Inhibition of p53 DNA binding by a small molecule protects mice from radiation toxicity

Abstract

Transcription factors are attractive therapeutic targets that are considered non-druggable because they do not have binding sites for small drug-like ligands. We established a cell-free high-throughput screening assay to search for small molecule inhibitors of DNA binding by transcription factors. A screen was performed using p53 as a target, resulting in the identification of NSC194598 that inhibits p53 sequence-specific DNA binding in vitro (IC50 = 180 nm) and in vivo. NSC194598 selectively inhibited DNA binding by p53 and homologs p63/p73, but did not affect E2F1, TCF1, and c-Myc. Treatment of cells with NSC194598 alone paradoxically led to p53 accumulation and modest increase of transcriptional output owing to disruption of the MDM2-negative feedback loop. When p53 was stabilized and activated by irradiation or chemotherapy drug treatment, NSC194598 inhibited p53 DNA binding and induction of target genes. A single dose of NSC194598 increased the survival of mice after irradiation. The results suggest DNA binding by p53 can be targeted using small molecules to reduce acute toxicity to normal tissues by radiation and chemotherapy.

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Fig. 1: A positive readout assay for p53 DNA-binding inhibitors.
Fig. 2: Adaptation of binding assay to other transcriptional factors.
Fig. 3: NSC194598 inhibits p53 DNA binding in vitro.
Fig. 4: Structure and activity of NSC194598 analogs.
Fig. 5: NSC194598 inhibits p53 DNA binding in cell culture.
Fig. 6: NSC194598 interacts with p53 DNA-binding domain.
Fig. 7: NSC194598 protects mice from radiation toxicity.

References

  1. 1.

    Bushweller JH. Targeting transcription factors in cancer - from undruggable to reality. Nat Rev Cancer. 2019;19:611–24.

    CAS  PubMed  Google Scholar 

  2. 2.

    Berg T. Small-molecule modulators of c-Myc/Max and Max/Max interactions. Curr Top Microbiol Immunol. 2011;348:139–49.

    CAS  PubMed  Google Scholar 

  3. 3.

    Hoggard LR, Zhang Y, Zhang M, Panic V, Wisniewski JA, Ji H. Rational design of selective small-molecule inhibitors for beta-catenin/B-cell lymphoma 9 protein-protein interactions. J Am Chem Soc. 2015;137:12249–60.

    CAS  PubMed  Google Scholar 

  4. 4.

    Zhang X, Yue P, Page BD, Li T, Zhao W, Namanja AT, et al. Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts. Proc Natl Acad Sci USA. 2012;109:9623–8.

    CAS  PubMed  Google Scholar 

  5. 5.

    He F, Borcherds W, Song T, Wei X, Das M, Chen L, et al. Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding. Proc Natl Acad Sci USA. 2019;116:8859–68.

    CAS  PubMed  Google Scholar 

  6. 6.

    Retzlaff M, Rohrberg J, Kupper NJ, Lagleder S, Bepperling A, Manzenrieder F, et al. The regulatory domain stabilizes the p53 tetramer by intersubunit contacts with the DNA binding domain. J Mol Biol. 2013;425:144–55.

    CAS  PubMed  Google Scholar 

  7. 7.

    Wassman CD, Baronio R, Demir O, Wallentine BD, Chen CK, Hall LV, et al. Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53. Nat Commun. 2013;4:1407.

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8:275–83.

    CAS  PubMed  Google Scholar 

  9. 9.

    Khoo KH, Verma CS, Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov. 2014;13:217–36.

    CAS  PubMed  Google Scholar 

  10. 10.

    Gudkov AV, Komarova EA. Prospective therapeutic applications of p53 inhibitors. Biochem Biophys Res Commun. 2005;331:726–36.

    CAS  PubMed  Google Scholar 

  11. 11.

    Komarova EA, Kondratov RV, Wang K, Christov K, Golovkina TV, Goldblum JR, et al. Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice. Oncogene. 2004;23:3265–71.

    CAS  PubMed  Google Scholar 

  12. 12.

    Westphal CH, Hoyes KP, Canman CE, Huang X, Kastan MB, Hendry JH, et al. Loss of atm radiosensitizes multiple p53 null tissues. Cancer Res. 1998;58:5637–9.

    CAS  PubMed  Google Scholar 

  13. 13.

    Jiang M, Wei Q, Wang J, Du Q, Yu J, Zhang L, et al. Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis. Oncogene. 2006;25:4056–66.

    CAS  PubMed  Google Scholar 

  14. 14.

    Wei Q, Dong G, Yang T, Megyesi J, Price PM, Dong Z. Activation and involvement of p53 in cisplatin-induced nephrotoxicity. Am J Physiol Ren Physiol. 2007;293:F1282–91.

    CAS  Google Scholar 

  15. 15.

    Carvajal D, Tovar C, Yang H, Vu BT, Heimbrook DC, Vassilev LT. Activation of p53 by MDM2 antagonists can protect proliferating cells from mitotic inhibitors. Cancer Res. 2005;65:1918–24.

    CAS  PubMed  Google Scholar 

  16. 16.

    Checler F, Alves, da Costa C. p53 in neurodegenerative diseases and brain cancers. Pharmacol Ther. 2014;142:99–113.

    CAS  PubMed  Google Scholar 

  17. 17.

    Crumrine RC, Thomas AL, Morgan PF. Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J Cereb Blood Flow Metab. 1994;14:887–91.

