Abstract
With the recent interest in targeting the DNA damage response (DDR) and DNA repair, new screening methodologies are needed to broaden the scope of targetable proteins beyond kinases and traditional enzymes. Many of the proteins involved in the DDR and repair impart their activity by making specific contacts with DNA. These protein–nucleic acid interactions represent a tractable target for perturbation with small molecules. We describe a high throughput, solution-based equilibrium binding fluorescence polarization assay that can be applied to a wide array of protein–nucleic acid interactions. The assay is sensitive, stable, and able to identify small molecules capable of blocking DNA–protein interactions.
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References
Gavande NS, VanderVere-Carozza PS, Hinshaw HD, Jalal SI, Sears CR, Pawelczak KS, Turchi JJ (2016) DNA repair targeted therapy: the past or future of cancer treatment? Pharmacol Ther 160:65–83
Lord CJ, Tutt AN, Ashworth A (2015) Synthetic lethality and cancer therapy: lessons learned from the development of PARP inhibitors. Annu Rev Med 66:455–470
Brown JS, O’Carrigan B, Jackson SP, Yap TA (2017) Targeting DNA repair in cancer: beyond PARP inhibitors. Cancer Discov 7:20–37
Murai J (2017) Targeting DNA repair and replication stress in the treatment of ovarian cancer. Int J Clin Oncol 22(4):619–628
Bhullar KS, Lagaron NO, McGowan EM, Parmar I, Jha A, Hubbard BP, Rupasinghe HPV (2018) Kinase-targeted cancer therapies: progress, challenges and future directions. Mol Cancer 17:48
Arora S, Heyza J, Zhang H, Kalman-Maltese V, Tillison K, Floyd AM, Chalfin EM, Bepler G, Patrick SM (2016) Identification of small molecule inhibitors of ERCC1-XPF that inhibit DNA repair and potentiate cisplatin efficacy in cancer cells. Oncotarget 7:75104–75117
Chapman TM, Wallace C, Gillen KJ, Bakrania P, Khurana P, Coombs PJ, Fox S, Bureau EA, Brownlees J, Melton DW, Saxty B (2015) N-Hydroxyimides and hydroxypyrimidinones as inhibitors of the DNA repair complex ERCC1-XPF. Bioorg Med Chem Lett 25:4104–4108
Goglia AG, Delsite R, Luz AN, Shahbazian D, Salem AF, Sundaram RK, Chiaravalli J, Hendrikx PJ, Wilshire JA, Jasin M, Kluger HM, Glickman JF, Powell SN, Bindra RS (2015) Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors. Mol Cancer Ther 14:326–342
Klitgaard RN, Lobner-Olesen A (2018) A novel fluorescence based screen for inhibitors of the initiation of DNA replication in bacteria. Curr Drug Discov Technol. https://doi.org/10.2174/1570163815666180423115514
Liu W, Zhou M, Li Z, Li H, Polaczek P, Dai H, Wu Q, Liu C, Karanja KK, Popuri V, Shan SO, Schlacher K, Zheng L, Campbell JL, Shen B (2016) A selective small molecule DNA2 inhibitor for sensitization of human cancer cells to chemotherapy. EBioMedicine 6:73–86
Mohammed MZ, Vyjayanti VN, Laughton CA, Dekker LV, Fischer PM, Wilson DM III, Abbotts R, Shah S, Patel PM, Hickson ID, Madhusudan S (2011) Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines. Br J Cancer 104:653–663
Huang X, Aulabaugh A (2016) Application of fluorescence polarization in HTS assays. Methods Mol Biol 1439:115–130
Patrick SM, Turchi JJ (1999) Replication protein a (RPA) binding to duplex cisplatin-damaged DNA is mediated through the generation of single-stranded DNA. J Biol Chem 274:14972–14978
Hermanson IL, Turchi JJ (2000) Overexpression and purification of human XPA using a Baculovirus expression system. Protein Expr Purif 19:1–11
Andrews BJ, Turchi JJ (2004) Development of a high-throughput screen for inhibitors of replication protein A and its role in nucleotide excision repair. Mol Cancer Ther 3:385–391
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Turchi, J.J., VanderVere-Carozza, P.S. (2019). Small-Molecule Inhibitor Screen for DNA Repair Proteins. In: Balakrishnan, L., Stewart, J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9500-4_13
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DOI: https://doi.org/10.1007/978-1-4939-9500-4_13
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