Most drugs exert pharmacological effects through interaction with their target proteins.Therefore,drug target identification is a crucial step towards the understanding of the mechanism of drug action.It is also imperative to study the pharmacodynamics of a known drug,with an aim to discover the potentially unrevealed actions and thus refine its future clinical applications.Currently,drug-target identification is either through in vitro affinity chromatography-based approaches or in vivo activity-based protein profiling(ABPP)approaches.However,these approaches generally face difficulties discriminating specific drug targets from non-specific ones.To address this issue,we have come up with a strategy by coupling iTRAQTM(isobaric tags for relative and absolute quantitation)quantitative proteomics approach with clickable ABPP,to specifically and compre hensively identify drug targets in live cells.Using this approach,we identified the protein targets of andrographolide,a natural product with known anti-inflammation and anti-cancer effects,in live cancer cells.The identified target list not only confirmed the known functions of the drug but also revealed its potential novel application as a tumor metastasis inhibitor.We have also used this strategy,combining with a cleavable probe to identify the protein targets of aspirin and its binding sites.Our results revealed the roles of aspirin ininhibition of protein synthesis and induction of autophagy,which have been functionally validated.Our strategy is widely applicable to the identification of protein targets of covalent drugs.
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