Figure 7.

Screening of small molecules targeting the RBX1-CUL4A interaction. (A) In vitro pulldown result. The same concentrations (150 nM) of purified His-CUL4A and GST-RBX1 were mixed, and the protein mixture was incubated with GST beads or Ni-NTA beads. The pulldown proteins were stained with Coomassie blue. (B) A representative model of AlphaScreen. (C) The determination of optimal protein concentrations. Varying concentrations (0, 50, 100, 150, 200 and 250 nM) of GST-RBX1 were added to 25, 50, 100, 150, 200, and 250 nM His-CUL4A to generate AlphaScreen signals. (D) The chemical structures of PSSM0332, PSSM0856 and PSSM1437. (E) IC₅₀ value of PSSM0332. (F) IC₅₀ value of PSSM0856. (G) IC₅₀ value of PSSM1437. (H) Comparison of the inhibitory abilities of PSSM0332, PSSM0856 and PSSM1437 at the same concentration. The same concentrations (4 µM) of three small molecules were incubated with His-CUL4A and GST-RBX1 to determine AlphaScreen signals. The signal in the mixture without small molecule supplementation was set as the control and defined as 100%, and the signals in the mixtures containing small molecules were normalized to the control. * P < 0.05, ** P < 0.01 and *** P < 0.001.