Understanding the structure-activity relationship (SAR) and PK of PROTAC linkers

Proteolysis targeting chimeras (PROTACs) are a relatively new class of heterobifunctional molecules that have been identified as a potential anti-cancer therapy. They work by bringing a protein of interest (POI) and E3 ligase proteins in proximity and triggering ubiquitination and subsequent targeted degradation of the POI via the proteasome. PROTACs are comprised of a ligand to both the POI and the E3 ligase connected by a linker. The linker is not a directly functional part of the molecule and so many different varieties exist, with varying lengths and physical properties.

However, limited research has been done on whether the linker has an indirect effect on the function and pharmacokinetics of the PROTAC. This project looks to explore the effect of varying the linker on metabolism and activity, with an aliphatic and an ethylene glycol linker tested for their stability in human blood plasma, human liver microsomes and human hepatocytes, with metabolite identification also performed.

This project shows that changing the linker does have a minor effect on stability, with different linkers performing better in different matrices. Stability of the PROTACs was higher in plasma than in HLMs, with anomalous results in hepatocytes. Metabolite identification showed that the likely route of metabolism in plasma was via hydrolysis, and via oxidation in HLMs. However, in HLMs the POI ligand was the primary point of metabolism, meaning that conclusions about linker metabolism couldn't be properly inferred.