Structures of human ENT1 in complex with adenosine reuptake inhibitors

The human Equilibrative Nucleoside Transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in human. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are clinically used. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited due to the absence of structural information on hENT1. Here we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-Nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1, while giving insight into adenosine recognition and transport. Our studies provide the platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.