Macromolecular Modeling Blog ™

A virtual docking screen discovers a small molecule activator of the pro-apoptotic protein BAX. This novel approach could lead to a ‘new generation’ of apoptotic modulators that directly activate BCL-2 killer proteins. Moreover – it’s cool!

BAX is one of the cell’s apoptosis executioners. When activated, it undergoes a conformational change from an inactive monomer to a deadly oligomer creating a pore in the mitochondrial membrane and starting the cascade of apoptosis.
Until not long ago it was thought that BAX was kept in check by protein-protein interactions with anti-apoptotic BCL-2 family proteins, and indeed small molecules and stapled peptides that disrupt these interactions are in different stages of pre-clinical and clinical investigations.
Recently, however, it was shown that BIM BH3 domain, an endogenous ‘death ligand’ – a helical peptide that disrupts said protein-protein interactions to drive the cell towards apoptosis, was also binding BAX directly at a secondary site that when engaged propels BAX into action.
This novel activation site, discovered by the Walensky lab, inspired them to search for small molecules selective activators of BAX. Using BAX conformations from molecular dynamics simulations, Gavathiotis et al. virtually screened (using Glide 4.0) 750,000 small molecules (commercially available, as a subset of ZINC). Out of the top 2000 scoring molecules from docking to both open and closed receptor conformations, they have selected 100 compounds for experimental validation.
Using a fluorescence polarization based competition assay (displacing a FITC-labeled stabilized BIM peptide) the authors found that 11 out of 78 compounds (that did not have auto-fluorescence) achieved >55% displacement at a concentration of 100uM. The most effective competitor, BAM7 (see figure) had an IC50 of 3.3uM – impressive when considering it is displacing a helix 6 times its size.

Direct and selective small molecule activation of BAX

The authors continue with a thorough characterization of BAM7. BAM, by the way, is short for “BAX activator molecule”. Using NMR they verified that BAM7 indeed binds to the secondary activation domain and not to the canonical BH3 binding groove. An additional (albeit weaker) support was the testing of analogs of BAM7 predicted by the docking pose to clash with the receptor as negative controls, that indeed showed weaker activities. BAM7 was also shown to be selective to BAX and did not show binding to BAK (a close homolog) at up to 50uM.
As for function, BAM7 was shown to trigger in-vitro BAX oligomerization (by size exclusion chromatography) BAX-mediated pore-formation (by liposomal assays that track release of a trapped fluorophore) and finally BAX mediated cell death (in BAK-less maurine embryonic fibroblasts).
This is a very elegant body of work, and moreover, a nice win for virtual screening. The authors note it is the first example of the discovery of a ‘gain of function’ small molecule. While I’m not sure there weren’t prior examples (if you know of any, let us know in the comments) ignoring ligand operated GPCR’s etc. this is still an impressive campaign and will enable a whole new level of investigation of BAX mediated apoptosis.

Gavathiotis E, Reyna DE, Bellairs JA, Leshchiner ES, & Walensky LD (2012). Direct and selective small-molecule activation of proapoptotic BAX. Nature chemical biology PMID: 22634637

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