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Pharmacological folding chaperones act as allosteric ligands of Frizzled4

Abstract

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt–β-catenin pathway and for drug discovery.

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Figure 1: Rescue of Fz4-FEVR PM localization as biological platform toward the identification of Fz4 modulators.
Figure 2: FzM1 binding site and mechanism of action as folding chaperone.
Figure 3: Molecular mechanism behind FzM1 inhibition of Wnt pathway.
Figure 4: Effect of FzM1 treatment on U87MG and CaCo-2 cells.

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Acknowledgements

We thank R. Polishchuk (Telethon Institute of Genetics and Medicine) and B.A. Stanton (Dartmouth Center for the Environmental Health Sciences) for sharing ATP-7B and CFTR expression constructs. We thank J. Hausmann for the fruitful discussions. We thank R. Carmelo for the technical help and A. Ciogli and A. Sansone for MS spectral analyses. This work was financed by Progetti di Rilevante Interesse Nazionale (PRIN) 2012–2015 (grant no. 2012C5YJSK_003) to L.M., PRIN2010-2011 (grant no. 2010W7YRLZ_001) to R.S., Bando Futuro in Ricerca 2010 (grant no. RBFR10ZJQT) to G.L.R. and Fondazione Telethon (grant no. GGP14002) to S.B.

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S.F.G. and M.S. performed all of the experiments with the assistance of S.B. and M.M. for HDX, and S.D.M. for LC/MS. M.D. performed the experiments on CaCO-2 cells. D.S. performed confocal imaging. M.G., G.L.R., S.P., H.C., A.B. and L.M. collected and synthesized the compounds of the library and performed chemical characterization. M.S. planned the work and analyzed the results. M.S. wrote the manuscript with assistance from E.N., S.B. and R.S.

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Correspondence to Mariano Stornaiuolo.

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The authors declare no competing financial interests.

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Generoso, S., Giustiniano, M., La Regina, G. et al. Pharmacological folding chaperones act as allosteric ligands of Frizzled4. Nat Chem Biol 11, 280–286 (2015). https://doi.org/10.1038/nchembio.1770

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