In Vitro ADME Profiling of PROTACs: Successes, Challenges, and Lessons Learned from Analysis of Clinical PROTACs from a Diverse Physicochemical Space

Established in vitro assays for ADME properties often struggle with compounds outside of the rule-of-5 space such as PROTACs. These bifunctional molecules, due to high lipophilicity and molecular weight, frequently exhibit low solubility, high nonspecific binding, and poor translation of assay data, necessitating empirical validation. This study evaluates the performance of standard ADME screening assays exemplified by 10 PROTACs in clinical development, with varying lipophilicity, in the context of the AstraZeneca data set. Assessment of in vitro metabolic clearance, plasma protein and incubation binding, blood-to-plasma ratio, Caco-2 permeability, and efflux was performed. Additionally, due to the poor performance of the aqueous solubility assay, the impact of biorelevant media was investigated on solubility. The primary objective was to evaluate the suitability of traditional ADME assays for PROTACs and understand the lipophilicity limits. This work aims to guide discovery teams on assay reliability and performance and inform decision-making regarding compound progression in this challenging area.