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. 2021 Apr 12;28(9):2690–2707. doi: 10.1038/s41418-021-00778-z

Fig. 6. Docking pose of NCI677397.

Fig. 6

The NCI677397 molecule (A). NCI677397 (blue) is docked into USP24 (B(a)) and USP7 (B(b)). The different residues are highlighted by a gray circle. The dashed green lines denote hydrogen bonds. The binding sites of USP24 and USP7 are shown in yellow and pink, respectively. The binding site residues are labeled and shown as lines. The levels of the USP24 substrates, Bax and BRD7 in A549 cells treated with various candidate USP24 inhibitors were determined by IB with anti-Bax and anti-BRD7 antibodies (C). The ubiquitination of p300 by IP with p300 antibodies in A549 cells with or without USP24 purified recombinant protein or USP24 inhibitor treatment was studied by Western blot analysis with anti-ubiquitin antibodies (D). The enzyme activity of purified recombinant USP24 (a), USP7 (b), and USP9X (c) proteins in the absence or presence of NCI677397 or NCI158067 was studied with an in vitro enzyme assay using biotin-tetra-ubiquitin as the substrate (E). The effect of NCI677397 was studied by an in vitro enzyme assay with or without NCI677397 in the presence of an HA-warhead (WH). Active and inactive USP24 were studied by IB with anti-USP24 and anti-HA antibodies (F).