Recent advances in protein precipitation-based methods for drug-target screening

Abstract

Drug targets are biological macromolecules that bind drug molecules in vivo. Therefore, the system-wide identification of drug targets plays a vital role in fully understanding the mechanism of drug action, efficacy, and side effects. The unbiased screening of drug targets may accelerate the process of drug discovery and candidate screening. Mass spectrometry is a key tool for large-scale protein identification and accurate quantification owing to its high acquisition speed, resolution, and sensitivity. Mass spectrometry-based proteomics has been widely used for drug-target screening. It can systematically identify the protein-target landscape of a drug and elucidate drug-protein interactions. Commonly used drug-target characterization methods, such as labeling-based affinity enrichment, require the chemical derivatization of drug molecules, which is not only time-consuming but may also affect the affinity of the drug towards its targets. Furthermore, the spatial effects of the derivatization groups may block interactions between the drug and its targets. Considering the disadvantages of affinity-enrichment methods, strategies that do not require chemical derivatization have received widespread attention. Proteins may undergo denaturation, unfolding, and precipitation under different conditions such as high temperatures, extreme pH, denaturants, and mechanical stress. Binding to small-molecule drugs may alter the folding balance of target proteins. The conformational stability of target proteins can be stabilized by binding with drugs, and protein-drug complexes are more resistant than free proteins to the precipitation induced by different conditions. Based on this mechanism, various large-scale drug-target identification methods using protein precipitation have been developed by combining proteomics and mass spectrometry analysis, including thermal proteome profiling and solvent-, mechanical stress-, and pH-induced protein precipitation. These methods have been successfully applied to the characterization of small-molecule drug targets. In this review, we describe the protein precipitation-based methods used for the high-throughput discovery of drug targets and elucidation of the interactions between drugs and proteins in the past decade. We also summarize the characteristics of each method and discuss their application potential in drug-efficacy evaluation and drug discovery.