Discovery of AHCY as an Off-Target of Doxorubicin by Integrative Analysis of Photoaffinity Labeling Chemoproteomics and Untargeted Metabolomics

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.