Applications of contemporary tools to measure plasma protein binding of targeted protein degraders

Assessing the fraction unbound in plasma (fu,p) for highly bound compounds (fu,p < 1%) is challenging, in part due to the compound-specific factors that can influence assay variability and performance. This study investigates human plasma protein binding obtained using the presaturation technique, and the results are compared with the standard equilibrium dialysis method and an orthogonal approach for an emerging class of compounds known as proteolysis targeting chimeras (PROTACs). Four exemplary PROTACs, cereblon-based ARV-110 and dTAG-13, and VHL-based ARV-771 and ERRα-degrader-3, were used to illustrate the challenges of obtaining accurate protein-binding results. The results reveal a protein-binding range from 0.03% to 3%. ARV-110 and dTAG-13 are highly bound to plasma proteins (fu,p ≤ 0.004). Under the standard equilibrium dialysis method, all compounds, except for ARV-771, had high variability or were unstable. Following presaturation and temperature adjustment (37 °C or 4 °C), fu,p values were obtained for ARV-771 (0.020, 95% confidence interval [CI]: 0.019-0.021), dTAG-13 (0.0028, 95% CI: 0.0024-0.0033), and ARV-110 (0.00042, 95% CI: 0.00033-0.00054). ERRα-degrader-3 remained unstable under all conditions. A comparative analysis of the binding values measured for ARV-110, dTAG-13, and ARV-771 by different techniques revealed good concordance (1.1-fold to 2.7-fold difference) between the methods evaluated. Utilizing a presaturation technique with low temperature is effective for measuring protein binding for challenging unstable degraders. Presaturation can reduce assay failures due to nonspecific binding to equipment by ensuring the system has reached equilibrium. This study provides valuable insights into the challenges encountered when measuring protein binding for lipophilic and highly bound PROTACs. SIGNIFICANCE STATEMENT: This paper details the evaluation of plasma protein binding for complex proteolysis targeting chimeras (PROTACs) molecules using contemporary binding assays. Given the higher protein-binding assay failure rates with challenging compounds such as PROTACs, this study outlines strategies to improve assay efficiency. The results show improved efficiency in measuring plasma protein binding with PROTACs and provide insights for enhancing widely accepted protein-binding methods currently employed in drug research.

Equilibrium dialysis; Nonspecific binding; PROTACs; Plasma protein binding; Presaturation.