Proteome‐Wide Identification of On‐ and Off‐Targets of Bcl‐2 Inhibitors in Native Biological Systems by Using Affinity‐Based Probes (AfBPs)

Abstract Selective inhibition of proteins of the Bcl‐2 family by small‐molecule inhibitors is a promising new approach in drug discovery. However, information about how these molecules interact with their cellular targets (on‐ and off‐) is highly limited. We have designed and synthesized photoreactive and “clickable” affinity‐based probes (AfBPs)—Nap‐2 and Nap‐5—by introducing photo‐crosslinkers onto Nap‐1, a fluorescent derivative of small‐molecule Bcl‐2 inhibitor S1‐6. The resulting trifunctional probes Nap‐2 and Nap‐5 can enrich, visualize, and enable the identification of cellular on‐ and off‐targets of Bcl‐2 inhibitors both in vitro and in situ. Tubulin was validated as an off‐target of Bcl‐2 inhibitors (Nap‐1 and S1‐6) by large‐scale cell‐based proteome profiling and pull‐down/western blotting (PD/WB) with Nap‐2 and Nap‐5. It was preliminarily illustrated to be a BH3‐containing protein because some well‐known Bcl‐2 inhibitors can block the labeling of tubulin by Nap‐2.