Survival of cells and the faithful propagation of the genome depend on elaborate mechanisms of detecting and repairing DNA damage. Treatment of advanced cancer relies on radiation therapy or chemotherapy, which kill cancer cells by causing extensive DNA damage. It is often found, that cancer cells develop resistance to therapy through enhanced activity of DNA repair functions;this has led to an increased interest in developing drugs that interfere with DNA repair, which could sensitise cancer cells to conventional therapy. This application involves two related human proteins, RECQ1 and BLM, which are important in resolving abnormal DNA structures formed during replication or homologous recombination. The BLM gene is defective in patients of Bloom's syndrome, which is associated with an increased incidence of cancer. Shutting down the expression of RECQ1 or BLM leads to chromosomal instability and higher radiation sensitivity in cultured cells. Remarkably, some cancer cell lines, but not normal cells, exhibit reduced growth and an increase in cell death when RECQ1 expression is inhibited by RNAi. The goal of this project is to develop inhibitors of RECQ1 and BLM activity, which can be used in cell and animal models to examine the consequences of inhibition on the survival of cancer cells. Initial candidates will be identified by a quantitative high-throughput screen of the MLSMR compound library, using a fluorescence-based in vitro biochemical assay that reveals inhibitors of RECQ1 and BLM DNA unwinding activity. The resulting compounds will then be subject to orthogonal, secondary biochemical assays, to triage the initial hits, to classify compounds based on mode of action, and to derive structure-activity relationships (SARs) of candidate effectors. SAR and protein structural information will be used in further chemical development to improve the potency and selectivity of the compounds. Cell-based assays will then be applied as the first step in utilizations of the verified inhibitors, examining their effects on cancer cell survival and sensitivity to radiation and chemotherapeutics. This project is based on extensive preliminary studies carried out by the applicants in collaboration with the NCGC. A fluorescence-based primary assay for helicase activity has been optimized for the 1536-well screening format. The screen has been validated against a 1280-compound library (LOPAC), and showed acceptable reproducibility, stability, and Z-factor. Significantly, the screen yielded only a handful of significant hits, indicating that the assay is not dominated by false-positives. Three secondary assays, all adopted for high throughput, have been implemented for verification and classification: ATP binding, DNA binding (both by fluorescence polarization), and protein stabilization (thermal denaturation). Standard biochemical assays and a battery of cell-based assays have been used to evaluate the effects of inhibition of RECQ1 and BLM expression, so provide the appropriate benchmarks for specific effects of chemical inhibition.

Public Health Relevance

Small-molecule inhibitors of the human RECQ1 and Bloom proteins Advanced cancer is usually treated by chemotherapy or radiation therapy, which kill cancer cells by damaging their DNA. Many cancer cells are resistant to such therapy, partly because of an elevated ability to repair DNA damage using a battery of cellular enzymes. This project aims to find chemicals that can inhibit DNA repair in cancer cells. It is hoped, that such inhibition will make the cancer cells more sensitive to DNA- damaging agents, thus allowing better treatment options for patients with resistant forms of cancer.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-BST-J (50))
Program Officer
Yao, Yong
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University of Oxford
United Kingdom
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OX1 2-JD
Rosenthal, Andrew S; Dexheimer, Thomas S; Gileadi, Opher et al. (2013) Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase. Bioorg Med Chem Lett 23:5660-6
Nguyen, Giang Huong; Dexheimer, Thomas S; Rosenthal, Andrew S et al. (2013) A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem Biol 20:55-62