A novel in vitro permeability and efflux classification system for proteolysis targeting chimeras

Proteolysis targeting chimeras (PROTACs) have emerged as a promising modality over the past decade, with only a few advancing to clinical trials. As "beyond rule-of-five" compounds, PROTACs pose significant challenges for absorption, distribution, metabolism, and excretion, often necessitating iterative in vitro absorption, distribution, metabolism, and excretion assay optimization or reliance on in vivo pharmacokinetic studies, particularly to understand oral absorption. In this study, in vitro-in vivo correlations from clinically advanced PROTACs were leveraged to develop a novel classification system with results from optimized Madin-Darby Canine kidney cell line type 1 transfected with human multidrug resistance 1 and colorectal adenocarcinoma cell permeability and efflux assays that prioritizes compounds based on estimated mouse oral absorption. Receiver operating characteristic analysis was applied to define data-driven thresholds for effective permeability (P_eff) and efflux ratio (ER), enabling classification of compounds into mouse absorption-relevant buckets with good discriminative performance. In the Madin-Darby Canine kidney cell line type 1 transfected with human multidrug resistance 1 assay, both individual cutoffs for normalized P_eff (0.36) and normalized ER (1.72) and combined continuous score of log₁₀ normalized P_eff - 1.3∗log₁₀ normalized ER yielded reasonable classification of PROTACs with F_aF_g > 20%. In the colorectal adenocarcinoma assay, both individual cutoffs for normalized P_eff (0.021) and normalized ER (0.22) and combined continuous score of log₁₀ normalized P_eff - 1.2∗log₁₀ normalized ER yielded reasonable classification of PROTACs with F_aF_g > 20%. This approach aims to streamline in vitro workflows and minimize the need for extensive in vivo pharmacokinetic studies when evaluating novel PROTACs in preclinical discovery studies. SIGNIFICANT STATEMENT: This work establishes optimized assay conditions to assess permeability and a novel classification strategy that enables more reliable in vitro assessment of proteolysis targeting chimeras oral absorption potential. By streamlining workflows, it reduces the need for extensive in vivo pharmacokinetic studies when evaluating novel proteolysis targeting chimeras in drug discovery.