Journal: Journal of Medicinal Chemistry

Article Title: Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor

doi: 10.1021/acs.jmedchem.3c01030

Figure Lengend Snippet: Inhibition of GST-tagged Hs PRMT9 and Hs PRMT7 (panels a and b, respectively) or Ce PRMT7 and Ce PRMT9 (panels c and d, respectively) by compounds 1a – c as detected by a radioisotope-based assay. The experiments were performed as reported in the Experimental procedures section. GST- Hs PRMT9 (a) and GST- Hs PRMT7 (b) were incubated with human GST-SF3B2 (401–550) peptide or recombinant Hs H2B, respectively (1 μg of enzyme, 5 μg of substrate), 0.14 μM [ 3 H]SAM, and the indicated concentrations of tested compounds at the corresponding optimal reaction temperature (37 °C for Hs PRMT9, 15 °C for Hs PRMT7). C. elegans GST-tagged enzymes PRMT9 (c) and PRMT7 (d) were incubated with GST- Ce SFTB-2 (99–248) fragment or recombinant Hs H2B, respectively (1 μg of enzyme, 5 μg of substrate), 0.14 μM [ 3 H]SAM, and the indicated concentrations of tested compounds at the corresponding optimal reaction temperature (25 °C for Ce PRMT9, 15 °C for Ce PRMT7). After SDS-PAGE, the gels were treated as previously described and densitometry analysis was done using ImageJ software, and data was plotted as normalized activity to the no inhibitor controls.

Article Snippet: Then, each well was added with 0.5 μL of the biotinylated substrate peptide SF3B2 (aa 500–519) (Pepmic, custom synthesis) (final concentration, 100 nM), 1 μL of SAM 250 μM, 1.5 μL of water, and 2 μL of 4× HMT assay buffer 2A to reach the final volume of 10 μL.

Techniques: Inhibition, Incubation, Recombinant, SDS Page, Software, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor

doi: 10.1021/acs.jmedchem.3c01030

Figure Lengend Snippet: Testing the effects of compounds 1a (EML734), 1b (EML736), 1e (EML979), and 1f (EML980) on PRMT9 activity in MCF7 (top) and MDA-MB-436 breast cancer cell lines. MCF7 and MDA-MB-436 cells were treated with 4 candidate inhibitors at indicated concentrations for 72 h. The total cell lysates were harvested in RIPA buffer and the levels of SF3B2 R508me2s, SF3B2, and PRMT9 were detected by using Western blot assays. Anti-Tubulin antibody was used as a loading control.

Article Snippet: Then, each well was added with 0.5 μL of the biotinylated substrate peptide SF3B2 (aa 500–519) (Pepmic, custom synthesis) (final concentration, 100 nM), 1 μL of SAM 250 μM, 1.5 μL of water, and 2 μL of 4× HMT assay buffer 2A to reach the final volume of 10 μL.

Techniques: Activity Assay, Western Blot, Control