Researchers in Singapore have used microarray technology to study the enzyme family linked to diseases such as SARS.

3CL protease is an important enzyme for the SARS virus, the cause of severe acute respiratory syndrome. It is a cysteine protease, an enzyme that hydrolyses peptide bonds using a cysteine residue in its active site. Now Shao Yao and colleagues at the National University of Singapore have made an array to identify cysteine protease activity and used it to study protease inhibitors; a method that could eventually be used to find drugs to target 3CL protease. 

The group made a series of fluorescent probes, each containing a vinyl sulfone group, a group known to inhibit cysteine proteases. They varied the structures by changing the amino acid to be recognised by the protease in each compound. As a general test for cysteine proteases, a cocktail of these probes was used to screen an array of enzymes. Any cysteine proteases could be identified because they were the only enzymes that the probes bound to and so labelled, giving a fluorescent signal.

Another microarray was used to screen each of the identified cysteine proteases with each of the probes, separately; this gave a fingerprint for each enzyme. Some of the proteases act only on substrates with specific amino acids in the peptide bond to be hydrolysed; these enzymes showed the strongest fluorescent signal with probes containing those amino acids. Others are more general and were labelled equally well by many of the probes. 'The resulting fingerprints reveal unique signatures for individual proteins, providing biological insights into their functional roles,' Yao said.

The group used a third array to screen small molecule inhibitors against the identified proteases to find inhibitor fingerprints. They added solutions of varying inhibitor concentration to each protease, followed by a vinyl sulfone probe. Increased inhibition was shown by lower probe fluorescence as the inhibitor blocked the probe from the enzyme.

Yao explained that future challenges for the field would include developing microarrays with detection methods that could be used to follow an enzyme as it reacts. His group hopes to extend its research to other kinds of enzymes, such as kinases and phosphatases.

Rachel Warfield