Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress

Abstract

Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex-binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response.

Fig. 1.

NSC 19630 inhibits cell proliferation in a WRN-specific manner and impairs cell growth and DNA synthesis. HeLa cells (A) or siRNA-transfected HeLa cells (B) were treated with DMSO (control) or the indicated concentration of specified compound for 0–3 d. Percent cell proliferation determined by WST-1 was calculated as the ratio of OD₄₅₀ values obtained for HeLa cells grown in the presence of a small-molecule inhibitor compared with the presence of DMSO. Day 0 represents effect of treatment on cell proliferation after 4 h. (C) Effect of NSC 19630 on cell growth. HeLa cells were treated with DMSO or the indicated concentration of NSC 19630 for 3 d. Cells were washed and allowed to grow in fresh medium lacking DMSO or NSC 19630 for 7 d and stained with methylene blue. (D) Percent colony-forming units was calculated as the ratio of colonies formed from cultures grown in the presence of NSC 19630 to cultures grown in the presence of DMSO, which was considered 100%. (E) Effect of NSC 19630 on DNA synthesis. HeLa cells were treated with DMSO or 2 μM NSC 19630 for 3 d. Cells were processed for EdU staining as described in SI Materials and Methods. Merged picture shows cells stained with EdU (red) or DAPI (blue). (F) Percent EdU-staining cells was calculated as the ratio of cells showing EdU staining compared with total number of cells with DAPI-stained nuclei.

Fig. 2.

NSC 19630 induces apoptosis and γ-H2AX foci. (A) Effect of NSC 19630 on apoptosis. Untransfected or WRN siRNA-transfected HeLa cells were treated with DMSO or 3 μM NSC 19630 for 3 d. Cells were assayed for histone-associated DNA fragments indicative of apoptosis as described in SI Materials and Methods. (B and C) NSC 19630 induces γ-H2AX foci in a WRN-dependent manner. Untransfected or WRN siRNA-transfected HeLa cells were treated with DMSO or 2 μM NSC 19630 for 3 d. Cells were stained for γ-H2AX as described in SI Materials and Methods. (B) Merged picture shows cells stained with anti–γ-H2AX antibody (green) and DAPI (blue). (C) Number of γ-H2AX foci per cell in cells treated with DMSO or 2 μM NSC 19630.

Fig. 3.

Elevated PCNA staining of NSC 19630-treated cells is WRN dependent. (A) Untransfected or WRN siRNA-transfected HeLa cells were treated with DMSO or 2 μM NSC 19630 for 3 d. Cells were stained for PCNA as described in SI Materials and Methods. Merged picture shows cells stained with PCNA antibody (green) and DAPI (blue). (B) Number of PCNA foci per cell in cells treated with DMSO or 2 μM NSC 19630.

Fig. 4.

NSC19630 exposure enhances the cells’ sensitivity to agents that induce replicational stress or DNA damage. (A) U2OS cells were treated with NSC 19630 (1 μM), TMS (0.6 μM), or both compounds for 3 d. (B) HeLa cells were treated with NSC 19630 (1 μM), PARP inhibitor KU0058948 (1 nM), or both compounds for 3 d. (C) HeLa cells were treated with NSC 19630 (1 μM), TPT (0.1 μM), or both compounds for 3 d. Cell proliferation was determined with WST-1 reagent as described in Materials and Methods. Percent proliferation was calculated as the ratio of OD₄₅₀ values obtained for HeLa cells grown in the presence of the indicated compounds compared with values for cells grown in the presence of DMSO. Day 0 represents the effect of treatment on cell proliferation after 4 h. (D) Cells were stained for γ-H2AX. Merged picture shows cells stained with anti–γ-H2AX antibody (green) or DAPI (blue). (E) Number of foci per cell in cells treated with NSC 19630 (1 μM), TPT (0.1 μM), or both NSC 19630 (1 μM) and TPT (0.1 μM).