title: Novel inhibitors of Rad6 ubiquitin conjugating enzyme: design, synthesis, identification, and functional characterization.
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.
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan.|Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan.
authors: Matthew A
authors: MA
authors: Sanders
authors: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales, UK.
authors: Ghali
authors: G
authors: Brahemi
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan.|Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan.
authors: Pratima
authors: P
authors: Nangia-Makker
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan.|Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan.
authors: Vitaly
authors: V
authors: Balan
authors: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales, UK.
authors: Matteo
authors: M
authors: Morelli
authors: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales, UK.|Faculty of Pharmacy, Zagazig University, Sharkeya, Egypt.
authors: Hend
authors: H
authors: Kothayer
authors: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales, UK.
authors: Andrew D
authors: AD
authors: Westwell
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan.|Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan.|Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan.
authors: Malathy P V
authors: MPV
authors: Shekhar
mesh_terms: D001483:Base Sequence; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017124:Conserved Sequence; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D014227:Triazines; D051176:beta Catenin; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes