Inhibiting Hdm2 and Ubiquitin-Activating Enzyme: Targeting the Ubiquitin Conjugating System in Cancer

  • A. M. Weissman
  • Y. Yang
  • J. Kitagaki
  • C. A. Sasiela
  • J. A. Beutler
  • B. R. O'Keefe
Conference paper
Part of the Ernst Schering Foundation Symposium Proceedings book series (SCHERING FOUND, volume 2008/1)


The ubiquitin conjugating system represents a rich source of potential molecular targets for cancer and other diseases. One target of great interest is the RING finger ubiquitin ligase (E3) Hdm2/Mdm2, which is frequently overexpressed in cancer and is a critical E3 for the tumor suppressor p53. For those 50% of tumors that express wild-type p53, agents that inhibit Hdm2 have great potential clinical utility. We summarize our ongoing efforts to identify inhibitors of Hdm2 E3 activity by high-throughput screening of both defined small molecules and natural product extracts. Employing a strategy using both enzymatic and cell-based assays, we have identified inhibitors that block the E3 activity of Hdm2, activate a p53 response, preferentially kill p53-expressing cells, and have the capacity to differentially cause death of transformed cells. Therefore, screening for inhibitors of Hdm2 ubiquitin ligase activity through in vitro assays represents a powerful means of identifying molecules that activate p53 in cancer cells to induce apoptosis. We also discuss the potential of inhibitors of ubiquitin-activating enzyme (E1) that were discovered during these screens. E1 inhibitors may similarly serve as the basis for novel therapeutics. Additionally, they represent unique tools for providing new insights into the ubiquitin conjugating system.


Ubiquitin Ligase Proteasome Inhibitor Ring Finger Ubiquitin Ligase Activity Potential Molecular Target 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank our collaborators at the National Cancer Institute, the Beatson Institute for Cancer Research, and at Meso Scale Discovery. This work was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, and by the Japanese Society for the Promotion of Science.


