Hetero-bifunctional PROteolysis TArgeting Chimeras (PROTACs) represent a new emerging class of small molecules designed to induce polyubiquitylation and proteasomal-dependent degradation of a target protein. Despite the increasing number of publications about the synthesis, biological evaluation, and mechanism of action of PROTACs, the characterization of the pharmacokinetic properties of this class of compounds is still minimal. Here, we report a study on the metabolism of a series of 40 PROTACs in cryopreserved human hepatocytes at multiple time points. Our results indicated that the metabolism of PROTACs could not be predicted from that of their constituent ligands. Their linkers’ chemical nature and length resulted in playing a major role in the PROTACs’ liability. A subset of compounds was also tested for metabolism by human cytochrome P450 3A4 (CYP3A4) and human aldehyde oxidase (hAOX) for more in-depth data interpretation, and both enzymes resulted in active PROTAC metabolism.

Significance: The metabolism of xenobiotics is a critical aspect of drug discovery; nowadays, aldehyde oxidase (AOX) has emerged as a key metabolic enzyme having a pivotal role in the failures of several clinical candidates. The lack of homogenous data on possible substrates and not substrates of this enzyme represents a serious limit for the development of an in silico model for metabolism prediction. Here, we present a database of 270 chemically diverse compounds containing aza-aromatic and/or amide moieties (susceptible to human AOX), experimentally tested in vitro. The results herein reported should be useful in the development of a reliable prediction model, which should be of wide interest in chemistry, biology, biotechnology, and medicine.