Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Direct and selective small-molecule activation of proapoptotic BAX

Nature Chemical Biology volume 8, pages 639–645 (2012)Cite this article

Subjects

Abstract

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: In silico screen for small-molecule binders of the BAX trigger site identifies BAM7.
Figure 2: BAM7 directly engages the BAX trigger site.
Figure 3: BAM7 triggers in vitro BAX oligomerization, BAX-mediated pore formation and BAX-dependent cell death.
Figure 4: BAM7 induces the biochemical and morphologic features of BAX-mediated apoptosis in Bak−/− MEFs.

References

  1. Danial, N.N. & Korsmeyer, S.J. Cell death: critical control points. Cell 116, 205–219 (2004).

    CAS  PubMed  Google Scholar 

  2. Sattler, M. et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 275, 983–986 (1997).

    Article  CAS  PubMed  Google Scholar 

  3. Nguyen, M. et al. Small molecule obatoclax (GX15–070) antagonizes MCL-1 and overcomes MCL-1–mediated resistance to apoptosis. Proc. Natl. Acad. Sci. USA 104, 19512–19517 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Oltersdorf, T. et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Wang, G. et al. Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins. J. Med. Chem. 49, 6139–6142 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Stewart, M.L., Fire, E., Keating, A.E. & Walensky, L.D. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat. Chem. Biol. 6, 595–601 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Walensky, L.D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305, 1466–1470 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wilson, W.H. et al. Navitoclax, a targeted high-affinity inhibitor of BCL-2, in lymphoid malignancies: a phase 1 dose-escalation study of safety, pharmacokinetics, pharmacodynamics, and antitumour activity. Lancet Oncol. 11, 1149–1159 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. O'Brien, S.M. et al. Phase I study of obatoclax mesylate (GX15–070), a small molecule pan–Bcl-2 family antagonist, in patients with advanced chronic lymphocytic leukemia. Blood 113, 299–305 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gandhi, L. et al. Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients with small-cell lung cancer and other solid tumors. J. Clin. Oncol. 29, 909–916 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gavathiotis, E., Reyna, D.E., Davis, M.L., Bird, G.H. & Walensky, L.D. BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol. Cell 40, 481–492 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Liu, X., Kim, C.N., Yang, J., Jemmerson, R. & Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997).

    Article  CAS  PubMed  Google Scholar 

  14. Du, C., Fang, M., Li, Y., Li, L. & Wang, X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33–42 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Wei, M.C. et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727–730 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Youle, R.J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076–1081 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Walensky, L.D. et al. A stapled BID BH3 helix directly binds and activates BAX. Mol. Cell 24, 199–210 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Friesner, R.A. et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 47, 1739–1749 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Friesner, R.A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49, 6177–6196 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Hsu, Y.T. & Youle, R.J. Nonionic detergents induce dimerization among members of the Bcl-2 family. J. Biol. Chem. 272, 13829–13834 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Wei, M.C. et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 14, 2060–2071 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang, K., Yin, X.M., Chao, D.T., Milliman, C.L. & Korsmeyer, S.J. BID: a novel BH3 domain-only death agonist. Genes Dev. 10, 2859–2869 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. García-Sáez, A.J., Mingarro, I., Perez-Paya, E. & Salgado, J. Membrane-insertion fragments of Bcl-xL, Bax, and Bid. Biochemistry 43, 10930–10943 (2004).

