Probing Enzyme Function in Cells

A systematic approach to find substrates, products, functional roles, and selective inhibitors of previously unknown enzymes has been developed. The technique, devised by professor of cell biology and chemistry Benjamin F. Cravatt and coworkers at Scripps Research Institute (Chem. Biol. 2006, 13, 1041), could ease the discovery of small-molecule drugs that target newly found enzymes, which is normally quite difficult.

The study began when Cravatt and coworkers used activity-based protein profiling (ABPP), a chemical proteomics method that measures enzyme activity, to identify KIAA1363, a previously unknown enzyme that showed elevated activity in highly aggressive cancer cells. "A lot of times when such an associative relationship is found, that's the last you ever hear of the protein," Cravatt says, "because no one knows what to do next with an unannotated [functionally uncharacterized] protein. It's a very hard problem."

To overcome this problem, his group used a competitive-binding version of ABPP to discover a small molecule that inhibits KIAA1363 potently and selectively in cells. They used that inhibitor to turn off the enzyme in cells and employed liquid chromatography/mass spectrometry to screen for corresponding changes in cell metabolite levels. The concentrations of a series of monoalkylglycerol ethers (MAGEs) went down about fivefold when KIAA1363 was inhibited. Further cellular experiments confirmed 2-acetyl MAGEs as the enzyme's substrates, other MAGEs as its products, and ether lipid metabolism as its functional role.

Cravatt and coworkers "show that ABPP studies can lead to the development of a potent and selective inhibitor for an enzyme with an unknown function, normally a nearly impossible task," write Ariel G. Herman and Brent R. Stockwell of Columbia University in a commentary. The technique "should allow for annotation of large numbers of enzymes with currently unknown functions," they predict.

This type of approach provides "a fairly systematic strategy for placing enzymes on metabolic pathways in cells and discovering the roles of those pathways in human disease," Cravatt says.