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.

  • Article
  • Published:

A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy

Abstract

Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration–approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Selectivity profile of SAR405.
Figure 2: Co-crystallization of SAR405 with Vps34 protein.
Figure 3: SAR405 affects vesicle trafficking.
Figure 4: SAR405 prevents starvation-induced autophagy.
Figure 5: SAR405 prevents autophagy induced by mTOR inhibitor and synergizes with everolimus.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Mizushima, N., Levine, B., Cuervo, A.M. & Klionsky, D.J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Boya, P., Reggiori, F. & Codogno, P. Emerging regulation and functions of autophagy. Nat. Cell Biol. 15, 713–720 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Choi, A.M.K., Ryter, S.W. & Levine, B. Autophagy in human health and disease. N. Engl. J. Med. 368, 651–662 (2013).

    CAS  PubMed  Google Scholar 

  4. White, E. Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Rubinsztein, D.C., Codogno, C. & Levine, B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat. Rev. Drug Discov. 11, 709–730 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Laplante, M. & Sabatini, D.M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Kim, J. et al. Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy. Cell 152, 290–303 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Russell, R.C. et al. ULK1 induces autophagy by phosphorylating Beclin-1 and activating Vps34 lipid kinase. Nat. Cell Biol. 15, 741–750 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Sasaki, T. et al. Mammalian phosphoinositide kinases and phosphatases. Prog. Lipid Res. 48, 307–343 (2009).

    CAS  PubMed  Google Scholar 

  10. Vanhaesebroeck, B., Guillermet-Guibert, J., Graupera, M. & Bilanges, B. The emerging mechanisms of isoform-specific PI3K signalling. Nat. Rev. Mol. Cell Biol. 11, 329–341 (2010).

    CAS  PubMed  Google Scholar 

  11. Backer, J.M. The regulation and function of class III PI3Ks: novel roles for Vps34. Biochem. J. 410, 1–17 (2008).

    CAS  PubMed  Google Scholar 

  12. Lindmo, K. & Stenmark, H. Regulation of membrane traffic by phosphoinositide 3-kinases. J. Cell Sci. 119, 605–614 (2006).

    CAS  PubMed  Google Scholar 

  13. Hnia, K., Vaccari, I., Bolino, A. & Laporte, J. Myotubularin phosphoinositide phosphatases: cellular functions and disease pathophysiology. Trends Mol. Med. 18, 317–327 (2012).

    CAS  PubMed  Google Scholar 

  14. Vergne, I. & Deretic, V. The role of PI3P phosphatases in the regulation of autophagy. FEBS Lett. 584, 1313–1318 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Dall'Armi, C., Devereaux, K.A. & Di Paolo, G. The role of lipids in the control of autophagy. Curr. Biol. 23, R33–R45 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Lamb, C.A., Yoshimori, T. & Tooze, S.A. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 (2013).

    CAS  PubMed  Google Scholar 

  17. Herman, P.K. & Emr, S.D. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 6742–6754 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Funderburk, S.F., Wang, Q.J. & Yue, Z. The beclin 1–Vps34 complex—at the crossroads of autophagy and beyond. Trends Cell Biol. 20, 355–362 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Peppard, J.V. et al. Identifying small molecules which inhibit autophagy : a phenotypic screen using image-based high-content cell analysis. Curr. Chem. Genomics. Transl. Med. 8, 3–15 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Gillooly, D.J., Simonsen, A. & Stenmark, H. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins. Biochem. J. 355, 249–258 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Kutateladze, T.G. Mechanistic similarities in docking of the FYVE and PX domains to phosphatidylinositol 3-phosphate containing membranes. Prog. Lipid Res. 46, 315–327 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Gillooly, D.J. et al. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Stuffers, S. et al. Time resolved ultrastructural detection of phosphatidylinositol 3-phosphate. J. Histochem. Cytochem. 58, 1025–1032 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Patricelli, M.P. et al. In situ kinase profiling reveals functionally relevant properties of native kinases. Chem. Biol. 18, 699–710 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhang, H. et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J. Clin. Invest. 112, 1223–1233 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Brumbaugh, K.M. et al. The mRNA surveillance protein hSMG-1 functions in genotoxic stress response pathways in mammalian cells. Mol. Cell 14, 585–598 (2004).

    CAS  PubMed  Google Scholar 

  27. Certal, V. et al. Discovery and optimization of new benzimidazole- and benzoxazole-pyrimidone selective PI3Kβ inhibitors for the treatment of phosphatase and TENsin homologue (PTEN)-deficient cancers. J. Med. Chem. 55, 4788–4805 (2012).

    CAS  PubMed  Google Scholar 

  28. Miller, S. et al. Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34. Science 327, 1638–1642 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Berndt, A. et al. The p110δ structure: mechanisms for selectivity and potency of new PI(3)K inhibitors. Nat. Chem. Biol. 6, 117–124 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kornev, A.P., Haste, N.M., Taylor, S.S. & Ten Eyck, L.F. Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc. Natl. Acad. Sci. USA 103, 17783–17788 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Johnson, E.E., Overmeyer, J.H., Gunning, W.T. & Maltese, A. Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes. J. Cell Sci. 119, 1219–1232 (2006).

