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New screening technique yields elusive compounds to block immune-regulating enzyme

gene from lab mice as an alternative way to study its functions was problematic, since mouse embryos can't survive without the protein.

Inspiration Close By

The inspiration for the new PRMT1-selective screening technique came from research performed in the neighboring laboratory of co-author Benjamin F. Cravatt III, who chairs the Scripps Research Department of Chemical Physiology. As reported in 2010 in the journal Nature, Cravatt and his team screened tens of thousands of human and mouse proteins for the presence of hyper-reactive cysteine amino acids, which almost certainly mark functional sites on those proteins. PRMT1 was found to be one of the reactive-cysteine-containing proteinsand all but one other, comparatively rare PRMT enzyme was known to lack that cysteine.

"We took that discovery a step further, and we were able to find a probe that specifically would recognize that cysteine in PRMT1," said Mowen, who was one of Cravatt's collaborators on the 2010 study.

She and her colleagues first verified that the reactive cysteine in PRMT1 is in the active site of the enzyme. They then found a fluorescent probe that would bind to that cysteine. If a test compound acted as an inhibitor by fastening to PRMT1's active site, it should interfere with the probe's binding, and the probe's fluorescence-based signal therefore should be lower. By contrast, if a test compound failed to bind to PRMT1's active site, the probe should bind normally and its signal should remain elevated.

"We were able to verify, using available non-specific inhibitors of PRMT enzymes, that they did indeed bind to PRMT1 and prevent the probe from binding, and that was the proof-of-concept that enabled us to go ahead with a screen," said Myles B. C. Dillon, a graduate student in Mowen's lab who was lead author of the study.

Exploring Libraries of Potential

Dillon and Mowen turned to collaborator Scripps Research Professo

Contact: Mika Ono
Scripps Research Institute

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