Intracellular exposure and membrane interactions of bifunctional protein degraders and small molecules

Abstract

Proteolysis-targeting chimeras (PROTACs) have emerged as a promising therapeutic modality, capable of inducing potent degradation of disease-driving proteins. However, their complex physicochemical properties challenge conventional assumptions regarding cellular permeability and druglikeness. While most research focuses on improving degradation efficiency, important knowledge gaps remain regarding the links between efficiency and intracellular exposure, which is likely to influence the pharmacological response. The overall aim was therefore to investigate the molecular mechanisms governing intracellular exposure of PROTACs, using computational and experimental approaches, including molecular dynamics simulations, passive cell permeability measurements, determination of intracellular total and unbound exposure, target engagement assays, and binding kinetics. Two major classes of PROTACs, recruiting either cereblon or von Hippel- Lindau (VHL), displayed distinct physicochemical and pharmacological profiles, with cereblon-binding PROTACs consistently showing higher target engagement and intracellular exposures. Computational analyses suggested that VHL-binding PROTACs can be limited by poor solubility and extensive non-specific binding to cell membranes. Furthermore, differences in observed target engagement were largely explained by intracellular unbound PROTAC exposure, and different extraction methods used to quantify intracellular exposure were found to sample different intracellular compound pools. Collectively, the findings in this thesis improve the understanding of how physicochemical properties influence intracellular exposure of PROTACs. The work further highlights important methodological considerations when quantifying intracellular exposure and provides insights that may support the rational design of PROTACs with improved intracellular availability.