Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo
10.1158/1535-7163.mct-05-0199
Crossref journal-article
American Association for Cancer Research (AACR)
Molecular Cancer Therapeutics (1086)
Abstract

Abstract The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC50s of 14 and 0.7 μmol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage–independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21waf1/cip1 levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors. [Mol Cancer Ther 2006;5(1):160–9]

Bibliography

Koblish, H. K., Zhao, S., Franks, C. F., Donatelli, R. R., Tominovich, R. M., LaFrance, L. V., Leonard, K. A., Gushue, J. M., Parks, D. J., Calvo, R. R., Milkiewicz, K. L., Marugán, J. J., Raboisson, P., Cummings, M. D., Grasberger, B. L., Johnson, D. L., Lu, T., Molloy, C. J., & Maroney, A. C. (2006). Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo. Molecular Cancer Therapeutics, 5(1), 160–169.

Authors 19
  1. Holly K. Koblish (first)
  2. Shuyuan Zhao (additional)
  3. Carol F. Franks (additional)
  4. Robert R. Donatelli (additional)
  5. Rose M. Tominovich (additional)
  6. Louis V. LaFrance (additional)
  7. Kristi A. Leonard (additional)
  8. Joan M. Gushue (additional)
  9. Daniel J. Parks (additional)
  10. Raul R. Calvo (additional)
  11. Karen L. Milkiewicz (additional)
  12. Juan José Marugán (additional)
  13. Pierre Raboisson (additional)
  14. Maxwell D. Cummings (additional)
  15. Bruce L. Grasberger (additional)
  16. Dana L. Johnson (additional)
  17. Tianbao Lu (additional)
  18. Christopher J. Molloy (additional)
  19. Anna C. Maroney (additional)
References 66 Referenced 137
  1. Jones SN, Sands AT, Hancock AR, et al. The tumorigenic potential and cell growth characteristics of p53-deficient cells are equivalent in the presence or absence of Mdm2. Proc Natl Acad Sci U S A 1996;93:14106–11. (10.1073/pnas.93.24.14106)
  2. Keshelava N, Zuo JJ, Chen P, et al. Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines. Cancer Res 2001;61:6185–93.
  3. Johnston JB, Daeninck P, Verburg L, et al. p53, MDM-2, BAX and BCL-2 and drug resistance in chronic lymphocytic leukemia. Leuk Lymphoma 1997;26:435–49. (10.3109/10428199709050881)
  4. Buttitta F, Marchetti A, Gadducci A, et al. p53 alterations are predictive of chemoresistance and aggressiveness in ovarian carcinomas: a molecular and immunohistochemical study. Br J Cancer 1997;75:230–5. (10.1038/bjc.1997.38)
  5. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol 2001;3:973–82. (10.1038/ncb1101-973)
  6. Coukos G, Rubin SC. Chemotherapy resistance in ovarian cancer: new molecular perspectives. Obstet Gynecol 1998;91:783–92. (10.1016/S0029-7844(98)00054-4)
  7. Harada T, Ogura S, Yamazaki K, et al. Predictive value of expression of p53, Bcl-2 and lung resistance-related protein for response to chemotherapy in non-small cell lung cancers. Cancer Sci 2003;94:394–9. (10.1111/j.1349-7006.2003.tb01453.x)
  8. Yamazaki Y, Chiba I, Hirai A, et al. Radioresistance in oral squamous cell carcinoma with p53 DNA contact mutation. Am J Clin Oncol 2003;26:e124–9. (10.1097/01.coc.0000091352.60347.f8)
  9. Beroud C, Soussi T. p53 gene mutation: software and database. Nucleic Acids Res 1998;26:200–4. (10.1093/nar/26.1.200)
  10. Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification database. Nucleic Acids Res 1998;26:3453–9. (10.1093/nar/26.15.3453)
  11. Chene P. Targeting p53 in cancer. Curr Med Chem Anti-Canc Agents 2001;1:151–61. (10.2174/1568011013354741)
  12. Chene P. Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat Rev Cancer 2003;3:102–9. (10.1038/nrc991)
  13. Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatory feedback loop. Genes Dev 1993;7:1126–32. (10.1101/gad.7.7a.1126)
