by ecancer reporter Clare Sansom

Many tumour cells survive chemotherapy by evading apoptosis.

The protein BCL-2 and some other related proteins which are often over-expressed in solid tumours act to protect tumour cells from apoptosis; these are known as pro-survival proteins.

BCL-XL, or B-cell lymphoma extra large, is a pro-survival protein related to BCL-2 that is located on the mitochondrial membrane and that prevents the release of cytochrome C from the mitochondria, thus preventing caspase activation and apoptosis.

Very few small-molecule inhibitors of BCL-2 and its relatives have yet been discovered, mainly because of the difficulties inherent in designing molecules that target protein-protein interactions.

No specific inhibitor of a BCL-2 family protein has yet entered clinical trials.

Guillaume Lessene from the Walter and Eliza Hall Institute of Medical Research, Parkville, Australia and his co-workers sought to design a specific inhibitor of BCL-XL using structure-guided methods.

In vivo, BCL-XL is inhibited when a pro-apoptotic protein such as BIM, BAD or NOXA binds its BH3 domain – an amphipathic alpha helix – into a long, shallow, lipophilic groove on the BCL-XL surface.

In principle, a small molecule that can mimic the activity of the BH3 domain and bind to this groove would also inhibit BCL-XL; however, designing a molecule with properties like these is always challenging.

A library of about 100,000 compounds was screened against BCL-W, a close relative of BCL-XL, and hit compounds were validated in an assay that tested for disruption of BH3- BCL-XL binding.

This screen identified a series of lead compounds containing a benzothiazole-hydrazone core.

Structural studies of BH3–BCL protein binding have shown that the helical inhibitors bind tightly to four hydrophobic pockets that form a groove on the surface of pro-survival BCL family proteins; these have been named P1 to P4.

Lessene and his co-workers obtained the crystal structure of BCL-XL bound to a benzothiazole-hydrazone inhibitor, revealing particularly tight binding to the P2 pocket on the protein surface.

The researchers used insights gained from this structure and those of BCL-XL bound to similar compounds to design a small number of phenol analogues of their original compound series.

The most potent compound in this new series had an IC50 of 1.1 nM against BCL-XL; this compound, which had a distinctive binding mode, was named WEHI-539 and taken forward for further analysis.

This compound and some others in the series were found to be selective for BCL-XL over other family members, with WEHI-539 being about 500 times more potent against BCL-XL than against BCL-2.

Other pro-survival BCL family proteins bound WEHI-539 slightly more tightly than BCL-2 did, which was surprising because BCL-XL is considered to be more closely related to BCL-2 than to some other BCL proteins.

The researchers next studied the effect of WEHI-539 on cells, and found that it induced apoptosis in mouse embryonic fibroblasts that lacked expression of another pro-survival protein, MCL-1.

Other compounds in the series also promoted apoptosis in these cells, and their activity was found to correlate with the strength of their binding to BCL-XL.

WEHI-539 was found to induce apoptosis mediated through BAK, which is a pro-apoptotic member of the BCL-2 family, but not through the related protein BAX.

Platelets are known to depend on BCL-XL for their survival, and WEHI-539 was found to induce apoptosis in both mouse and human platelets; this could be inhibited by a broad-spectrum caspase inhibitor.

Taken together, these results suggest that the novel compound WEHI-539 is a potent and specific inhibitor of the pro-survival protein BCL-XL that can selectively induce apoptosis in cells that express the pro-apoptotic protein BAK.

Compounds that can inhibit BCL-XL selectively are likely to prove useful against tumours that have developed drug resistance through evasion of apoptosis, and should also provide important insights into the specific pro-apoptotic mechanism and roles of this protein.

Reference:

Lessene, G., Czabotar, P. E., Sleebs, B. E. and 18 others (2013). Structure-guided design of a selective BCL-XL inhibitor. Nature Chemical Biology, published online ahead of print 21 April 2013. doi:10.1038/nchembio.1246