Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1

Abstract

Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets but their role in physiological and pathological pathways is far from being clear, due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone- (H3) or non-histone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators.

A TR‐FRET‐Based Functional Assay for Screening Activators of CARM1

“…[29] Adox inhibits S-adenosylhomocysteine hydrolase, leading to the elevated levels of adenosylhomocysteine that block utilization of S-adenosylmethionine by methyltransferases. [30] As shown in Figure 6A, the Me-GFPPABP1 TR-FRET assay in MCF7 cells exhibited a dose-dependent response to the addition of Adox, with a representative IC 50 value for Adox calculated as 5.2 μM.…”

“…Uracandolates (aryl ureido acetamido indole carboxylates) 1k , 3c , and 3g were recently shown able to increase the methylation of PABP1 induced by CARM1 at 50 μM in in vitro methylation assays and in cells, although the activation mechanism is still unclear. [29] As shown in Figure 6B, the TR-FRET assays in the defined genetic background exhibited dose-dependent responses to all the three activators, although the EC 50 values for these activators are determined to be 259 μM, 272 μM, and 306 μM, respectively. Taken together, these results validated that the TR-FRET assay responds appropriately to the addition of a chemical, ensuring a reliable readout for HTS.…”

“…Although compounds 1k , 3c and 3g have been shown to increase CARM1-mediated methylation of PABP1, both in the in vitro methylation assay and in the TR-FRET assay here, these compounds do not bind to CARM1 in the absence of substrates or cofactor and do not increase histone H3 methylation catalyzed by CARM1. [29] Moreover, the activation effects of these compounds were achieved at high concentrations (Figure 6B), which are likely to have off-target effects. Therefore it is advantageous to screen for more potent, selective activators of CARM1 which bind to allosteric site(s) on CARM1 in order to study the epigenetic regulation of CARM1 in breast cancer and other diseases.…”

A continuous-flow synthesis of 1,4-benzodiazepin-5-ones, privileged scaffolds for drug discovery

“…The term, first coined by Evans in the late 1980s, 3 was originally referring to the 1,4-benzodiazepine nucleus and, indeed, numerous 1,4-benzodiazepine derivatives endowed with selective activities against a variety of enzymes and receptors have been identified thus far. 4,5 Being interested in the development of small-molecule modulators of epigenetic targets, 6,7 we planned the synthesis of the 3,4-dihydro-5H-benzo[e] [1,4]diazepin-5-one scaffold (1) which could be easily manipulated to provide a number of highly functionalized potential ligands (2) (Figure 1). …”

Patent Highlights

“…Over 30 arginine methylation sites have been identified on core histones; these modifications alter protein structure, impact interactions with DNA and also generate docking sites for effector molecules [27]. Small-molecule enhancers [28] as well as inhibitors [29] of CARM1 have been reported. The novel CARM1 inhibitors presented here are mostly structured around an imidazopyridinyl, pyrazolopyridinyl, pyrrolopyrimidinyl or pyrazolotriazinyl core moiety.…”