    CAS  PubMed  Google Scholar 

  32. Jaber, N. et al. Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function. Proc. Natl. Acad. Sci. USA 109, 2003–2008 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Komatsu, M., Kageyama, S. & Ichimura, Y. p62/SQQTM1/A170: physiology and pathology. Pharmacol. Res. 66, 457–462 (2012).

    CAS  PubMed  Google Scholar 

  34. Zaidi, N., Maurer, A., Nieke, S. & Kalbacher, H. Cathepsin D: a cellular roadmap. Biochem. Biophys. Res. Commun. 376, 5–9 (2008).

    CAS  PubMed  Google Scholar 

  35. Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Klionsky, D.J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. García-Echeverria, C. Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment. Bioorg. Med. Chem. Lett. 20, 4308–4312 (2010).

    PubMed  Google Scholar 

  38. Benjamin, D., Colombi, M., Moroni, C. & Hall, M.N. Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat. Rev. Drug Discov. 10, 868–880 (2011).

    CAS  PubMed  Google Scholar 

  39. Straetemans, R. et al. Design and analysis of drug combination experiments. Biom. J. 47, 299–308 (2005).

    PubMed  Google Scholar 

  40. Zhou, X., Takatoh, J. & Wang, F. The mammalian class 3 PI3K (PI3KC3) is required for early embryogenesis and cell proliferation. PLoS ONE 6, e16358 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhou, X. et al. Deletion of PI3KC3/Vps34 in sensory neurons causes rapid neurodegeneration by disrupting the endosomal but not the autophagic pathway. Proc. Natl. Acad. Sci. USA 107, 9424–9429 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. McLeod, I.X., Zhou, X., Li, Q.J., Wang, F. & He, Y.W. The class III kinase Vps34 promotes T lymphocyte survival through regulating IL-7Rα surface expression. J. Immunol. 187, 5051–5061 (2011).

    CAS  PubMed  Google Scholar 

  43. Willinger, T. & Flavell, R.A. Canonical autophagy dependent on the class III phosphoinositide-3 kinase Vps34 is required for naïve T-cell homeostasis. Proc. Natl. Acad. Sci. USA 109, 8670–8675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Parekh, V.V. et al. Impaired autophagy, defective T cell homeostasis, and a wasting syndrome in mice with a T cell specific deletion of Vps34. J. Immunol. 190, 5086–5101 (2013).

    CAS  PubMed  Google Scholar 

  45. Lipinski, M.M. et al. A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions. Dev. Cell 18, 1041–1052 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Codogno, P., Mehrpour, M. & Proikas-Cezanne, T. Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat. Rev. Mol. Cell Biol. 13, 7–12 (2012).

    CAS  Google Scholar 

  47. Wiczer, B.M. & Thomas, G. Phospholipase D and mTORC1: nutrients are what bring them together. Sci. Signal. 5, pe13 (2012).

    PubMed  Google Scholar 

  48. Pike, K.G. et al. Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014. Bioorg. Med. Chem. Lett. 23, 1212–1216 (2013).

    CAS  PubMed  Google Scholar 

  49. Stauffer, F., Maira, S.M., Furet, P. & Garcia-Echeverria, C. Imidazo[4,5-c]quinolines as inhibitors of the PI3K/PKB-pathway. Bioorg. Med. Chem. Lett. 18, 1027–1030 (2008).

    CAS  PubMed  Google Scholar 

  50. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Capdevila, N. Michot, S. Plantard, C. Valtre, M. Lowinski, A. Rak, C. Castell, V. Bazin, C. Delorme, H. Robbe, J.-P. Ridoux, A. Casse, F. Windenberger, A.-C. Kerangueven, V. Onado, S. El Batti, J.-P. Letallec, V. Sonnefraud, F. Gay, M. Brollo, L. Delbarre, V. Loyau, P. Richepin, L. Bertin, F. Pilorge and G. McCort for their valuable input and discussion.

Author information

Authors and Affiliations

Authors

Contributions

O.F., L.V., L.D. and C.D. performed all the cellular assays; M.-F.B., A.L. and F.F. performed the biochemical assays; J.-P.M., M.M. and T.B. performed the co-crystallization and structural analysis; B.R., Y.E.-A., B.F.-R. designed and contributed to the synthesis of chemical compounds; L.S., C.G.-E. and H.G. contributed to discussions and revised the manuscript; B.P. designed the biological experiments, directed the project and wrote the manuscript.

Corresponding author

Correspondence to Benoit Pasquier.

Ethics declarations

Competing interests

The authors have competing interests. All of the authors are employees of Sanofi, and all are or have been shareholders of Sanofi.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–21 and Supplementary Tables 1 and 2. (PDF 2606 kb)

Supplementary Data Set

Kinase profiling of SAR405 (PDF 1340 kb)

Supplementary Note

Supplementary Note (DOCX 48 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ronan, B., Flamand, O., Vescovi, L. et al. A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy. Nat Chem Biol 10, 1013–1019 (2014). https://doi.org/10.1038/nchembio.1681

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1681

This article is cited by

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