Identifying Protein–Drug Interactions in Cell Lysates Using Histidine Hydrogen Deuterium Exchange

Identifying
the targets of a drug is critical to understand the
mechanism of action and predicts possible side effects. The conventional
approach is capturing interacting proteins by affinity purification.
However, it requires drugs to be immobilized to a solid support or
derivatized with chemical moieties used for pulling down interacting
proteins. Such covalent modifications to drugs may mask a critical
recognition site for or alter the binding affinity to their targets.
To overcome the drawback, several methods that do not require covalent
modifications to drugs have been developed. These methods identify
targets by detecting proteins whose thermodynamic stability is enhanced
in the presence of drugs. Although the utility of these methods has
been demonstrated, the difficulty in identifying low abundant targets
is the common problem of these methods. We have developed a new target
identification method that increases the likelihood of identifying
low abundant targets. The method uses histidine-hydrogen deuterium
exchange (His-HDX) as a readout technique to probe the changes in
protein stability induced by drugs. The workflow involves incubating
cell lysates in various concentrations of a protein denaturant in
the presence and absence of a drug in D2O followed by digestion
of the proteins, enrichment of His-containing peptides, and analysis
of the enriched His-peptides by liquid chromatography–tandem
mass spectrometry (LC–MS/MS). The developed method was successfully
applied to identify the interaction between endogenously expressed
MAPK14 and its inhibitor in HEK293 cell lysates. The implementation
of selective enrichment of histidine-containing peptides in the workflow
was a key that enabled identifying the MAPK14–inhibitor interaction.