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, MDAMB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.

Gold nanoparticle conjugated Rad6 inhibitor induces cell death in triple negative breast cancer cells by inducing mitochondrial dysfunction and PARP-1 hyperactivation: Synthesis and characterization

“…SMI#9 was synthesized as previously described (12). The steps for GNP and SMI#9 nanoconjugation are described in Scheme 1 (19-23), and details are provided under Supplementary Materials.…”

“…A minimum of six fields with at least 50 cells/field were scored for determination of dye uptake (12), and experiments were repeated at least three times.…”

“…Conversely, Rad6B silencing renders cells chemosensitive (11), implicating the relevance of Rad6 in transformation and drug resistance, and the potential therapeutic benefit of inhibiting Rad6. We have recently reported the development of a novel Rad6-selective small molecule inhibitor SMI#9 that inhibits Rad6 UBC activity (12). SMI#9 treatment suppresses proliferation and migration, and induces apoptosis in breast cancer cells but spares normal breast cells (12).…”

“…We have recently reported the development of a novel Rad6-selective small molecule inhibitor SMI#9 that inhibits Rad6 UBC activity (12). SMI#9 treatment suppresses proliferation and migration, and induces apoptosis in breast cancer cells but spares normal breast cells (12). However, SMI#9 has poor aqueous solubility that limits its therapeutic efficacy.…”

A Systematic Review on Antitumor Agents with 1, 3, 5-triazines

“…TZ8 ( Figure 19) and TZ9 ( Figure 18) were formerly reported Rad6B-inhibitory anticancer agents MDA-MB-231 cells [146]. The triazine carboxamide (Figure 19) was the highest active compound against MCF-7 cell line.…”