Target identification is critically important for understanding
the mechanism of action of drugs. Here, we reported a new strategy
for deconvolution of drug targets (or off-targets) with photoaffinity
labeling chemoproteomics in combination with untargeted metabolomics
by using doxorubicin (DOX) as a model. The DOX-derived photoaffinity
probes were prepared and applied to capture DOX-interacting proteins
in living cells. The captured DOX-interacting proteins were then identified
by label-free quantitative proteomics. Totally, 151 significant proteins
were identified with high confidence (fold change >4, p-value < 0.005). The gene ontology enrichment analysis suggested
that the proteins were mainly involved in carbon metabolism, citrate
cycle, fatty acid metabolism, and metabolic pathways. Therefore, untargeted
metabolomics was applied to quantify the significantly altered metabolites
in cells upon drug treatment. The pathway enrichment analysis suggested
that DOX mainly interrupted with the processes of pyrimidine and purine
metabolism, carbon metabolism, methionine metabolism, and phosphatidylcholine
biosynthesis. Integrative analysis of chemoproteomics and metabolomics
indicated that adenosylhomocysteinase (AHCY) is a new target (off-target)
of DOX leading to the accumulation of S-adenosyl homocysteine. This
deduced DOX target was confirmed by the cellular thermal shift assay,
affinity competitive pull-down assay, biochemical assay, and siRNA
knock down experiments. Our result suggested that AHCY is the uncovered
off-target of DOX.
Profiling drug–protein interactions is critical
for understanding
a drug’s mechanism of action and predicting the possible adverse
side effects. However, to comprehensively profile drug–protein
interactions remains a challenge. To address this issue, we proposed
a strategy that integrates multiple mass spectrometry-based omics
analysis to provided global drug–protein interactions, including
physical interactions and functional interactions, with rapamycin
(Rap) as a model. Chemoproteomics profiling reveals 47 Rap binding
proteins including the known target protein FKBP12 with high confidence.
Gen Ontology enrichment analysis suggested that the Rap binding proteins
are implicated in several important cellular processes, such as DNA
replication, immunity, autophagy, programmed cell death, aging, transcription
modulation, vesicle-mediated transport, membrane organization, and
carbohydrate and nucleobase metabolic processes. The phosphoproteomics
profiling revealed 255 down-regulated and 150 up-regulated phosphoproteins
responding to Rap stimulation; they mainly involve the PI3K-Akt-mTORC1
signaling axis. Untargeted metabolomic profiling revealed 22 down-regulated
metabolites and 75 up-regulated metabolites responding to Rap stimulation;
they are mainly associated with the synthesis processes of pyrimidine
and purine. The integrative multiomics data analysis provides deep
insight into the drug–protein interactions and reveals Rap’s
complicated mechanism of action.
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