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Playing it cool with PRMT1
LA JOLLA, Calif.—Recent work at the Scripps Research Institute has led to the discovery of the first chemical compounds that work to block an enzyme linked to inflammatory conditions such as asthma, arthritis and some inflammation-promoted cancers. The study, "Novel Inhibitors for PRMT1 Discovered by High-Throughput Screening Using Activity-Based Fluorescence Polarization," was published in the journal ACS Chemical Biology.
Until now, standard screening techniques have been unsuccessful in distinguishing between compounds that inhibit PRMT1 (protein arginine methyltransferase 1) and compounds that inhibit other PRMT family enzymes. In the new study, however, Scripps scientists developed the first PRMT1-specific screening technique, resulting in two promising candidates.
"Aberrant PRMT1 activity has been associated with cardiovascular, infectious, autoimmune and malignant disease, making it a viable therapeutic target for several indications," says Scripps Research assistant 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, DNA repair, RNA processing and signal transduction. Mowen notes that their lab has also found that PRMT1 "can promote the expression of select cytokines by T cells," having demonstrated in 2004 that the enzyme helps drive the production of interferon-gamma and interleukin-4, key immune-stimulating proteins. PRMT1 has been linked to lung, colon, pancreatic, gastric and bladder cancer, she adds, and "the estrogen receptor is hyper-arginine methylated in a subset of breast cancers. "
Additionally, Mowen says that in 2007, Eric So's lab discovered that the enzyme is also a key component "for transformation mediated by the MLL-EEN fusion protein, which causes mixed lineage leukemia, a very aggressive 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 all nearly identical, structurally and biochemically. But Mowen and the other researchers found a way to overcome that challenge with a creative and highly effective screening technique inspired by the research of fellow Scripps scientist Benjamin F. Cravatt III, chair of the Scripps Research Department of Chemical Physiology. In 2010, Cravatt and his team screened tens of thousands of human and mouse proteins for the presence of hyper-reactive cysteine amino acids, which almost always mark functional sites on proteins. PRMT1 ended up containing the reactive cysteine, and, even better, only one other (rather rare) PRMT enzyme was known to have the cysteine as well.
After verifying that the cysteine was in the active site of PRMT1, the researchers found a fluorescent probe that would bind to the cysteine, so that if a test compound fastened to PRMT1's active site like an inhibitor, it would interfere with the probe's binding and result in a lower fluorescence-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-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," Myles B. C. Dillon, a graduate student in Mowen's lab and lead author of the study, said in a press release.
Mowen and Dillon then sought out Prof. Hugh Rosen, a Scripps researcher who curates the Maybridge Hitfinder Collection, a library of 16,000 chemical compounds. The researchers tested the compounds one by one, and with Rosen's help, were able to adapt the setup as an automated, high-throughput screening technique, ending up with two inhibitor candidates that demonstrated "good efficacy and specificity," that the team "might be able to modify … to make them even better," Dillon said.
The team recently received a grant from the National Institutes of Health (NIH) to use their new screening technique on a 300,000-compound NIH library, which was also curated at Scripps.
The next step, Mowen says, will be to improve the efficacy and bioavailability of the hit compounds they discovered, to screen the NIH's compound library and to test the hits in animal models of inflammation.
"Biologic-based therapies have provided the pharmaceutical industry with blockbuster drugs, but they have notable shortcomings as they are not effective in all patients (about 30 percent of RA patients fail to respond to TNFa blocking agents), can induce anti-drug immune responses and are tremendously expensive," says Mowen. "Our lab and others have shown that PRMT1 can modify and regulate several critical immunomodulatory proteins, which suggests that PRMT1 small molecular inhibitors may be valuable for the treatment of inflammation and autoimmunity."
Additional authors of the study were Daniel A. Bachovchin of the Cravatt laboratory, Steven J. Brown of the Scripps Research Molecular Screening Center and M. G. Finn, professor in the Scripps Research Department of Chemistry.