RT Journal Article SR Electronic T1 Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP MOLPHARM-AR-2021-000333 DO 10.1124/molpharm.121.000333 A1 Siennah R. Miller A1 Meghan E. McGrath A1 Kimberley M. Zorn A1 Sean Ekins A1 Stephen H. Wright A1 Nathan J. CHERRINGTON YR 2021 UL http://molpharm.aspetjournals.org/content/early/2021/09/09/molpharm.121.000333.abstract AB Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs remdesivir, molnupiravir and its active metabolite, EIDD-1931 and four non-nucleoside molecules repurposed as antivirals for COVID-19. The study used 3D pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H] uridine uptake (ENT1 IC50: 38.65 mM; ENT2 IC50: 76.72 mM), followed by EIDD-1931 (ENT1 IC50: 258.9 mM; ENT2 IC50: 467.3 mM), while molnupiravir was a modest inhibitor (ENT1 IC50: 701.0 mM; ENT2 IC50: 851.4 mM). Other proposed antivirals failed to inhibit ENT-mediated [3H] uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of NBMPR by 30% in ENT1 cells (p = 0.0248) and 27% in ENT2 cells (p = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (p = 0.0463 ) and by 64% in ENT2 cells (p = 0.0132), supporting computational predictions that both are ENT substrates which may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. Significance Statement Significance statement: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.