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.