  14. Jones SN, Roe AE, Donehower LA, Bradley A. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 1995;378:206–8. (10.1038/378206a0)
  15. Montes de Oca Luna R, Wagner DS, Lozano G. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 1995;378:203–6. (10.1038/378203a0)
  16. Bottger A, Bottger V, Garcia-Echeverria C, et al. Molecular characterization of the hdm2-p53 interaction. J Mol Biol 1997;269:744–56. (10.1006/jmbi.1997.1078)
  17. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 1997;420:25–7. (10.1016/S0014-5793(97)01480-4)
  18. Suzuki A, Toi M, Yamamoto Y, Saji S, Muta M, Tominaga T. Role of MDM2 overexpression in doxorubicin resistance of breast carcinoma. Jpn J Cancer Res 1998;89:221–7. (10.1111/j.1349-7006.1998.tb00552.x)
  19. Zhou M, Gu L, Abshire TC, et al. Incidence and prognostic significance of MDM2 oncoprotein overexpression in relapsed childhood acute lymphoblastic leukemia. Leukemia 2000;14:61–7. (10.1038/sj.leu.2401619)
  20. Ikeguchi M, Ueda T, Fukuda K, Yamaguchi K, Tsujitani S, Kaibara N. Expression of the murine double minute gene 2 oncoprotein in esophageal squamous cell carcinoma as a novel marker for lack of response to chemoradiotreatment. Am J Clin Oncol 2002;25:454–9. (10.1097/00000421-200210000-00006)
  21. Chen L, Agrawal S, Zhou W, Zhang R, Chen J. Synergistic activation of p53 by inhibition of MDM2 expression and DNA damage. Proc Natl Acad Sci U S A 1998;95:195–200. (10.1073/pnas.95.1.195)
  22. Chen L, Lu W, Agrawal S, Zhou W, Zhang R, Chen J. Ubiquitous induction of p53 in tumor cells by antisense inhibition of MDM2 expression. Mol Med 1999;5:21–34. (10.1007/BF03402136)
  23. Tortora G, Caputo R, Damiano V, et al. A novel MDM2 anti-sense oligonucleotide has anti-tumor activity and potentiates cytotoxic drugs acting by different mechanisms in human colon cancer. Int J Cancer 2000;88:804–9. (10.1002/1097-0215(20001201)88:5<804::AID-IJC19>3.0.CO;2-Z)
  24. Bottger A, Bottger V, Sparks A, Liu WL, Howard SF, Lane DP. Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo. Curr Biol 1997;7:860–9. (10.1016/S0960-9822(06)00374-5)
  25. Wang H, Nan L, Yu D, Agrawal S, Zhang R. Antisense anti-MDM2 oligonucleotides as a novel therapeutic approach to human breast cancer: in vitro and in vivo activities and mechanisms. Clin Cancer Res 2001;7:3613–24.
  26. Pearson S, Jia H, Kandachi K. China approves first gene therapy. Nat Biotechnol 2004;22:3–4. (10.1038/nbt0104-3)
  27. Lane DP, Lain S. Therapeutic exploitation of the p53 pathway. Trends Mol Med 2002;8:S38–42. (10.1016/S1471-4914(02)02309-2)
  28. Grasberger BL, Lu T, Schubert C, et al. Discovery and cocrystal structure of benzodiazepinedione Hdm2 antagonists that activate p53 in cells. J Med Chem 2005;48:909–12. (10.1021/jm049137g)
  29. Parant J, Chavez-Reyes A, Little NA, et al. Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat Genet 2001;29:92–5. (10.1038/ng714)
  30. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55. (10.1016/0065-2571(84)90007-4)
  31. Pantoliano MW, Petrella EC, Kwasnoski JD, et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 2001;6:429–40. (10.1177/108705710100600609)
  32. Parks DJ, Lafrance LV, Calvo RR, et al. 1,4-Benzodiazepine-2,5-diones as small molecule antagonists of the HDM2-p53 interaction: discovery and SAR. Bioorg Med Chem Lett 2005;15:765–70. (10.1016/j.bmcl.2004.11.009)
  33. Wang H, Zeng X, Oliver P, et al. MDM2 oncogene as a target for cancer therapy: an antisense approach. Int J Oncol 1999;15:653–60. (10.3892/ijo.15.4.653)
  34. Milczarek GJ, Martinez J, Bowden GT. p53 Phosphorylation: biochemical and functional consequences. Life Sci 1997;60:1–11. (10.1016/S0024-3205(96)00479-1)
