in mice: Mice received intraperitoneal (i.p.) injections starting on post natal day 1 (P1) defined as the day of birth. All treated litters were culled at P4 to a total of six pups. RG3039 was dissolved in distilled water (or saline in Ko laboratory) at appropriate concentrations for 2.5 µl volume/gram injection volume. Mice treated with RG3039 at 10 mg/kg/day showed increased weights and survival, which were not further increased by the 20 mg/kg dose.

To verify the ability of RG3039 to inhibit DcpS, it was examined DcpS enzyme activity in protein extracts isolated from a P10 SMA mouse brain treated in vitro with various doses of the drug. DcpS activity. Mouse brains were homogenized in lysis buffer (PBS, pH 7.4, 1% BSA, 0.1% IGEPAL CA-630 with protease and phosphatase inhibitors). For the DcpS enzyme decapping assay, 20 µg of tissue extract was mixed with 3 µm of biotinylated CAP substrate m7GpppA(N6) in the decapping buffer (50 mm Tris, pH 7.9, 100 mm MgCl2, 150 mm (NH4)2SO4) with a 10 µl total reaction volume. The reaction was terminated by adding 10 µl of 10 µm RG3039 to the reaction mixture. The resulting enzymatic reaction product, biotinylated ADP, was quantified using ELISA. As expected, RG3039 robustly inhibited DcpS enzyme activity in a dose-dependent manner with an IC50 = 3.4 nm.