Advertisement

If you have an ACS member number, please enter it here so we can link this account to your membership. (optional)

ACS values your privacy. By submitting your information, you are gaining access to C&EN and subscribing to our weekly newsletter. We use the information you provide to make your reading experience better, and we will never sell your data to third party members.

ENJOY UNLIMITED ACCES TO C&EN

Biological Chemistry

Chemists Report First Inhibitors Of AMPylation

Chemical Biology: Molecules blocking bacterial enzymes that transfer AMP could lead to new antibiotics

by Melissae Fellet
December 5, 2013

AMPed Up
[+]Enlarge
Credit: ACS Chem. Biol.
These two molecules inhibit VopS, a bacterial enzyme that transfers adenosine monophosphate to other proteins.
Chemical structures of VopS inhibitors.
Credit: ACS Chem. Biol.
These two molecules inhibit VopS, a bacterial enzyme that transfers adenosine monophosphate to other proteins.

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.

Advertisement

Article:

This article has been sent to the following recipient:

0 /1 FREE ARTICLES LEFT THIS MONTH Remaining
Chemistry matters. Join us to get the news you need.