SGC707 is a potent, selective, and cell-active allosteric inhibitor of PRMT3.

PRMT3:31 nM, PRMT3:53 nM(Kd)

在细胞中，SGC707与PRMT3结合，并降低PRMT3依赖性的H4R3me2a。SGC707还能在HEK293和A549细胞中稳定PRMT3，其EC50值分别为1.3 μM和1.6 μM。[1]

在CD-1雄性小鼠中，SGC707（30 mg/kg，i.p.）在6小时内展现了良好的血浆暴露度，峰值血浆浓度达到38000 nM，这表明SGC707适用于动物研究。[1]

PRMT3 biochemical assay: A radioactivity-based assay is optimized and used to determine the inhibition of PRMT3 in vitro. In a scintillation proximity assay (SPA), tritiated S-adenosylmethionine (3H-SAM) served as a methyl donor to methylate biotinylated histone peptide substrate. After completion of the reaction, the reaction mixture is transferred to the wells of a streptavidin / scintillant-coated microplate. The Biotinylated peptides binding to streptavidin coated resin, brings the incorporated 3H-methyl and the scintillant to close proximity. The amount of methylated peptide is quantified by tracing the radioactivity (counts per minute) as measured by the TopCount NXT? Microplate Scintillation and Luminescence Counter. Due to the very acidic nature of the 3H-SAM solution, non-tritiated (cold) SAM is used to supplement the reactions. The C-terminally biotinylated peptide composed of the first 24 amino acids residues of histone H4 (H4 1-24) is used as substrate. The typical assay mixture contained 0.01% Tween-20, 5 mM DTT, 20 nM PRMT3, 0.3 μM B-H4 1-24 and 28 μM (5 μM 3H-SAM plus 23 μM cold SAM) SAM in 20 mM Tris-HCl (pH 7.5) and a final volume of 20 μL. The IC50 values are determined at Km concentrations of both substrates by titration of the compound in the reaction mixture in a range between 2000-0.2 nM.

Cells are seeded in 96-well plates and treated with different concentrations of SGC707 for 72 h. Cell viability is determined using Alamar blue 0.01 mg/mL in the media. Resazurin reduction is monitored with 544 nm excitation, measuring fluorescence at 590 nm. (Only for Reference)