Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

Abstract

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure–function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling

“…There is a need to understand and clarify the interactions between the equilibrative nucleoside transporters (ENTs) and nucleoside analogs. These ubiquitously expressed transporters play a role in the pharmacokinetics and drug-drug interactions (DDIs) of nucleoside analogs, which share a similar chemical structure to endogenous substrates of these transporters (Atlas, 2020;Huber-Ruano and Pastor-Anglada, 2009;Koczor et al, 2012;Pastor-Anglada and Pérez-Torras, 2015;Rehan et al, 2019). Although there is clear value in heterologous expression systems to study the ENTs, the resulting models can be complicated to interpret; the complexity owing to a variety of issues, including the influence of endogenous nucleoside transport activity (Boswell-Casteel and Hays, 2017;Sundaram, 2001;Ward et al, 2000;Yao et al, 2001).…”

Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1

“…The kinetics of endogenous substrates for nucleoside transporters has been extensively studied, predominately with ENT1 (Boleti et al, 1997;Ward et al, 2000;Bone and Hammond, 2007;Boswell-Casteel and Hays, 2017;Miller et al, 2020;Miller et al, 2021) and recent advancements include the determination of the ENT1 crystal structure bound to two nontransported ENT1 inhibitors, 6-nitrobenzylthioinosine and dilazep (Wright and Lee, 2019), but further research is necessary to understand the roles that human ENT1 and ENT2 play in the pharmacokinetics of currently prescribed therapeutics and therapeutics currently in development. Identifying, predicting and validating drug interactions with these transporters may inform and facilitate the drug development process for new chemotherapeutics, antivirals, and male contraceptives and fertility agents (Mruk et al, 2011;Ekins et al, 2012;Pastor-Anglada and Pérez-Torras, 2018;Ekins et al, 2019;Rehan et al, 2019).…”

Decreased Equilibrative Nucleoside Transporter 1 (ENT1) Activity Contributes to the High Extracellular Adenosine Levels in Mesenchymal Glioblastoma Stem-Like Cells

“…The CNT family is composed of three members (CNT1-CNT3); they actively transport substrates against a chemical gradient in a strict sodium-dependent manner. ENTs are passive transporters specific to eukaryotes, this family is composed of four members (ENT1–4) [ 30 , 34 ]. CNTs and ENTs regulate the physiological cellular uptake of purine and pyrimidine nucleosides and nucleobases, the precursors of nucleotides that are essential for DNA and RNA synthesis [ 29 , 30 , 35 ].…”