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 anticancer 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 multiprotein 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 anticancer 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 anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

Figure 1

Initial screening results. (A) Optimized assay parameters provide a robust, reproducible disruption of the Rev1-CT/FAM-Polκ-RIR complex (100 nM) by unlabeled Polκ-RIR. Graph shows a single representative experiment that was reproduced at least three separate times. (B) Scatter plot of fluorescence polarization values (mP) from the initial pilot screen. Each compound (4,800) was evaluated at 10 μM. Rev1-CT/FAM-Polκ-RIR concentration was 100 nM.

Figure 2

Initial hits identified from the pilot screen.

Figure 3

Compound 5 binds directly to Rev1-CT at the RIR interface. (A) Regions of ¹H-¹⁵N HSQC NMR spectrum of the Rev1-CT domain upon its titration with increasing amount of compound 5, showing gradual shifts for selected peaks toward their positions in the compound bound state. (B) Cumulative ¹H and ¹⁵N NMR chemical shift changes for the backbone and Trp side-chain HN groups of the Rev1-CT domain induced by binding compound 5 mapped onto Rev1-CT structure (PDB: 2LSY; chemical shift changes Δϖ are proportional to sphere radii). (C) ‘Hot spot’ residues of the Polη-RIR motif (purple) and corresponding RIR binding pocket on Rev1-CT (PDB: 2LSK).

Figure 4

Predicted binding of compound 5 in the Rev1-CT/RIR ‘hot spot’. (A) Three-dimensional representation showing key Rev1-CT amino acid residues. (B) Two-dimensional representation of binding showing key interactions between the scaffold and Rev1-CT. Green line, pi-pi interaction; dotted purple arrow, side chain hydrogen bond; gray sphere, solvent exposed.

Figure 5

Compounds 4 and 5 sensitize HT1080 cells to killing by cisplatin and reduce mutagenesis. (A) Compounds 4 and 5 enhance sensitivity to cisplatin in a colony survival assay. Cells were incubated with cisplatin (0.6 μM) for 24 hrs, followed by the addition of either 4 or 5 (1.5 μM) and colonies were allowed to form for seven days. Colonies were stained and counted with coomassie blue. *P<0.05 and ***P<0.001 were calculated with the Student t-test using comparisons as indicated. (B) Compounds 4 and 5 reduce the cisplatin-induced HPRT mutations in HT1080 cells. Data are the Ave ± SD of two separate experiments with 3 replicates each. All experiments were performed at least two separate times and results were comparable across all replicates. **P<0.01 and ***P<0.001 versus cisplatin treated cells, Student t-test.

Figure 6

Compounds 4 and 5 sensitize MEF cells to killing by cisplatin in a REV1^+/+-dependent manner and reduce mutagenesis. (A) Compounds 4 and 5 enhance sensitivity of WT MEFs to cisplatin in a colony survival assay and not Rev1^−/− cells. Cells were incubated with cisplatin (0.6 μM) for 24 hrs, followed by the addition of either 4 or 5 (1.5 μM) and colonies were allowed to form for seven days. Colonies were stained and counted with coomassie blue. Data are the Ave ± SD of two separate experiments performed in triplicate. NS = Not significant. (B) Compounds 4 and 5 reduce cisplatin-induced HPRT mutations in the WT MEF cells. Data are the Ave ± SD of two separate experiments with 6 replicates each. ***P<0.001 versus cisplatin treated cells, Student t-test.

Figure 7

Enhanced sensitivity to UV light in HT1080 cells treated with Rev1-CT/RIR PPI inhibitors. In (A) and (B) cells were plated and exposed to UV light (5 J/m²) or left untreated. The next day, 4 or 5 was added and cell viability was measured 24 hrs later. **P<0.01 and ***P<0.001 versus cells treated with compound alone, Student t-test. (C) Cells were treated with UV light followed by dosing with 4 or 5 (1.5 μM). Cells were incubated to allow for the formation of colonies that were counted with commassie blue. *P<0.05 were calculated with the Student t-test using comparisons as indicated. Data are the Ave ± SD of two separate experiments performed in triplicate. Experiments were performed at least two separate times and results were comparable across all replicates.