Cell‐ and Tissue‐Based Proteome Profiling and Dual Imaging of Apoptosis Markers with Probes Derived from Venetoclax and Idasanutlin

Abstract

Venetoclax (ABT-199) and idasanutlin (RG7388) are efficient anticancer drugs targeting two essential apoptosis markers, Bcl-2 and MDM2, respectively. Recent studies have shown that the combination of these two drugs leads to remarkable enhancement of anticancer efficacy, both in vitro and in vivo. In an attempt to disclose the relationships of their protein targets, competitive affinity-based proteome profiling coupled with bioimaging was employed to characterize their protein targets in the same cancer cell line and tumor tissue. A series of protein hits, including ITPR1, GSR, RER1, PDIA3, Apoa1, and Tnfrsf17 were simultaneously identified by pull-down/LC-MS/MS with the two sets of affinity-based probes. Dual imaging was successfully carried out, with the simultaneous detection of Bcl-2 and MDM2 expression in various cancer cells. This could facilitate the novel diagnostic and therapeutic strategies of dual targeting of Bcl-2/MDM2.

Cell‐ and Tissue‐Based Proteome Profiling and Bioimaging with Probes Derived from a Potent AXL Kinase Inhibitor

“…Importantly, these approaches can be integrated with phenotypic screening as reported by our group and others, to accelerate the mechanistic characterization of screening hits. Moreover, we have endeavored to apply these approaches to study the protein targets of combination drugs, which provided useful information in understanding their synergistic effects . Meanwhile, a suite of fluorescent probes was developed to image cancer/apoptosis markers .…”

“…[12] Meanwhile, as uite of fluorescent probesw as developed to image cancer/apoptosis markers. [10,12] These effortsh ave improved the applications of AfBP and bioimaging approaches in drug discovery and cancer-related diagnoses.…”

“…Subsequently,b ioimaging experiments were carried out to assess whether the affinity-based probe (AX-1)c an track the cellular distribution of the parent inhibitor.M DA-MB-231c ells were treated with AX-1,a nd this was followed by UV irradiation (20 mino ni ce) to initiate photo-crosslinking. Subsequently,t he cells were fixed, permeabilized, and clicked with TAMRA-N 3 under previously established optimal click chemistryc onditions, [7][8][9]12] and then were imaged. Strongf luorescence signals were observed, mainly in the cell membranes and cytosol of probe-treated cells( Figure2Da nd S1 A).…”

Applications of Activity‐Based Protein Profiling (ABPP) and Bioimaging in Drug Discovery

“…[43] Recent work also demonstratedt hat ABPP can be appliedi n the study of underlying mechanism of combination drugs. [44] Venetoclax (ABT-199) and idasautlin (RG7388) are two first-inclass anticancer drugs targeting two essential apoptosis markers, Bcl2a nd MDM2. Recently,s everals tudies have shown that the combination of these drugs leads to remarkable synergistic anticancer activity.I no rder to understandt he underlying mechanism of this therapeutic strategy ande xplore their cellular off-targets, as uite of probes weres ynthesized by introducing am inimalist photocrosslinker or fluorophore directly into the original compounds (Figure 4, AB1/2 and RG-1), enabling simultaneous ABPP and bioimaging studies in cancerc ells and tumor tissues.As eries of protein hits including ITPR1, GSR, RER1, PDIA3,A poa1 and Tnfrs17 were successfully identified by pull-down/LC-MS/MS and confirmed by pull-down/WB and enzymatic assays,w hich can identify potentialo ff-targets and the reasons underlying synergistic effects.…”

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

“…So far, probes with PGs or “minimal” linkers, so called affinity‐based probes (AfBPs), have succeeded in labeling, enriching, and enabling the identification of many noncovalently interacting enzyme targets, including methyltransferases and kinases . However, in contrast with the success associated with creating many kinase‐labeling probes, the probes for profiling protein–protein‐interaction (PPI) targets are rather limited …”