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Monoamine oxidase A (MAO-A) plays a critical role in the development of glioma and other neurological disorders. Specific analysis of MAO-A activities and its drug interactions in intact tissue is important for biological and pharmacological research, but highly challenging with current chemical tools. Fluorogenic-inhibitor-based probes offer improved selectivity, sensitivity, and effectiveness to image and profile endogenous targets in an activity-based manner from mammalian cells, which are however rare. Herein, we report HD1 as the first fluorogenic-inhibitor-based probe that can selectively label endogenous MAO-A from various mammalian cells and clinical tissues. The probe was delicately designed based on N-propargyl tetrahydropyridine, a small MAO-A-specific fluorogenic and inhibitory war-head, so that the probe becomes fluorescent upon in situ enzymatic oxidation and covalent labeling of MAO-A. With the excellent binding affinity (vin itro Ki = 285 nM) and fluorogenic properties, HD1 offers a promising approach to simultaneously image endogenous MAO-A activities by super-resolution fluorescence microscopy and study its drug interactions by subsequent activity-based protein profiling, in both live cells and human glioma tissues. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]