Efficacy and specificity of inhibitors of BCL-2 family protein interactions assessed by affinity measurements in live cells

Abstract

Cytoplasmic and membrane-bound BCL-2 family proteins regulate apoptosis, a form of programmed cell death, via dozens of binary protein interactions confounding measurement of the effects of inhibitors in live cells. In cancer, apoptosis is frequently dysregulated, and cell survival depends on antiapoptotic proteins binding to and inhibiting proapoptotic BH3 proteins. The clinical success of BH3 mimetic inhibitors of antiapoptotic proteins has spawned major efforts by the pharmaceutical industry to develop molecules with different specificities and higher affinities. Here, quantitative fast fluorescence lifetime imaging microscopy enabled comparison of BH3 mimetic drugs in trials and preclinical development by measuring drug effects on binding affinities of interacting protein pairs in live cells. Both selectivity and efficacy were assessed for 15 inhibitors of four antiapoptotic proteins for each of six BH3 protein ligands. While many drugs target the designed interaction, most also have unexpected selectivity and poor efficacy in cells.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins

“…1 D ). As expected, binding to the wildtype V BCL-XL and V BCL-2, known to bind BIK in live cells ( 52 ), resulted in binding curves with higher FRET efficiency that also saturate when the acceptor:donor ratio is greater than 1. In contrast, primarily collisions were detected for the negative binding control, C BIK-L61G.…”

“…Further delineation of how and where these interactions occur is an important step for understanding BCL-2 family protein function. When expressed in live cells, BIK with an N-terminally fused fluorescent protein remains functional ( 52 ). We recently demonstrated that in live cells a fluorescent protein fusion of BIK binds to BCL-2, BCL-XL, and BCL-W with high affinity and MCL-1 with lower affinity ( 52 ).…”

“…When expressed in live cells, BIK with an N-terminally fused fluorescent protein remains functional ( 52 ). We recently demonstrated that in live cells a fluorescent protein fusion of BIK binds to BCL-2, BCL-XL, and BCL-W with high affinity and MCL-1 with lower affinity ( 52 ). Here, we investigate where these interactions occur in cells.…”

“…We recently published a quantitative Fast FLIM–FRET (qF 3 ) method, which enables live-cell measurements of apparent K d values ( 52 ). However, the expression levels for the fluorescence protein–tagged query proteins required for qF 3 exceed the actual K d s of Bcl-2 family interactions.…”

“…However, the expression levels for the fluorescence protein–tagged query proteins required for qF 3 exceed the actual K d s of Bcl-2 family interactions. Thus, the apparent K d s measured by qF 3 can be most effectively used to measure the effects of point mutations and small molecules on binding interactions ( 52 ). For the studies reported here, we used a simpler version of FLIM–FRET that enables determining relative binding efficiencies ( 62 , 63 ).…”