PROTACs meet chemical proteomics: Comprehensive understanding of time- and concentration-dependent protein kinase degradability (protACtion)

• Applicant: Dr. Stephanie Wilhelm
• Subject Area: Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525132892

Degrader molecules such as proteolysis targeting chimeras (PROTACs) have revolutionized chemical biology and drug discovery. In particular, the field of protein kinases has produced some successful candidates bearing this novel mode-of-action. Yet, synthesizing such molecules continues to be an inefficient trial-and-error process. The molecular design principles of degraders and the factors influencing the efficiency of intracellular degradation remain largely unknown and fundamental questions are still unsolved: When and why does binding lead, or not lead, to degradation? How does time and concentration influence kinase degradation and PROTAC selectivity? Does protein ubiquitination play a role in the selectivity of a degrader compound? Systematic and mechanistic data are scarce but may aid in shedding light on these questions; also most PROTAC studies miss important characteristics such as potency (DC50), maximal degradation (Dmax) or proteome-wide selectivity. In this project, mass-spectrometry based proteomics will be combined with classical molecular biology to address three main objectives: 1. to characterize the binding and degradation behavior of a kinase-focused PROTAC toolset; 2. to evaluate the time- and concentration dependency of kinome degradability; 3. to gain a mechanistic understanding of PROTAC-mediated target protein ubiquitination. Initially, chemoproteomic selectivity profiling will be performed with 64 multi-kinase targeting PROTACs. This assay will systematically characterize the proteome-wide binding affinities and selectivities of the PROTACs (work package 1). Concomitantly, time- and concentration-dependent cellular degradation induced by these molecules will be monitored by quantitative proteomics. This will address the key question of which PROTAC molecule degrades which kinase at what timepoint and concentration. The combination of the binding and degradation data will reveal which binding events actually lead to productive degradation, and how binding selectivity differs from degradation selectivity (work package 2). Based on these findings, the localization and timing of PROTAC-mediated ubiquitination will be investigated on selected examples (work package 3). Overall, the proposed project will provide fundamental insights into the selectivity and degradation of (protein kinase) PROTACs.

• DFG Programme: Research Grants