  1. Adams J, Kauffman M (2004) Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest 22:304–311CrossRefPubMedGoogle Scholar
  2. Aylon Y, Oren M (2007) Living with p53, dying of p53. Cell 130:597–600CrossRefPubMedGoogle Scholar
  3. Davydov IV, Woods D, Safiran YJ, Oberoi P, Fearnhead HO, Fang S, Jensen JP, Weissman AM, Kenten JH, Vousden KH (2004) Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2. J Biomol Screen 9:695–703CrossRefPubMedGoogle Scholar
  4. Dimond PF (2005) Using nanotechnologies In biotech and medicine: nanomaterials are more than the sum of tiny parts. Gen Eng News 25:21–28Google Scholar
  5. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825CrossRefPubMedGoogle Scholar
  6. Fang S, Weissman AM (2004) A field guide to ubiquitylation. Cell Mol Life Sci 61:1546–1561CrossRefPubMedGoogle Scholar
  7. Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM (2000) Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 275:8945–8951CrossRefPubMedGoogle Scholar
  8. Fang S, Lorick KL, Jensen JP, Weissman AM (2003) RING finger ubiquitin protein ligases: implications for tumorigenesis, metastasis and for molecular targets in cancer. Semin Cancer Biol 13:5–14CrossRefPubMedGoogle Scholar
  9. Glass DJ (2003) Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol 5:87–90CrossRefPubMedGoogle Scholar
  10. Haas AL, Rose IA (1982) The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem 257:10329–10337PubMedGoogle Scholar
  11. Haas AL, Warms JV, Hershko A, Rose IA (1982) Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem 257:2543–2548PubMedGoogle Scholar
  12. Hakli M, Lorick KL, Weissman AM, Janne OA, Palvimo JJ (2004) Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity. FEBS Lett 560:56–62CrossRefPubMedGoogle Scholar
  13. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479CrossRefPubMedGoogle Scholar
  14. Honda R, Yasuda H (2000) Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase. Oncogene 19:1473–1476CrossRefPubMedGoogle Scholar
  15. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, Pramanik A, Selivanova G (2004) Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med 10:1321–1328CrossRefPubMedGoogle Scholar
  16. Joazeiro CA, Weissman AM (2000) RING finger proteins: mediators of ubiquitin ligase activity. Cell 102:549–552CrossRefPubMedGoogle Scholar
  17. Kitagaki J, Agama KK, Pommier Y, Yang Y, Weissman AM (2008) Targeting tumor cells expressing p53 with a water soluble inhibitor of Hdm2. Mol Cancer Ther 7:2445–2454CrossRefPubMedGoogle Scholar
  18. Kumar S, Harvey KF, Kinoshita M, Copeland NG, Noda M, Jenkins NA (1997) cDNA cloning, expression analysis, and mapping of the mouse Nedd4 gene. Genomics 40:435–443CrossRefPubMedGoogle Scholar
  19. Lai Z, Yang T, Kim YB, Sielecki TM, Diamond MA, Strack P, Rolfe M, Caligiuri M, Benfield PA, Auger KR, Copeland RA (2002) Differentiation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitination activity by small molecular weight inhibitors. Proc Natl Acad Sci U S A 99:14734–14739CrossRefPubMedGoogle Scholar
  20. Leonard JP, Furman RR, Coleman M (2006) Proteasome inhibition with bortezomib: a new therapeutic strategy for non-Hodgkin's lymphoma. Int J Cancer 119:971–979CrossRefPubMedGoogle Scholar
  21. Levine AJ, Hu W, Feng Z (2006) The P53 pathway: what questions remain to be explored? Cell Death Differ 13:1027–1036CrossRefPubMedGoogle Scholar
  22. Li WD, Wang MJ, Ding F, Yin DL, Liu ZH (2005) Cytotoxic effect of a non-peptidic small molecular inhibitor of the p53-HDM 2 interaction on tumor cells. World J Gastroenterol 11:2927–2931PubMedGoogle Scholar
  23. Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc Natl Acad Sci U S A 96:11364–11369CrossRefPubMedGoogle Scholar
  24. Lorick KL, Tsai YC, Yang Y, Weissman AM (2006) RING fingers and relatives: determinators of protein fate. In: Mayer RJ, Ciechanover A, Rechsteiner M (eds) Ubiquitin and the chemistry of life. Wiley, Weinhein, pp 44–104Google Scholar
  25. Lowe SW, Ruley HE, Jacks T, Housman DE (1993) p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957–967CrossRefPubMedGoogle Scholar
  26. Momand J, Jung D, Wilczynski S, Niland J (1998) The MDM2 gene amplification database. Nucleic Acids Res 26:3453–3459CrossRefPubMedGoogle Scholar
  27. Nalepa G, Rolfe M, Harper JW (2006) Drug discovery in the ubiquitin-proteasome system. Nat Rev Drug Discov 5:596–613CrossRefPubMedGoogle Scholar
  28. Newman DJ, Cragg GM (2007) Natural products as sources of new drugs over the last 25 years. J Nat Prod 70:461–477CrossRefPubMedGoogle Scholar
  29. Prudden J, Pebernard S, Raffa G, Slavin DA, Perry JJ, Tainer JA, McGowan CH, Boddy MN (2007) SUMO-targeted ubiquitin ligases in genome stability. EMBO J 26:4089–4101CrossRefPubMedGoogle Scholar
  30. Salvesen GS, Duckett CS (2002) IAP proteins: blocking the road to death's door. Nat Rev Mol Cell Biol 3:401–410CrossRefPubMedGoogle Scholar
  31. Sasiela CA, Stewart D, Kitagaki J, Safiran YJ, Yang Y, Weissman AM, Oberoi P, Davydov IV, Goncharova E, Beutler JA, McMahon JB, O'Keefe BR (2008) Identification of inhibitors for MDM2 ubiquitin ligase activity from natural product extracts by a novel high-throughput electrochemiluminescent screen. J Biomol Screen 13:229–237CrossRefPubMedGoogle Scholar
  32. Sun H, Leverson JD, Hunter T (2007) Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J 26:4102–4112CrossRefPubMedGoogle Scholar
  33. Ulrich HD (2005) Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest. Trends Cell Biol 15:525–532CrossRefPubMedGoogle Scholar
  34. Vassilev LT (2007) MDM2 inhibitors for cancer therapy. Trends Mol Med 13:23–31CrossRefPubMedGoogle Scholar
  35. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303:844–848CrossRefPubMedGoogle Scholar
  36. Vousden KH, Lane DP (2007) p53 in health and disease. Nat Rev Mol Cell Biol 8:275–283CrossRefPubMedGoogle Scholar
  37. Wilson JM, Henderson G, Black F, Sutherland A, Ludwig RL, Vousden KH, Robins DJ (2007) Synthesis of 5-deazaflavin derivatives and their activation of p53 in cells. Bioorg Med Chem 15:77–86CrossRefPubMedGoogle Scholar
  38. Yang Y, Li CC, Weissman AM (2004) Regulating the p53 system through ubiquitination. Oncogene 23:2096–2106CrossRefPubMedGoogle Scholar
  39. Yang Y, Ludwig RL, Jensen JP, Pierre SA, Medaglia MV, Davydov IV, Safiran YJ, Oberoi P, Kenten JH, Phillips AC, Weissman AM, Vousden KH (2005) Small molecule inhibitors of HDM 2 ubiquitin ligase activity stabilize and activate p53 in cells. Cancer Cell 7:547–559CrossRefPubMedGoogle Scholar
  40. Yang Y, Kitagaki J, Dai RM, Tsai YC, Lorick KL, Ludwig RL, Pierre SA, Jensen JP, Davydov IV, Oberoi P, Li CC, Kenten JH, Beutler JA, Vousden KH, Weissman AM (2007) Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res 67:9472–9481CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • A. M. Weissman
    • 1
  • Y. Yang
    • 1
  • J. Kitagaki
    • 1
  • C. A. Sasiela
    • 2
  • J. A. Beutler
    • 2
  • B. R. O'Keefe
    • 2
  1. 1.Laboratory of Protein Dynamics and SignalingNational Cancer Institute at FrederickFrederickUSA
  2. 2.Molecular Targets Development ProgramNational Cancer Institute at FrederickFrederickUSA

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