  35. Meek DW. Post-translational modification of p53. Semin Cancer Biol 1994;5:203–10.
  36. Thompson T, Tovar C, Yang H, et al. Phosphorylation of p53 on key serines is dispensable for transcriptional activation and apoptosis. J Biol Chem 2004;279:53015–22. (10.1074/jbc.M410233200)
  37. Lee TK, Lau TC, Ng IO. Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines. Cancer Chemother Pharmacol 2002;49:78–86. (10.1007/s00280-001-0376-4)
  38. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323–31. (10.1016/S0092-8674(00)81871-1)
  39. Slee EA, O'Connor DJ, Lu X. To die or not to die: how does p53 decide? Oncogene 2004;23:2809–18. (10.1038/sj.onc.1207516)
  40. Flatt PM, Polyak K, Tang LJ, et al. p53-dependent expression of PIG3 during proliferation, genotoxic stress, and reversible growth arrest. Cancer Lett 2000;156:63–72. (10.1016/S0304-3835(00)00441-9)
  41. Kaeser MD, Iggo RD. Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo. Proc Natl Acad Sci U S A 2002;99:95–100. (10.1073/pnas.012283399)
  42. Yeh PY, Chuang SE, Yeh KH, Song YC, Chang LL, Cheng AL. Phosphorylation of p53 on Thr55 by ERK2 is necessary for doxorubicin-induced p53 activation and cell death. Oncogene 2004;23:3580–8. (10.1038/sj.onc.1207426)
  43. Fei P, Bernhard EJ, El-Deiry WS. Tissue-specific induction of p53 targets in vivo. Cancer Res 2002;62:7316–27.
  44. Bartel F, Harris LC, Wurl P, Taubert H. MDM2 and its splice variant messenger RNAs: expression in tumors and down-regulation using antisense oligonucleotides. Mol Cancer Res 2004;2:29–35. (10.1158/1541-7786.29.2.1)
  45. Kawabe S, Munshi A, Zumstein LA, Wilson DR, Roth JA, Meyn RE. Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts. Int J Radiat Biol 2001;77:185–94. (10.1080/09553000010008540)
  46. Prasad G, Wang H, Agrawal S, Zhang R. Antisense anti-MDM2 oligonucleotides as a novel approach to the treatment of glioblastoma multiforme. Anticancer Res 2002;22:107–16.
  47. Wang H, Yu D, Agrawal S, Zhang R. Experimental therapy of human prostate cancer by inhibiting MDM2 expression with novel mixed-backbone antisense oligonucleotides: in vitro and in vivo activities and mechanisms. Prostate 2003;54:194–205. (10.1002/pros.10187)
  48. Zhang Z, Wang H, Prasad G, et al. Radiosensitization by antisense anti-MDM2 mixed-backbone oligonucleotide in in vitro and in vivo human cancer models. Clin Cancer Res 2004;10:1263–73. (10.1158/1078-0432.CCR-0245-03)
  49. Cristofanilli M, Khrisnamurthy S, Guerra L, et al. Advexin® (Ad5CMV-p53) combined with docetaxel (T) and doxorubicin (D) as induction chemotherapy (IC): efficacy of a novel gene-therapy approach for patients with locally advanced breast cancer (LABC). 27th Annual San Antonio Breast Cancer Symposium; San Antonio TX; 2004.
  50. Haapajarvi T, Pitkanen K, Laiho M. Human melanoma cell line UV responses show independency of p53 function. Cell Growth Differ 1999;10:163–71.