    CAS  PubMed  Google Scholar 

  18. 18.

    Komarov PG, Komarova EA, Kondratov RV, Christov-Tselkov K, Coon JS, Chernov MV, et al. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science. 1999;285:1733–7.

    CAS  PubMed  Google Scholar 

  19. 19.

    Komarova EA, Neznanov N, Komarov PG, Chernov MV, Wang K, Gudkov AV. p53 inhibitor pifithrin alpha can suppress heat shock and glucocorticoid signaling pathways. J Biol Chem. 2003;278:15465–8.

    CAS  PubMed  Google Scholar 

  20. 20.

    Walton MI, Wilson SC, Hardcastle IR, Mirza AR, Workman P. An evaluation of the ability of pifithrin-alpha and -beta to inhibit p53 function in two wild-type p53 human tumor cell lines. Mol Cancer Ther. 2005;4:1369–77.

    CAS  PubMed  Google Scholar 

  21. 21.

    Zhu J, Singh M, Selivanova G, Peuget S. Pifithrin-alpha alters p53 post-translational modifications pattern and differentially inhibits p53 target genes. Sci Rep. 2020;10:1049.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA. 2004;101:12288–93.

    CAS  PubMed  Google Scholar 

  23. 23.

    Segal DJ, Crotty JW, Bhakta MS, Barbas CF 3rd, Horton NC. Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. J Mol Biol. 2006;363:405–21.

    CAS  PubMed  Google Scholar 

  24. 24.

    Mandell JG, Barbas CF 3rd. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 2006;34:W516–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Jiang BH, Rue E, Wang GL, Roe R, Semenza GL. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem. 1996;271:17771–8.

    CAS  PubMed  Google Scholar 

  26. 26.

    van de Wetering M, Cavallo R, Dooijes D, van Beest M, van Es J, Loureiro J, et al. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell. 1997;88:789–99.

    PubMed  Google Scholar 

  27. 27.

    Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA. 1995;92:5510–4.

    CAS  PubMed  Google Scholar 

  28. 28.

    Khoo KH, Andreeva A, Fersht AR. Adaptive evolution of p53 thermodynamic stability. J Mol Biol. 2009;393:161–75.

    CAS  PubMed  Google Scholar 

  29. 29.

    Hall-Jackson CA, Eyers PA, Cohen P, Goedert M, Boyle FT, Hewitt N, et al. Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol. 1999;6:559–68.

    CAS  PubMed  Google Scholar 

  30. 30.

    Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature. 2010;464:427–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ross FA, Hawley SA, Auciello FR, Gowans GJ, Atrih A, Lamont DJ, et al. Mechanisms of paradoxical activation of AMPK by the kinase inhibitors SU6656 and sorafenib. Cell Chem Biol. 2017;24:813–24. e814.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature. 1997;387:296–9.

    CAS  Google Scholar 

  33. 33.

    Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 1993;7:1126–32.

    CAS  PubMed  Google Scholar 

  34. 34.

    Pochampally R, Li C, Lu W, Chen L, Luftig R, Lin J, et al. Temperature-sensitive mutants of p53 homologs. Biochem Biophys Res Commun. 2000;279:1001–10.

    CAS  PubMed  Google Scholar 

  35. 35.

    Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 1993;362:847–9.

    CAS  PubMed  Google Scholar 

  36. 36.

    Pant V, Xiong S, Jackson JG, Post SM, Abbas HA, Quintas-Cardama A, et al. The p53-Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity. Genes Dev. 2013;27:1857–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Shin YJ, Kumarasamy V, Camacho D, Sun D. Involvement of G-quadruplex structures in regulation of human RET gene expression by small molecules in human medullary thyroid carcinoma TT cells. Oncogene. 2015;34:1292–9.

    CAS  PubMed  Google Scholar 

  38. 38.

    Goh AM, Xue Y, Leushacke M, Li L, Wong JS, Chiam PC, et al. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice. Oncotarget. 2015;6:17968–80.

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Midgley CA, Lane DP. p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding. Oncogene. 1997;15:1179–89.

    CAS  PubMed  Google Scholar 

  40. 40.

    Lilyestrom W, Klein MG, Zhang R, Joachimiak A, Chen XS. Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor. Genes Dev. 2006;20:2373–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Tisato V, Voltan R, Gonelli A, Secchiero P, Zauli G. MDM2/X inhibitors under clinical evaluation: perspectives for the management of hematological malignancies and pediatric cancer. J Hematol Oncol. 2017;10:133.

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors wish to thank the National Cancer Institute Developmental Therapeutics Program for providing key compounds. This work is supported in part by grants from the National Institutes of Health (CA141244, CA186917, CA208363 to J.C. GM115556 to D.G. R50CA211447 to H. R. L.). H. Lee Moffitt Cancer Center & Research Institute is an NCI designated Comprehensive Cancer Center (P30-CA076292).

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Study conception: J.C. experiment design and execution: Q.L., R.M.K., M.C., M.D., L.C., C.Z., H.L. Data analysis and intellectual contribution: D.G., E.S., H.J. Manuscript writing: J.C., E.S., H.L.

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Correspondence to Jiandong Chen.

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Li, Q., Karim, R.M., Cheng, M. et al. Inhibition of p53 DNA binding by a small molecule protects mice from radiation toxicity. Oncogene 39, 5187–5200 (2020). https://doi.org/10.1038/s41388-020-1344-y

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