the activity of CYP3A4 should be forthcoming now that the long-awaited crystal structure is available.

References

ORIGINAL RESEARCH PAPER Williams, P. A. et al. Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science 15 July 2004 (doi:10.1126/science.1099736).

WEB SITE

Astex Technology http://www.astex-technology.com/index.html

http://www.astex-technology.com/home.jsp