Abstract
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.
Highlights
Rational drug design represents an essential approach to optimize time and cost in drug discovery and development,[1] but it remains a challenging task
This study represents the first analysis of the metabolic stability of Proteolysis Targeting Chimeras (PROTACs) applied to a collection of compounds with large chemical variability
Metabolism assays were first performed in cryopreserved human hepatocytes that, containing all hepatic drug-metabolizing enzymes and cofactors at physiological levels, represent the “gold standard” even for the early screening of metabolic stability
Summary
Rational drug design represents an essential approach to optimize time and cost in drug discovery and development,[1] but it remains a challenging task. In the comfortable space of small molecules, the accuracy and sensitivity of the models are usually very good.[10−14] Nowadays, the chemical space of the drugs is quickly expanding, ranging from peptides or peptidomimetics[15] to Proteolysis Targeting Chimeras (PROTACs)[16−26] and their analogues.[27−29] PROTACs can be defined as hetero-bifunctional molecules that induce a ligand to bind with the protein of interest (POI), another ligand to recruit an E3 ubiquitin ligase, and a linker to concatenate the two ligands.[17] The formation of the ternary complex composed of the POI, the PROTAC, and the E3 ligase allows the E2 ubiquitin-conjugating enzyme to transfer ubiquitin to the surface of the POI, inducing its proteasomal-dependent degradation.[30] One of the main advantages of PROTACs is that they can degrade proteins regardless of their function, turning into druggable the “undruggable”, due to their innovative mechanism of action.[16] Degradation by PROTACs is a catalytic process, due to the dissociation of the complex after polyubiquitination of the POI, indicating that PROTACs can be recycled for successive rounds of degradation and used at reduced doses.[31] PROTACs represent an innovative class of compounds that overcome traditional limitations, opening a new therapeutic modality and, at the same time, breaking the rules used so far with the potential to revolutionize drug discovery. The entry in phase I clinical trial in 2019 of the first two oral PROTACs (ARV-110 and ARV-471) for the treatment of metastatic
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