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

Abstract

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Recently, our group has developed a lead compound, 9im, capable of displaying potent and specific inhibition of AXL. To further identify the cellular on/off targets, in this study, competitive affinity-based proteome profiling was carried out, leading to the discovery of several unknown cellular targets such as BCAP31, LPCAT3, POR, TM9SF3, SCCPDH and CANX. In addition, trans-cyclooctene (TCO) and acedan-containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of AXL in various biosystems.

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

“…[41] To further uncover its potential on/off-targets, an affinity-based probe was synthesized aiming to provide useful information for subsequent structure optimization and understanding of the drug action (Figure 4, AX-1). [42] Chemoproteomics experiments were then carriedo ut in cells and in tumor tissues. After rational analysis,4 4p rotein hits from live cells and 20 from tumor tissues including RYK, PCK, ATP1A3, EIF4A1 and Ptpm were identified.…”

“…We recently reported a lead compound (9im) showing highly potent and specific inhibition of Axl . To further uncover its potential on/off‐targets, an affinity‐based probe was synthesized aiming to provide useful information for subsequent structure optimization and understanding of the drug action (Figure , AX‐1) . Chemoproteomics experiments were then carried out in cells and in tumor tissues.…”

Quantitative Proteomics Reveals Cellular Off-Targets of a DDR1 Inhibitor

“…The DDR1 was not shown in the protein list, which could be accounted for by low solubility of DDR1, a membrane-located protein, in lysis buffer. 3 The top hit, Cathepsin D (CTSD), plays crucial roles in metabolic degradation of intracellular proteins and is also involved in antigen processor activation and regulation of programmed cell death. GPR107 was proposed as a receptor for neuronostatin and may play an important role in the central control of cardiovascular function.…”

“…As a result, comprehensive knowledge of the full target spectrum of lead compounds can provide a molecular basis with which to evaluate the possible side effects. It can guide subsequent structural optimization and may offer novel therapeutic applications . Over the past decades, affinity-based protein profiling (A f BP) coupled with bioimaging has been widely used in the investigation of genuine drug target engagement in native cellular environments. − To facilitate the synthesis of high-quality probes, a suite of bioorthogonal-handle containing linkers were developed for reversible and irreversible inhibitors, respectively, which enable simultaneous proteome profiling and bioimaging studies to improve the accuracy in target identification. − …”

“…These protein hits were also detected in enrichment experiments (DR-1/DMSO), indicating their high reliability (Figure B). The DDR1 was not shown in the protein list, which could be accounted for by low solubility of DDR1, a membrane-located protein, in lysis buffer . The top hit, Cathepsin D (CTSD), plays crucial roles in metabolic degradation of intracellular proteins and is also involved in antigen processor activation and regulation of programmed cell death.…”