Journal: ACS Chemical Biology

Article Title: Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

doi: 10.1021/cb500883h

Figure Lengend Snippet: Identification and characterization of compound 1 as a novel CDC25B ligand. (A) A portion of the 1 H– 15 N HSQC spectrum for the CDC25B catalytic domain in the presence (red) and absence (black) of 2 mM 1 . (B) Crystal structure of 1 bound to CDC25B. Dark gray surface denotes the enzymatic active site. Two arginine residues involved in interaction with CDK2/Cyclin A substrate are labeled and shown in red. The distance between the catalytic cysteine and 1 is shown. (C) Molecular details of the interaction of 1 with CDC25B binding pocket. 1 binds in two equally populated orientations with symmetry along CN, OH axis. Distance between position 6 of 1 and the sulfate ion is given (PDB ID: 4WH7). The hydrogen bond network between the hydroxyl of 1 and four waters in the binding pocket is also shown. (D) AlphaLISA signal due to the protein–protein interaction between CDC25B and the CDK2/Cyclin A complex. CDC25B WT is shown in black, and the hotspot mutation R492L is shown in red.

Article Snippet: Proteins were incubated together at a final concentration of 10 nM each for 1 h prior to incubation with compound for 1 h, followed by addition of Ni-chelate AlphaScreen donor beads (PerkinElmer) and Anti-Flag AlphaLISA acceptor beads (PerkinElmer) at a final dilution of 1:1000 for 1 h. Protein–protein interaction assays were quantified using a PheraStar plate reader with excitation at 680 nm wavelength and emission at 615 nm in 20 uL volumes in an uncoated, white, low-volume, 384-well plate (Corning).

Techniques: Labeling, Binding Assay, Mutagenesis

Journal: ACS Chemical Biology

Article Title: Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

doi: 10.1021/cb500883h

Figure Lengend Snippet: Small molecule ligand binding to the protein–protein interaction site inhibits CDC25B activity. (A) Activity of compound 1 and 7 in an AlphaLISA-based protein–protein interaction assay. (B) In vitro phosphatase assay utilizing phosphorylated CDK2/Cyclin A as a substrate for CDC25B in the presence or absence of 1 or 7 . Remaining phosphorylated CDK2/Cyclin A is shown as detected by Western blot.

Article Snippet: Proteins were incubated together at a final concentration of 10 nM each for 1 h prior to incubation with compound for 1 h, followed by addition of Ni-chelate AlphaScreen donor beads (PerkinElmer) and Anti-Flag AlphaLISA acceptor beads (PerkinElmer) at a final dilution of 1:1000 for 1 h. Protein–protein interaction assays were quantified using a PheraStar plate reader with excitation at 680 nm wavelength and emission at 615 nm in 20 uL volumes in an uncoated, white, low-volume, 384-well plate (Corning).

Techniques: Ligand Binding Assay, Activity Assay, Protein Protein Interaction Assay, In Vitro, Phosphatase Assay, Western Blot