US scientists are targeting an enzyme essential to bacterial metabolism in the search for new antibiotics.

Michael Burkart of the University of California, San Diego, and Anton Simeonov from the National Institutes of Health, Bethesda, and coworkers have developed a high-throughput kinetic assay to screen small molecules as inhibitors of surfactin-type phosphopantetheinyl transferase (Sfp-PPTase) enzymes.

Transferases are among a group of enzymes that can add and remove groups from proteins after their polypeptide backbone has been built - a process known as posttranslational modification. The enzymes are of biological and pharmaceutical interest as their inhibitors have been suggested as avenues for antibacterial, antifungal and anticancer therapeutic development. Sfp-PPTases in particular are known to activate biosynthetic pathways towards virulence factors in pathogens and so finding small molecules that reduce the enzymes' activity is of interest to researchers pursuing novel antibiotics. 'But transferase enzymes have been resistant to the development of simple methods for activity determination that do not require centrifugation, washing or separation steps,' explains Simeonov.

Thiadiazole SCH-202676 was the most active inhibitor of a Bacillus subtilis PPTase

The US researchers built upon their previous work which uses PPTases to transfer a fluorescent substrate analogue onto a protein. The protein contains a fluorescence quenching group, meaning that the PPTase activity can be monitored by observing a fluorescence decrease as the reaction proceeds. The team has now incorporated the reaction into an assay which identifies PPTase inhibitors by their effect on the fluorescence decrease. They were able to validate the new assay in a pilot screen of approximately 1200 bioactive molecules and found several potential PPTase inhibitors.

Hirotada Mori, of the Nara Institute of Science and Technology, Ikoma City, Japan, who specialises in bioinformatics, says that the development 'will help a lot in this field, by helping in the discovery of new chemical inhibitors. Such inhibitors will be the tools that allow further research into these enzymes,' he adds, 'so this work will open the gate to further research into PPTases.'

'We will be conducting a high throughput screen of the molecular libraries small molecule repository, a diverse

collection of around 300 000 small molecules,' says Simeonov. 'With access to chemical probes of PPTase enzymes, we hope to gain a further understanding of phosphopantetheinyl transfer events in a range of organisms from bacteria to humans. We also speculate that the methodology may be adaptable to other transferase enzyme classes,' he adds.

Mary Badcock

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