Proteolysis
targeting chimera (PROTAC) recruits an E3 ligase to
a target protein to induce its ubiquitination and subsequent degradation.
We reported success in the development of two PROTACs (C3 and C5) that potently and selectively induce the degradation
of Mcl-1 and Bcl-2 (DC50 = 0.7 and 3.0 μM), respectively,
by introducing the E3 ligase cereblon-binding ligand pomalidomide
to Mcl-1/Bcl-2 dual inhibitors S1-6 and Nap-1 with micromolar-range affinity. C3-induced Mcl-1 ubiquitination
translated into much more lethality in Mcl-1-dependent H23 cells than
the most potent Mcl-1 occupancy-based inhibitor A-1210477 with nanomolar-range affinity. Moreover, structure–activity
relationship analysis and molecular dynamic simulations discovered
the structural basis for turning nonselective or promiscuous Bcl-2
family ligands into selective PROTACs. C3 and C5 exhibited reversible depletion in living cells, which provides a
new potent toolkit for gain-of-function studies to probe the dynamic
roles of Bcl-2 and Mcl-1 in apoptosis networks.
A novel 1-oxo-1H-phenalene-2,3-dicarbonitrile (OPD)-based fluorescent probe was developed to sense and image GSH in HeLa cells, different imatinib-resistant K562 cells, D. magna and zebrafish embryos.
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.
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