Fig. 2
Novel covalent inhibition of SCP1 with small molecule compounds. A Chemical structures of lead SCP1 inhibitors. B–D Kinetic characterization of GR-28 against the phosphatase activity of SCP1. B Competitive assay against the pNPP substrate was performed with preincubation of GR-28 compound and SCP1 enzyme for 30, 60, 120, 180, 300, and 1260 min. Time-lapse IC50 curves were obtained. C The IC50 data of the pNPP substrate was converted to ln % remaining activity against time at different concentrations of GR-28 (0, 0.3125, 0.625, 1.25, and 2.5 μM). D The rate of inactivation (kobs) was plotted against inhibitor concentration and fitted to the equation: kobs = kinact × [I]/(KI + [I]). Each data point was obtained from three replicates, and the error bars indicate SD. E The rate of inorganic phosphate generation measured by malachite green assay: SCP1 was pre-incubated with DMSO/GR-28 (20 μM), then samples were incubated with phosphorylated p-Ser861-REST. Each data point was obtained from three replicates, and the error bars indicate SD. F SCP1 WT was incubated overnight with GR-28 compound and then analyzed by MALDI-TOF MS. The blue trace represents the DMSO control, while the orange trace represents the GR-28 treatment. G pNPP assay of phosphatase activity of SCP1 WT (orange) or SCP1 C181A mutant (blue) upon incubation with GR-28 for 5 h. Error bars indicate SD from three replicates. H SCP1 C181A was incubated overnight with GR-28 compound and then analyzed by MALDI-TOF MS. I Model of covalent inhibition of SCP1 by GR28 (PDB Code: 3PGL). A magnesium ion is shown in the active site of SCP1 as a green sphere. Key residues predicted to interact with GR28 are shown as sticks. GR-28 is shown as sticks colored by atoms with carbon atoms in violet, oxygen in red, and nitrogen in blue. Side chain and main chain interactions between SCP1 and GR28 are shown in dashed lines