Comprehensive Profiling of Rapamycin Interacting Proteins with Multiple Mass Spectrometry-Based Omics Techniques

Area of Science:

• Pharmacology and Chemical Biology
• Proteomics and Metabolomics
• Systems Biology

Background:

• Comprehensive profiling of drug-protein interactions is essential for understanding drug mechanisms and predicting side effects.
• Current methods face challenges in comprehensively mapping these interactions.
• Rapamycin (Rap) serves as a model compound to investigate drug-protein interactions.

Purpose of the Study:

• To develop and validate an integrated multi-omics strategy for global drug-protein interaction profiling.
• To elucidate the physical and functional interactions of rapamycin (Rap) using this novel approach.
• To gain deeper insights into the complex mechanism of action of Rap.

Main Methods:

• Integration of multiple mass spectrometry-based omics analyses: chemoproteomics, phosphoproteomics, and untargeted metabolomics.
• Chemoproteomics was employed to identify Rap binding proteins.
• Gene Ontology enrichment, phosphoproteomics, and metabolomics were used to analyze functional consequences.

Main Results:

• Chemoproteomics identified 47 Rap binding proteins, including the known target FKBP12.
• Gene Ontology analysis linked Rap binding proteins to key cellular processes like DNA replication, immunity, and autophagy.
• Phosphoproteomics revealed significant changes in phosphoproteins involved in the PI3K-Akt-mTORC1 signaling pathway, while metabolomics indicated alterations in pyrimidine and purine synthesis.

Conclusions:

• The integrated multi-omics strategy provides a comprehensive view of drug-protein interactions.
• This approach successfully elucidated the complex molecular interactions and cellular responses to Rap.
• The findings offer deep insights into Rap's mechanism of action and its involvement in various biological processes.