Abstract
Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein–protein interactions attractive therapeutic targets. Although these interactions have been extensively characterized biochemically, there is a paucity of tools to assess these interactions in cells. Here, we address this limitation by developing quantitative, high throughput microscopy assays to characterize Bcl-2 and BH3-only protein interactions in live cells. We use fluorescent proteins to label the interacting proteins of interest, enabling visualization and quantification of their mitochondria-localized interactions. Using tool compounds, we demonstrate the suitability of our assays to characterize the cellular activity of putative therapeutic molecules that target the interaction between pro-survival Bcl-2 and pro-death BH3-only proteins. In addition to the relevance of our assays for drug discovery, we anticipate that our work will contribute to an improved understanding of the mechanisms that regulate these important protein–protein interactions within the cell.
Highlights
Inhibition of the interactions between the Bcl-2 pro-survival proteins and their BH3-only counterparts is a popular therapeutic approach and several of the resulting BH3 mimetic inhibitors have entered clinical trials.[8,9] ABT-737 is a BH3 mimetic, small molecule inhibitor of BH3-only interactions with Bcl-2, Bcl-xL and Bcl-w that exemplifies this approach.[10]
We have developed microscopy-based assays that directly measure the interactions of Bcl-2 pro-survival with proapoptotic BH3-only proteins in live cells, preserving the interacting proteins in the mitochondrial membrane environment that is known to be critical for their activity.[15]
Consistent with previous reports,[18,19] we showed that Bcl-2 localized to the endoplasmic reticulum (ER), as determined by cotransfection of mCherry-Bcl-2 with eCFP-calreticulin (Supplementary Figure 1a)
Summary
Inhibition of the interactions between the Bcl-2 pro-survival proteins and their BH3-only counterparts is a popular therapeutic approach and several of the resulting BH3 mimetic inhibitors have entered clinical trials.[8,9] ABT-737 is a BH3 mimetic, small molecule inhibitor of BH3-only interactions with Bcl-2, Bcl-xL and Bcl-w that exemplifies this approach.[10] tools such as nuclear magnetic resonance-based screening along with fluorescence polarization and time resolved fluorescence resonance energy transfer measurements have proven invaluable for identification and characterization of the selectivity and potency of such inhibitors in a biochemical setting,[10,11] there is a lack of tools to evaluate the activity of such compounds in cells.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call