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Abstract
MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments.
Highlights
MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities
We have confirmed that, as an imaging probe, MLN-2 performed at least as well as the probe developed by Weissleder et al Together with another ‘‘minimalist’’ probe PU-1 which targets CDK127, we show, for the first time, small molecule-based multiplex bioimaging could be conducted for simultaneous in situ monitoring of different cell-cycle regulating protein kinases
We showed the terminal alkyne-containing diazirine-based probe, MLN-2, could be used to image endogenous AKA activity at least as well as a TCO-based probe, MLN-3
Summary
MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. In order to study potential cellular off-targets of a kinase inhibitor, including MLN8054, recent efforts have focused on high-throughput screening (HTS) using large panels of recombinant kinases as well as mass spectrometry (MS)-based, proteome-wide chemical profiling methods[11,12,13] Most of these methods, could not directly detect kinase-drug interaction in situ (i.e. in living cells, not lysates)[14]. Small molecule-based bioimaging approaches have in recent years become increasingly available for in situ monitoring of a variety of proteins including enzymes[31], but chemical proteomic strategies capable of simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, are still quite rare[27,28]. We have successfully discovered novel AKA interactions which are further validated by in situ immunoprecipitation (IP) experiments
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