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.

Designing Chimeric Molecules for Drug Discovery by Leveraging Chemical Biology

“…This method can be applied also to probes that bind proteins in a reversible fashion by the addition of a photoreactive group for UV detection of probe–protein interactions in cells (see photoaffinity labeling) . ABCP allows also off-targets detection in cells …”

“…166 ABCP allows also off-targets detection in cells. 167 Wong et al investigated the specificity for a series of ATPcompetitive bivalent kinase inhibitors targeting ABL1. 168 They proved the affinity and selectivity of bivalent inhibitors against Abl protein kinase with respect to other off-targets using dual functional chemical proteomics probes.…”

Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability

“…The large and shallow binding groove makes Mcl-1 intrinsically difficult to be targeted . Several groups have developed peptide Mcl-1 inhibitors. − However, during the last decade, medicinal chemists have contributed more to developing Mcl-1 small molecule inhibitors. − As shown in Figure , Zhang’s team has reported a series of phenalene-based Mcl-1 inhibitors, − of which the most potent 1 showed a K i of 5 nM . Indole-based Mcl-1 inhibitors have also been investigated in depth.…”

Proteolysis Targeting Chimeras for the Selective Degradation of Mcl-1/Bcl-2 Derived from Nonselective Target Binding Ligands

“…In this article, we constructed two series of PROTACs, H1 – H5 , and C1 – C6 , by introducing the E3 ligase cereblon (CRBN)-binding ligand pomalidomide to Bcl-2/Mcl-1 dual inhibitors S1-6 − and Nap-1 (Figure a), which exhibit a lower M w (approximately 500) and a lower binding affinity for both targets in the micromolar range, via alkyl and PEG linkers of different lengths. Among them, PROTACs C3 and C5 were observed to potently and selectively induce the ubiquitination and proteasomal degradation of Mcl-1 and Bcl-2 in cellulo, respectively, by hijacking a CRBN ubiquitin ligase to form a ternary complex with the target protein.…”