Navigating Drug-Protein Interactions with Site-Specific Precision Using Phosphonate Affinity Tags

Proteins are fundamental to cellular function, influencing various aspects of cellular behavior and ultimately defining the phenotype of cells and organisms. In diseases like cancer, aberrant protein behavior, such as mutations or overexpression, plays a significant role in promoting tumor growth. Analyzing proteins within tumors can offer insights crucial for diagnosis, prognosis, treatment, and drug development. Liquid chromatography-mass spectrometry (LC-MS)-based proteomics has revolutionized our ability to study proteins, allowing for the analysis of thousands of different proteins in biological samples rapidly. This technique has proven instrumental in advancing our understanding of diseases, including cancer. LC-MS-based proteomics is versatile and can be tailored to address specific scientific questions, such as the specific analysis of newly synthesized proteins during perturbations in biological systems. One emerging area is chemoproteomics, which focuses on understanding drug action by identifying the proteins targeted by drugs. Chemoproteomics techniques, such as activity-based protein profiling (ABPP), offer a means to monitor both on- and off-target interactions of small molecule drugs. This approach accelerates our understanding of disease mechanisms and aids in drug development. The thesis introduces a novel chemoproteomic technique called PhosID-ABPP, which enables the precise analysis of drug-protein interactions at a site-specific level using phosphonate affinity tags. These tags, initially developed for analyzing protein synthesis, allow for the selective enrichment of peptides bound to an ABP, thereby facilitating the identification of drug binding sites. By applying PhosID-ABPP, the thesis enhances our understanding of drug action through various investigations. Chapter 1 provides an overview of mass spectrometry-based proteomics and the role of chemoproteomic strategies in drug development. ABPP, as a chemoproteomic technique, offers a means to assess drug-protein interactions comprehensively. Chapter 2 reviews current methods in LC-MS-based proteomics for analyzing newly synthesized proteins. While traditional proteomic methods efficiently assess protein abundance, specialized techniques are required to detect and quantify rapid alterations in protein synthesis. Chapter 3 describes the development of PhosID-ABPP, demonstrating its efficacy in identifying drug binding sites with high precision. The strategy is applied to an Afatinib derivative, leading to the identification and quantification of over 500 binding sites in intact cells and cellular lysates. Chapter 4 further refines PhosID-ABPP, utilizing advanced mass spectrometry techniques for dual dose-dependent analysis of different ABPs within a single cellular proteome. This comparative analysis reveals ABP-specific binding preferences and off-target reactivity profiles, providing valuable insights into drug engagement. Chapter 5 showcases the application of PhosID-ABPP for site-specific kinase profiling, to elucidate on- and off-targets of protein kinase inhibitors. This approach enables the screening of over 100 kinase active sites in a single LC-MS run, aiding in the identification of inhibitor targets and off-targets. In summary, the thesis underscores the importance of chemoproteomic techniques, particularly PhosID-ABPP, in elucidating drug-protein interactions. The application of PhosID-ABPP offers a precise and comprehensive approach to navigating drug targets at the amino acid level, thereby advancing drug development efforts.