Skip to main content
. 2019 Sep 3;294(46):17654–17668. doi: 10.1074/jbc.RA119.010201

Figure 5.

Figure 5.

Primary and secondary screening of the NCATS Pharmaceutical Collection (NPC). (A), Sixty-one common hits were identified by the orthogonal WIP1 activity assays in the qHTS against the NPC library. The Rh-PBP assay (blue) had a higher overall hit rate than the RapidFire MS assay (green). (B), Among the 61 common hits in the primary screens, 58 compounds were selected as cherry picks. The cherry picks were further evaluated in 11-point dose response with the primary assays, the Rh-PBP assay with a P38 peptide substrate 1, and the Amplex Red assay to identify compounds capable of redox cycling. The number of compounds showing activity (hits) from each assay is indicated on the right. (C), Among the 58 cherry picks, 34 compounds were confirmed by both the Rh-PBP assay (69% confirmation rate, blue) and the RapidFire MS assay (64% confirmation rate, green). (D–I), Quantitative HTS data from the primary and secondary assays. None of the selected compounds show activity in the Amplex Red assay (red squares). (D), GSK2830371 inhibits WIP1 with a low nanomolar potency and was used as a control for the WIP1 activity assays. The data are shown as the mean value ± S.D. for the RapidFire MS assay with the P53 substrate 3 (green circles, IC50 = 3.5 nm, Hill slope = −1.9, n = 3) and the Rh-PBP assay with the P53 substrate 3 (blue diamonds, IC50 = 4.9 nm, Hill slope = −1.7, n = 2) or P38 substrate 1 (teal triangles, IC50 = 5 nm, Hill slope = −1.5, n = 2). (E), NCGC00244580 inhibits WIP1 activity toward the P53 substrate 3 with the RapidFire MS assay (IC50 = 7.3 μm, Hill slope = −2.1) or Rh-PBP assay (IC50 = 5.9 μm, Hill slope = −1.7). The Rh-PBP assay shows that NCGC00244580 also inhibits WIP1 activity against the P38 substrate 1 (IC50 = 42 μm, Hill slope = −3). (F), NCGC00015480 inhibits WIP1 activity similarly according to the RapidFire MS assay (IC50 = 14 μm, Hill slope = −3.1) or the Rh-PBP assay with the P53 substrate 3 (IC50 = 9.6 μm, Hill slope = −2) or P38 substrate 1 (ambiguous fit). (G), NCGC00162249 inhibits WIP1 activity against the P53 substrate 3 as shown by RapidFire MS (IC50 = 14 μm, Hill slope = −1.9) or Rh-PBP (IC50 = 13 μm, Hill slope = −2.4) assays, with partial activity at high concentrations with the P38 substrate 1. (H), The activity of WIP1 toward the P53 substrate 3 is inhibited by NCGC00161599 as shown by the RapidFire MS (IC50 = 11 μm, Hill slope = −0.28) and Rh-PBP (IC50 = 51 μm, Hill slope = −0.1) assays. The activity of WIP1 against the P38 substrate 1 was partially inhibited (IC50 = 44 μm, Hill slope = −2). (I), Although no inhibition was apparent with the P38 substrate 1, NCGC00185994 inhibited WIP1 activity against the P53 substrate 3 according to the RapidFire MS (IC50 = 13 μm, Hill slope = −1) and Rh-PBP (IC50 = 20 μm, Hill slope = −1.2) assays.