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Direct and selective small-molecule activation of proapoptotic BAX

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.

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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.

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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.

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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.

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Correspondence to Evripidis Gavathiotis or Loren D Walensky.

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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

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