Assays for Measuring the Cell Permeability of Proteolysis-Targeting Chimeras (PROTACs): Performance, Correlations, Applicability and Recommendations

Most Proteolysis-Targeting Chimeras (PROTACs) are beyond-Rule-of-Five compounds, and optimizing their ability to cross cell membranes is a central topic in PROTAC therapeutic research. In this study, we systematically investigate the performance and assay correlations of PROTAC cell permeability assays and the chemical space in which these assays provide reliable results. Our work builds on a comprehensive data set of 3271 PROTACs spanning diverse chemical and target spaces, providing unprecedented coverage of PROTAC cell permeability data. These compounds were measured in up to three different types of assays: the NanoBRET E3 ligase target engagement assay, the Caco-2 assay, and a P-glycoprotein (P-gp) efflux liability assay. We find a strong correlation between readouts from the NanoBRET E3 ligase target engagement assay (availability shift) and those from the Caco-2 assay (apparent intrinsic permeability, basolateral-to-apical permeability, and efflux ratio), while P-gp liability derived from the P-gp assay cannot easily be translated to the Caco-2 efflux ratio. In the process, we also investigate the impact of physicochemical properties included in the Rule-of-Five on the applicability of the assays. Based on these findings, we derive guidelines and data-driven recommendations for the use of cell permeability assays in PROTAC optimization. While this study focuses on PROTACs, some of the observations may be relevant to other modalities with beyond-Rule-of-Five physicochemical properties.