Playing it cool with PRMT1

LA JOLLA, Calif.—Recent work at the Scripps ResearchInstitute has led to the discovery of the first chemical compounds that work toblock an enzyme linked to inflammatory conditions such as asthma, arthritis andsome inflammation-promoted cancers. The study, "Novel Inhibitors for PRMT1Discovered by High-Throughput Screening Using Activity-Based Fluorescence Polarization,"was published in the journal ACS ChemicalBiology.

Until now, standard screening techniques have beenunsuccessful in distinguishing between compounds that inhibit PRMT1 (proteinarginine methyltransferase 1) and compounds that inhibit other PRMT familyenzymes. In the new study, however, Scripps scientists developed the firstPRMT1-specific screening technique, resulting in two promising candidates.

"Aberrant PRMT1 activity has been associated withcardiovascular, infectious, autoimmune and malignant disease, making it aviable therapeutic target for several indications," says Scripps Researchassistant professor Dr. Kerri Mowen, the study's principal investigator.

Mowen, who has been studying PRMT1 since graduate school,says the enzyme accounts for more than 85 percent of cellular PRMT activities,with PRMTs regulating many biological processes such as transcription, DNArepair, RNA processing and signal transduction. Mowen notes that their lab hasalso found that PRMT1 "can promote the expression of select cytokines by Tcells," having demonstrated in 2004 that the enzyme helps drive the productionof interferon-gamma and interleukin-4, key immune-stimulating proteins. PRMT1has been linked to lung, colon, pancreatic, gastric and bladder cancer, sheadds, and "the estrogen receptor is hyper-arginine methylated in a subset ofbreast cancers."

Additionally, Mowen says that in 2007, Eric So's labdiscovered that the enzyme is also a key component "for transformation mediatedby the MLL-EEN fusion protein, which causes mixed lineage leukemia, a veryaggressive and predominantly pediatric blood cancer."

Despite its existence as a promising therapeutic target,PRMT1 inhibitors have yet to be developed given that the PRMT enzymes are allnearly identical, structurally and biochemically. But Mowen and the otherresearchers found a way to overcome that challenge with a creative and highlyeffective screening technique inspired by the research of fellow Scrippsscientist Benjamin F. Cravatt III, chair of the Scripps Research Department ofChemical Physiology. In 2010, Cravatt and his team screened tens of thousandsof human and mouse proteins for the presence of hyper-reactive cysteine aminoacids, which almost always mark functional sites on proteins. PRMT1 ended upcontaining the reactive cysteine, and, even better, only one other (ratherrare) PRMT enzyme was known to have the cysteine as well.

After verifying that the cysteine was in the active site ofPRMT1, the researchers found a fluorescent probe that would bind to thecysteine, so that if a test compound fastened to PRMT1's active site like aninhibitor, it would interfere with the probe's binding and result in a lowerfluorescence-based signal. If a compound failed to bind, the probe would bind normally,denoting an ineffective inhibitor.

"We were able to verify, using available non-specificinhibitors of PRMT enzymes, that they did indeed bind to PRMT1 and prevent theprobe from binding, and that was the proof-of-concept that enabled us to goahead with a screen," Myles B. C. Dillon, a graduate student in Mowen's lab andlead author of the study, said in a press release.

Mowen and Dillon then sought out Prof. Hugh Rosen, a Scrippsresearcher who curates the Maybridge Hitfinder Collection, a library of 16,000chemical compounds. The researchers tested the compounds one by one, and withRosen's help, were able to adapt the setup as an automated, high-throughputscreening technique, ending up with two inhibitor candidates that demonstrated"good efficacy and specificity," that the team "might be able to modify … tomake them even better," Dillon said.

The team recently received a grant from the NationalInstitutes of Health (NIH) to use their new screening technique on a300,000-compound NIH library, which was also curated at Scripps.

The next step, Mowen says, will be to improve the efficacyand bioavailability of the hit compounds they discovered, to screen the NIH'scompound library and to test the hits in animal models of inflammation.

"Biologic-based therapies have provided the pharmaceuticalindustry with blockbuster drugs, but they have notable shortcomings as they arenot effective in all patients (about 30 percent of RA patients fail to respondto TNFa blocking agents), can induce anti-drug immune responses and aretremendously expensive," says Mowen. "Our lab and others have shown that PRMT1can modify and regulate several critical immunomodulatory proteins, whichsuggests that PRMT1 small molecular inhibitors may be valuable for the treatmentof inflammation and autoimmunity."

Additional authors of the study were Daniel A. Bachovchin ofthe Cravatt laboratory, Steven J. Brown of the Scripps Research MolecularScreening Center and M. G. Finn, professor in the Scripps Research Departmentof Chemistry.