Chemists Report First Inhibitors Of AMPylation

When Vibrio parahemolyticus, the bacterium responsible for most cases of food poisoning, infects a host, it injects a cocktail of proteins into the host’s cells. One of these proteins, VopS, transfers adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to the host’s proteins, which disrupts signaling pathways, causing the cells to morph in shape and eventually die. Now researchers have identified the first inhibitors of VopS, which could serve as a first step toward new antibiotics (ACS Chem. Biol. 2013, DOI: 10.1021/cb4006886).

The AMP transfer is called AMPylation (Science 2009, DOI: 10.1126/science.1166382). Proteins structurally similar to VopS are found in many pathogenic bacteria, yet the exact biochemical role of these AMPylating proteins in each species is unknown. To start learning more about these proteins, Paul R. Thompson, of Scripps Research Institute, Jupiter, Florida, and his colleagues wanted to find molecules that stop AMPylation. They developed a light-based, high-throughput assay to identify inhibitors of VopS.

In the assay, the researchers mix VopS with a fluorescently labeled ATP and a protein substrate called Cdc42. They then shine polarized light through the mixture, causing the ATP to fluoresce. The polarization of the emitted light increases as VopS transfers fluorescent AMPs to the substrates. That’s because the fluorescent molecules rotate more slowly when attached to Cdc42, so the fluorophores have a better chance of being properly aligned with the incoming light to emit polarized light.

But the fluorescence polarization drops when a molecule that inhibits VopS is added to the mixture. The inhibitor prevents the AMP transfer, so the fluorescent molecules tumble more easily in solution, which depolarizes the emitted light.

Using this polarized-light assay, the researchers screened a commercially available library of 1,280 compounds. Based on how much the molecules cut the polarization and other data, the team identified four candidates for further tests in human cells expressing VopS. Two inhibitors reduced AMPylation in the cells, but the downstream effects of the AMP transfers were still present: The cells still changed shape.

Using this assay, researchers can now look for more potent inhibitors of VopS or other AMPylating proteins, Thompson says.