  51. Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 1992;356:215–21. (10.1038/356215a0)
  52. Hollstein M, Hergenhahn M, Yang Q, Bartsch H, Wang ZQ, Hainaut P. New approaches to understanding p53 gene tumor mutation spectra. Mutat Res 1999;431:199–209. (10.1016/S0027-5107(99)00162-1)
  53. Hainaut P. Tumor-specific mutations in p53: the acid test. Nat Med 2002;8:21–3. (10.1038/nm0102-21)
  54. Zeimet A, Marth C. Why did p53 gene therapy fail in ovarian cancer? Lancet Oncol 2003;4:415–22. (10.1016/S1470-2045(03)01139-2)
  55. Sigalas I, Calvert AH, Anderson JJ, Neal DE, Lunec J. Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences: transforming ability and frequent detection in human cancer. Nat Med 1996;2:912–7. (10.1038/nm0896-912)
  56. Jones SN, Hancock AR, Vogel H, Donehower LA, Bradley A. Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis. Proc Natl Acad Sci U S A 1998;95:15608–12. (10.1073/pnas.95.26.15608)
  57. Boyd MT, Vlatkovic N, Haines DS. A novel cellular protein (MTBP) binds to MDM2 and induces a G1 arrest that is suppressed by MDM2. J Biol Chem 2000;275:31883–90. (10.1074/jbc.M004252200)
  58. Reinke V, Lozano G. The p53 targets mdm2 and Fas are not required as mediators of apoptosis in vivo. Oncogene 1997;15:1527–34. (10.1038/sj.onc.1201316)
  59. Ha L, Ceryak S, Patierno SR. Chromium (VI) activates ataxia telangiectasia mutated (ATM) protein. Requirement of ATM for both apoptosis and recovery from terminal growth arrest. J Biol Chem 2003;278:17885–94. (10.1074/jbc.M210560200)
  60. Wolf BB, Schuler M, Echeverri F, Green DR. Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J Biol Chem 1999;274:30651–6. (10.1074/jbc.274.43.30651)
  61. Isaacs JS, Saito S, Neckers LM. Requirement for HDM2 activity in the rapid degradation of p53 in neuroblastoma. J Biol Chem 2001;276:18497–506. (10.1074/jbc.M100638200)
  62. Fan S, Smith ML, Rivet DJ, II, et al. Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res 1995;55:1649–54.
  63. Bauer JA, Trask DK, Kumar B, et al. Reversal of cisplatin resistance with a BH3 mimetic, (−)-gossypol, in head and neck cancer cells: role of wild-type p53 and Bcl-xL. Mol Cancer Ther 2005;4:1096–104. (10.1158/1535-7163.MCT-05-0081)
  64. Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004;303:844–8. (10.1126/science.1092472)
  65. Issaeva N, Bozko P, Enge M, et al. Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med 2004;10:1321–8. (10.1038/nm1146)
  66. Krajewski M, Ozdowy P, D'Silva L, Rothweiler U, Holak TA. NMR indicates that the small molecule RITA does not block p53-MDM2 binding in vitro. Nat Med 2005;11:1135–6. (10.1038/nm1105-1135)
Dates
Type When
Created 19 years, 5 months ago (March 21, 2006, 1:48 p.m.)
Deposited 3 years, 2 months ago (June 8, 2022, 4:59 a.m.)
Indexed 1 month, 4 weeks ago (June 29, 2025, 4:20 p.m.)
Issued 19 years, 7 months ago (Jan. 1, 2006)
Published 19 years, 7 months ago (Jan. 1, 2006)
Published Online 19 years, 7 months ago (Jan. 23, 2006)
Published Print 19 years, 7 months ago (Jan. 1, 2006)
Funders 0

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@article{Koblish_2006, title={Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo}, volume={5}, ISSN={1538-8514}, url={http://dx.doi.org/10.1158/1535-7163.mct-05-0199}, DOI={10.1158/1535-7163.mct-05-0199}, number={1}, journal={Molecular Cancer Therapeutics}, publisher={American Association for Cancer Research (AACR)}, author={Koblish, Holly K. and Zhao, Shuyuan and Franks, Carol F. and Donatelli, Robert R. and Tominovich, Rose M. and LaFrance, Louis V. and Leonard, Kristi A. and Gushue, Joan M. and Parks, Daniel J. and Calvo, Raul R. and Milkiewicz, Karen L. and Marugán, Juan José and Raboisson, Pierre and Cummings, Maxwell D. and Grasberger, Bruce L. and Johnson, Dana L. and Lu, Tianbao and Molloy, Christopher J. and Maroney, Anna C.}, year={2006}, month=jan, pages={160–169} }