Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites

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 detect-able in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesi-vir, molnupiravir, and molnupiravir's active metabolite, b-D-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside mole-cules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmaco-phores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interac-tions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 mu M; ENT2 IC50: 77 mu M), followed by EIDD-1931 (ENT1 IC50: 259 mu M; ENT2 IC50: 467 mu M), whereas molnupiravir was a modest inhibi-tor (ENT1 IC50: 701 mu M; ENT2 IC50: 851 mu M). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (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), which supported computational pre-dictions that both are ENT substrates that 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 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.

Automated summary

This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2, providing a potential mechanism for uptake of these drugs into cells and highlighting their importance for antiviral potential in tissues expressing these transporters.