Inhibition of equilibrative nucleoside transporters 1 and 2 by proteolysis-targeting chimeras (PROTACs).

Proteolysis-targeting chimeras (PROTACs) are gathering considerable interest due to their ability to address previously undruggable targets. We were keen to understand the potential for these very large molecules to interact with transporters that may influence absorption, distribution, metabolism, and excretion or toxicity properties and to what extent this may be predictable using machine learning models. Consequently, we tested a set of PROTACs against several human drug transporters, namely the equilibrative nucleoside (ENT) family transporters ENT1 and ENT2, which have been directly implicated in the uptake of anticancer or antiviral drugs into target cells. We describe the dramatic inhibition observed for ENT1 and ENT2 but not for the unrelated transporter organic anion transporter 4. In addition, we report dose-response relationships for ENT1 to show some PROTACs are nanomolar inhibitors. We also explored the chemistry space of small molecules tested against ENT1 and ENT2 and compared them with PROTACs to illustrate that they are found on the periphery and close to other larger small molecules. While PROTACs are thought of as a dissimilar class to small molecules, it may be possible to bring them closer to those Food and Drug Administration-approved orally available large molecules, and in turn, increase their oral bioavailability. The outcomes of these combined in vitro and computational assessments could influence PROTAC development, be useful for their repurposing as ENT1 inhibitors for several disease indications beyond their primary one, and be used for transporter machine learning model generation and evaluation. SIGNIFICANCE STATEMENT: Proteolysis-targeting chimeras are an increasingly popular class of molecules for which we do not have a complete picture of their absorption, distribution, metabolism, and excretion or toxicity properties. For example, their interactions with uptake and efflux transporters are unknown. Here, we provide evidence that many proteolysis-targeting chimeras act as inhibitors of equilibrative nucleoside transporters 1 and 2. We hope to stimulate further study of their potential for inhibition of other transporters.