0.5, 2, and 5 mg once-daily for 4-week treatment

Human CP4H/Cofactor mix (4X final concentration in 50 mM HEPES, pH 7.5 with 50 mM KCl) was incubated at room temperature for 30 minutes with GSK1278863 (10 point, 3-fold serial dilution in 50% DMSO, 15X final concentration) or 50% DMSO. Substrate mix (1.4X final concentration in 50 mM HEPES, pH 7.5 with 50 mM KCl) was added to the plate yielding a 32 μL final reaction volume. After 20 minutes, the reaction was quenched by the addition of 0.67% final H3PO4. Product formation was detected using Reverse Phase High Performance Liquid Chromatography (HPLC; Ex 336nM and Em 517 nM). Final concentrations of the CP4H/Cofactor mix were 21.11 nM enzyme, 1 mg/mL BSA, 50 μM FeCl2, 2 mM ascorbic acid, 100 μM DTT, and 100 μg/mL catalase. The substrate mix consisted of 100 μM dansyl-GFPG-Oet, and 110 μM α-ketoglutarate. Percent inhibition data were determined from the ratio of product to substrate after a 20 minute reaction. The IC50 value was determined by fitting the percent inhibition data to a two-parameter IC50 equation.