Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein−Protein Interaction

Abstract

Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anti-cancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multi-protein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anti-cancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anti-cancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

Virtual Pharmacophore Screening Identifies Small‐Molecule Inhibitors of the Rev1‐CT/RIR Protein–Protein Interaction

“…[14] In this study,w e generated ap harmacophore model from as mall-molecule scaffold known to disrupt the Rev1-CT/RIR PPI. We previously demonstrated that small-molecule disruptiono ft he PPI between Rev1-CT and the RIRm otif presenti nm ultiple TLS polymerases inhibits TLS in cell culture.…”

“…[14] Compounds with IC 50 values less than 15 mm were classifieda so ur primary hits (4-11, Figure 5), while those with IC 50 values greater than 20 mm are shown in Supporting Information Table 2. [14] Compounds with IC 50 values less than 15 mm were classifieda so ur primary hits (4-11, Figure 5), while those with IC 50 values greater than 20 mm are shown in Supporting Information Table 2.…”

“…The replicative bypasso fc isplatin intra-strandc rosslinksr equires Rev1/polzmediated TLS and we have demonstrated previously that these cells are av alid model for evaluating TLSi nhibitors that functionv ia direct binding to Rev1-CT. [8,9,14,18] Each compound significantly enhanced the ability of cisplatin to kill the MEFs, albeit at concentrations (! The replicative bypasso fc isplatin intra-strandc rosslinksr equires Rev1/polzmediated TLS and we have demonstrated previously that these cells are av alid model for evaluating TLSi nhibitors that functionv ia direct binding to Rev1-CT. [8,9,14,18] Each compound significantly enhanced the ability of cisplatin to kill the MEFs, albeit at concentrations (!…”

“…[1,2,8,9] Severalo ft hese switching events are mediated by protein-protein interactions (PPIs) between the C-terminal domain of the Y-familyp olymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) from the Y-familyp olymerases polh,p olk,p oli,a nd polz. [14][15][16] These compounds increase sensitivity to cisplatin and decrease cisplatin-mediated mutagenesis in human cancer cells, highlighting their potential as combination therapies for variousforms of cancer. [14][15][16] These compounds increase sensitivity to cisplatin and decrease cisplatin-mediated mutagenesis in human cancer cells, highlighting their potential as combination therapies for variousforms of cancer.…”

“…[10][11][12][13] We recently identified the first small molecules( Figure1A, 1 and 2)t hat disrupt the Rev1-CT/ RIR PPI. [14][15][16] These compounds increase sensitivity to cisplatin and decrease cisplatin-mediated mutagenesis in human cancer cells, highlighting their potential as combination therapies for variousforms of cancer.…”