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. 2013 Apr;12(4):373-83.
doi: 10.1158/1535-7163.MCT-12-0793. Epub 2013 Jan 21.

Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

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Free PMC article

Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

Matthew A Sanders et al. Mol Cancer Ther. .
Free PMC article

Abstract

Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2-M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.

Figures

Figure 1
Figure 1
Schematic illustration of synthesis of substituted (4-amino-6-(phenylamino)-1,3,5-triazin-2-yl) methyl benzoates.
Figure 2
Figure 2
A, identification of TZs with Rad6 inhibitory activity. In vitro histone H2A ubiquitination assays were conducted as described in Materials and Methods. Rad6B-ubiquitin thioester (Rad6B~Ub) and monoubiquitinated histone H2A (Ub-H2A) were detected by ubiquitin antibody. B, in vitro BCA2 ubiquitination assays were conducted with UbcH5 or Rad6B, with and without SMI #9. BCA2 and ubiquitinated BCA2 (Ub-BCA2) were detected by BCA2 antibody. Control vector- and BCA2-transfected COS7 extracts are included for identification of expressed BCA2. C and D, docking model of SMIs #8 and 9, respectively, with human Rad6B showing interactions with the indicated amino acids surrounding the catalytic site Cys88. Predefined color codes of PyMol were used. E, Rad6-binding sites of SMIs #8 and 9.
Figure 3
Figure 3
Rad6 SMIs inhibit MDA-MB-231 cell proliferation and migration. A and C, MTT assays with MDA-MB-231 (A) or nontransformed MCF10A (C) cells. B, phase contrast micrographs of MDA-MB-231 cells. Magnification, ×10. D, migration of MDA-MB-231 cells measured in presence of the indicated TZs (top panel) or doses of SMIs #8 or 9 (bottom panel).*, P < 0.05.
Figure 4
Figure 4
SMI #9 treatment induces MDA-MB-231 cell death. A, acridine orange/ethidium bromide staining of cells treated with vehicle, 15 μmol/L cisplatin (CDDP), 10 μmol/L SMI #8 or TZ #15, or 5 μmol/L SMI #9. Magnification, ×40 (SMI#9), ×20 (all others). B, colony survival assays. Representative wells and magnified images of colonies are shown. C, graphic representation of colony-forming frequencies. *, P < 0.05.
Figure 5
Figure 5
Rad6 SMI #9 induces MDA-MB-231 G2–M-arrest and decreases β-catenin protein levels. A, cell-cycle measurements at the indicated SMI #8 or 9 concentrations (left panel) or 10 μmol/L SMI #8 or 9 (right panel). Results are mean ± SE from 2 independent experiments. *, P < 0.05. B, immunofluorescence (red) staining of cyclin B1 (a–d) and α-tubulin (e–g). Arrows in d indicate cells with pleiomorphic or multiple nuclei; arrows in f and g show retention of α-tubulin polymers at the dividing plane of SMI #9-treated cells compared with their absence (arrows in e) in vehicle controls. Magnification, ×20 (a, b, f), ×40 (c, d), ×100 (e), ×200 (g). C, top panel, analysis of endogenous Rad6 activity in MDA-MB-231 cells (see Materials and Methods); ubiquitinated (Ub)-histone H2A (arrow). Bottom panel, immunoblot analysis of Rad6 and PCNA. D, immunoblot analysis of β-catenin (right panel), and immunofluorescence staining (left panel) of Rad6 (FITC) and β-catenin (Texas Red) in MDA-MB-231 cells. Magnification, ×40.
Figure 6
Figure 6
Sequence alignment of Rad6B with representative E2 family members. Asterisks indicate Rad6B noncovalent interaction sites with SMIs #8 and 9, and those conserved in other E2s. Conserved amino acids in E2s are in upper case. E2 catalytic site cysteines are indicated by solid squares.

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