In the field of drug research and development, traditional small-molecule modification or immobilization strategies applied to drug target screening have limitations, such as easily altering the pharmacodynamic properties of compounds and difficulty in capturing weak interactions between drugs and targets. To address this issue, this study established a novel drug target screening method based on copper ion-induced protein precipitation. This method requires no labeling of drugs, maximally retains the original properties of compounds, and captures drug-target interactions with high sensitivity. The study first used methotrexate (MTX) and its target protein dihydrofolate reductase (DHFR) as a model, and verified through Western blot that copper ions can induce the precipitation of DHFR. Furthermore, the specific binding of MTX to DHFR changes the conformational stability of DHFR and reduces its precipitation amount under the action of copper ions, which confirms the feasibility of this method. Subsequently, the method was extended to the research of other drugs: for SNS-032, a selective cyclin-dependent kinase (CDK) inhibitor, the study not only verified its interaction with HSP90 but also screened out potential off-target proteins, such as NME2 through dimethyl labeling mass spectrometry. This method provides reliable technical support for the efficient screening and verification of drug targets, and is of great significance for promoting drug research and development as well as disease treatment research.
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