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PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling


Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein–membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2–Bragsin complex and structure–activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein–membrane interactions without disruption.

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Data availability

All data generated or analyzed during this study are available from the corresponding authors upon reasonable request. Uncropped western blot and gel are shown in Supplementary Fig. 6. Coordinates and structure factors have been deposited to the Protein Data Bank with entry code 6FNE.

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Change history

  • 04 March 2019

    In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.


  1. 1.

    Arkin, M. R., Tang, Y. & Wells, J. A. Small-molecule inhibitors of protein-protein interactions: progressing toward the reality. Chem. Biol. 21, 1102–1114 (2014).

  2. 2.

    Dang, C. V., Reddy, E. P., Shokat, K. M. & Soucek, L. Drugging the ‘undruggable’ cancer targets. Nat. Rev. Cancer 17, 502–508 (2017).

  3. 3.

    Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81, 153–208 (2001).

  4. 4.

    Papke, B. & Der, C. J. Drugging RAS: know the enemy. Science 355, 1158–1163 (2017).

  5. 5.

    Loirand, G., Sauzeau, V. & Pacaud, P. Small G proteins in the cardiovascular system: physiological and pathological aspects. Physiol. Rev. 93, 1659–1720 (2013).

  6. 6.

    Aktories, K. Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487–498 (2011).

  7. 7.

    Cherfils, J. & Zeghouf, M. Chronicles of the GTPase switch. Nat. Chem. Biol. 7, 493–495 (2011).

  8. 8.

    Cherfils, J. & Zeghouf, M. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269–309 (2013).

  9. 9.

    Appels, N. M., Beijnen, J. H. & Schellens, J. H. Development of farnesyl transferase inhibitors: a review. Oncologist 10, 565–578 (2005).

  10. 10.

    Donaldson, J. G. & Jackson, C. L. ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat. Rev. Mol. Cell Biol. 12, 362–375 (2011).

  11. 11.

    D’Souza, R. S. & Casanova, J. E. The BRAG/IQSec family of Arf GEFs. Small GTPases 7, 257–264 (2016).

  12. 12.

    Morishige, M. et al. GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion. Nat. Cell Biol. 10, 85–92 (2008).

  13. 13.

    Yoo, J. H. et al. ARF6 is an actionable node that orchestrates oncogenic GNAQ signaling in uveal melanoma. Cancer Cell. 29, 889–904 (2016).

  14. 14.

    Zhu, W. et al. Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy. J. Clin. Invest. 127, 4569–4582 (2017).

  15. 15.

    Aizel, K. et al. Integrated conformational and lipid-sensing regulation of endosomal ArfGEF BRAG2. PLoS Biol. 11, e1001652 (2013).

  16. 16.

    Jian, X., Gruschus, J. M., Sztul, E. & Randazzo, P. A. The pleckstrin homology (PH) domain of the Arf exchange factor Brag2 is an allosteric binding site. J. Biol. Chem. 287, 24273–24283 (2012).

  17. 17.

    Karandur, D., Nawrotek, A., Kuriyan, J. & Cherfils, J. Multiple interactions between an Arf/GEF complex and charged lipids determine activation kinetics on the membrane. Proc. Natl Acad. Sci. USA 114, 11416–11421 (2017).

  18. 18.

    Lee, A. Y. et al. Mapping the cellular response to small molecules using chemogenomic fitness signatures. Science 344, 208–211 (2014).

  19. 19.

    Volpicelli-Daley, L. A., Li, Y., Zhang, C. J. & Kahn, R. A. Isoform-selective effects of the depletion of ADP-ribosylation factors 1-5 on membrane traffic. Mol. Biol. Cell. 16, 4495–4508 (2005).

  20. 20.

    Moravec, R., Conger, K. K., D’Souza, R., Allison, A. B. & Casanova, J. E. BRAG2/GEP100/IQSec1 interacts with clathrin and regulates α5β1 integrin endocytosis through activation of ADP ribosylation factor 5 (Arf5). J. Biol. Chem. 287, 31138–31147 (2012).

  21. 21.

    Peurois, F. et al. Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering. Biochem. J. 474, 1259–1272 (2017).

  22. 22.

    Mouratou, B. et al. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf. BMC Genomics 6, 20 (2005).

  23. 23.

    Casanova, J. E. Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors. Traffic 8, 1476–1485 (2007).

  24. 24.

    Nastou, K. C., Tsaousis, G. N., Kremizas, K. E., Litou, Z. I. & Hamodrakas, S. J. The human plasma membrane peripherome: visualization and analysis of interactions. Biomed. Res. Int. 2014, 397145 (2014).

  25. 25.

    DiNitto, J. P. et al. Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. Mol. Cell 28, 569–583 (2007).

  26. 26.

    Kreso, A. & Dick, J. E. Evolution of the cancer stem cell model. Cell Stem Cell 14, 275–291 (2014).

  27. 27.

    Charafe-Jauffret, E. et al. ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program. Cancer Res. 73, 7290–7300 (2013).

  28. 28.

    Ginestier, C. et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 (2007).

  29. 29.

    Scholz, R. et al. AMPA receptor signaling through BRAG2 and Arf6 critical for long-term synaptic depression. Neuron 66, 768–780 (2010).

  30. 30.

    Dunphy, J. L. et al. The Arf6 GEF GEP100/BRAG2 regulates cell adhesion by controlling endocytosis of β1 integrins. Curr. Biol. 16, 315–320 (2006).

  31. 31.

    Dottermusch-Heidel, C., Groth, V., Beck, L. & Önel, S. F. The Arf-GEF Schizo/Loner regulates N-cadherin to induce fusion competence of Drosophila myoblasts. Dev. Biol. 368, 18–27 (2012).

  32. 32.

    Shafaq-Zadah, M. et al. Persistent cell migration and adhesion rely on retrograde transport of β1 integrin. Nat. Cell Biol. 18, 54–64 (2016).

  33. 33.

    Mana, G. et al. PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis. Nat. Commun. 7, 13546 (2016).

  34. 34.

    Lemmon, M. A. Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 9, 99–111 (2008).

  35. 35.

    Vonkova, I. et al. Lipid cooperativity as a general membrane-recruitment principle for PH domains. Cell Rep. 12, 1519–1530 (2015).

  36. 36.

    Picas, L., Gaits-Iacovoni, F. & Goud, B. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction. F1000Res. 5, 422 (2016).

  37. 37.

    Hopkins, A. L. & Groom, C. R. The druggable genome. Nat. Rev. Drug. Discov. 1, 727–730 (2002).

  38. 38.

    Segers, K. et al. Design of protein membrane interaction inhibitors by virtual ligand screening, proof of concept with the C2 domain of factor V. Proc. Natl Acad. Sci. USA 104, 12697–12702 (2007).

  39. 39.

    Odell, L. R. et al. Pyrimidine-based inhibitors of dynamin I GTPase activity: competitive inhibition at the pleckstrin homology domain. J. Med. Chem. 60, 349–361 (2017).

  40. 40.

    Joh, E. H., Hollenbaugh, J. A., Kim, B. & Kim, D. H. Pleckstrin homology domain of Akt kinase: a proof of principle for highly specific and effective non-enzymatic anti-cancer target. PLoS One 7, e50424 (2012).

  41. 41.

    Wu, W. I. et al. Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase inhibition. PLoS One 5, e12913 (2010).

  42. 42.

    Deyle, K. M. et al. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1. Nat. Chem. 7, 455–462 (2015).

  43. 43.

    Viaud, J. et al. Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes. Proc. Natl Acad. Sci. USA 104, 10370–10375 (2007).

  44. 44.

    Mossessova, E., Corpina, R. A. & Goldberg, J. Crystal structure of ARF1*Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism. Mol. Cell 12, 1403–1411 (2003).

  45. 45.

    Renault, L., Guibert, B. & Cherfils, J. Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426, 525–530 (2003).

  46. 46.

    Pommier, Y. & Cherfils, J. Interfacial inhibition of macromolecular interactions: nature’s paradigm for drug discovery. Trends Pharmacol. Sci. 26, 138–145 (2005).

  47. 47.

    Milroy, L. G., Grossmann, T. N., Hennig, S., Brunsveld, L. & Ottmann, C. Modulators of protein-protein interactions. Chem. Rev. 114, 4695–4748 (2014).

  48. 48.

    Jin, L., Wang, W. & Fang, G. Targeting protein-protein interaction by small molecules. Annu. Rev. Pharmacol. Toxicol. 54, 435–456 (2014).

  49. 49.

    Padovani, D. et al. EFA6 controls Arf1 and Arf6 activation through a negative feedback loop. Proc. Natl Acad. Sci. USA 111, 12378–12383 (2014).

  50. 50.

    Benabdi, S. et al. Family-wide analysis of the inhibition of Arf guanine nucleotide exchange factors with small molecules: evidence of unique inhibitory profiles. Biochemistry 56, 5125–5133 (2017).

  51. 51.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  52. 52.

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

  53. 53.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  54. 54.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, (213–221 (2010).

  55. 55.

    Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D. Biol. Crystallogr. 60, 2210–2221 (2004).

  56. 56.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

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Research in the J.C. laboratory was supported by the Institut National du Cancer (INCA) (grant number 2014-160) and the Fondation pour la Recherche Médicale (FRM) (grant number DEQ20150331694). Research in the R.R. laboratory is supported by the European Research Council (grant number 647973). We thank the scientists at synchrotron SOLEIL (Gif-sur-Yvette, France) for making the PX beamlines available to us and for their excellent advice. Plasmids encoding Arf Q/L mutants were kindly provided by J. Ménétrey (LEBS, CNRS, Gif-sur-Yvette France). We are grateful to F. Peurois, L. Akendengué, R. Hergesheimer and the other members of the Cherfils lab for their help and V. Henriot (LEBS, Imagif, Gif-sur-Yvette, France) for cloning.

Author information

A.N., S.B., S.N., M.-H.K., C.G., T.C., L.T., A.M., E.C.-J. and M.Z. designed and performed experiments and analyzed data, R.P.S.O., G.G., and C.N. provided small molecules, R.R., M.Z. and J.C. conceived and supervised the research, and J.C. wrote the manuscript with input from the other authors.

Competing interests

A patent application (EP18305962.5) has been filed on the small molecules presented in this study for the treatment of cancers.

Correspondence to Mahel Zeghouf or Jacqueline Cherfils.

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Supplementary Table 1, Supplementary Figures 1–6

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Fig. 1: Bragsin2 affects Arf pathways in cells.
Fig. 2: Bragsin is a specific inhibitor of the ArfGEF BRAG2.
Fig. 3: Bragsin targets the PH domain of BRAG2 in vitro and in cells.
Fig. 4: Bragsin is a noncompetitive inhibitor of protein–membrane interactions.
Fig. 5: Bragsin affects breast cancer stem cells.