    Article  PubMed  Google Scholar 

  25. Muchmore, S.W. et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381, 335–341 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Tse, C. et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 68, 3421–3428 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Ren, D. et al. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 330, 1390–1393 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cartron, P.F. et al. The first α helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA. Mol. Cell 16, 807–818 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Kim, H. et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat. Cell Biol. 8, 1348–1358 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Kim, H. et al. Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol. Cell 36, 487–499 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kuwana, T. et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol. Cell 17, 525–535 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Kuwana, T. et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111, 331–342 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Letai, A. et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183–192 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Lovell, J.F. et al. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 135, 1074–1084 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Takeuchi, O. et al. Essential role of BAX, BAK in B cell homeostasis and prevention of autoimmune disease. Proc. Natl. Acad. Sci. USA 102, 11272–11277 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Roberts, A.W. et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J. Clin. Oncol. 30, 488–496 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Certo, M. et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9, 351–365 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Walensky, L.D. From mitochondrial biology to magic bullet: navitoclax disarms BCL-2 in chronic lymphocytic leukemia. J. Clin. Oncol. 30, 554–557 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Walensky, L.D. & Gavathiotis, E. BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore. Trends Biochem. Sci. 36, 642–652 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Irwin, J.J. & Shoichet, B.K. ZINC—a free database of commercially available compounds for virtual screening. J. Chem. Inf. Model. 45, 177–182 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pitter, K., Bernal, F., Labelle, J. & Walensky, L.D. Dissection of the BCL-2 family signaling network with stabilized α-helices of BCL-2 domains. Methods Enzymol. 446, 387–408 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bernal, F. et al. A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell 18, 411–422 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Suzuki, M., Youle, R.J. & Tjandra, N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103, 645–654 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, 2002).

  45. Yethon, J.A., Epand, R.F., Leber, B., Epand, R.M. & Andrews, D.W. Interaction with a membrane surface triggers a reversible conformational change in Bax normally associated with induction of apoptosis. J. Biol. Chem. 278, 48935–48941 (2003).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank E. Smith for editorial and graphics assistance, M. Davis for help with establishing the competitive BAX binding assay, G. Bird for BIM SAHB production and characterization and CreaGen Biosciences for BAM7 resynthesis and characterization. This research program was supported by a grant from the William Lawrence and Blanche Hughes Foundation to L.D.W. Additional funding was provided by US National Institutes of Health (NIH) grant 4R00HL095929 to E.G. and NIH grant 5R01CA050239 and a Stand Up to Cancer Innovative Research Grant to L.D.W.

Author information

Authors and Affiliations

  1. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Evripidis Gavathiotis, Denis E Reyna, Joseph A Bellairs, Elizaveta S Leshchiner & Loren D Walensky

  2. Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Evripidis Gavathiotis, Denis E Reyna, Joseph A Bellairs, Elizaveta S Leshchiner & Loren D Walensky

  3. Division of Hematology and Oncology, Children's Hospital Boston, Boston, Massachusetts, USA

    Evripidis Gavathiotis, Denis E Reyna, Joseph A Bellairs, Elizaveta S Leshchiner & Loren D Walensky

  4. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

    Evripidis Gavathiotis, Denis E Reyna, Joseph A Bellairs, Elizaveta S Leshchiner & Loren D Walensky

  5. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA

    Evripidis Gavathiotis & Denis E Reyna

  6. Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA

    Evripidis Gavathiotis & Denis E Reyna

  7. Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, New York, USA

    Evripidis Gavathiotis & Denis E Reyna

  8. Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, New York, USA

    Evripidis Gavathiotis & Denis E Reyna

Authors
  1. Evripidis Gavathiotis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Denis E Reyna
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph A Bellairs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elizaveta S Leshchiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Loren D Walensky
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.G. performed the in silico screen; E.G., D.E.R., J.A.B. and E.S.L. conducted the binding assays; E.G. carried out the structural analyses; E.G. and D.E.R. performed the biochemical studies; and E.G., D.E.R. and J.A.B. conducted the cellular experiments, with guidance from L.D.W. L.D.W. wrote the manuscript, which was reviewed by all authors.

Corresponding authors

Correspondence to Evripidis Gavathiotis or Loren D Walensky.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 13020 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gavathiotis, E., Reyna, D., Bellairs, J. et al. Direct and selective small-molecule activation of proapoptotic BAX. Nat Chem Biol 8, 639–645 (2012). https://doi.org/10.1038/nchembio.995

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.995

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer