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

Abstract

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.

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Acknowledgements

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.