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 molnupiravir’s active metabolite, β-d-N⁴-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional 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: 39 μM; ENT2 IC₅₀: 77 μM), followed by EIDD-1931 (ENT1 IC₅₀: 259 μM; ENT2 IC₅₀: 467 μM), whereas molnupiravir was a modest inhibitor (ENT1 IC₅₀: 701 μM; ENT2 IC₅₀: 851 μM). 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 predictions 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.

中文翻译：

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Remdesivir 和 EIDD-1931 与人类平衡核苷转运蛋白 1 和 2 相互作用：对到达 SARS-CoV-2 病毒避难所的影响

平衡核苷转运蛋白 (ENT) 存在于血睾屏障 (BTB)，在那里它们可以促进抗病毒药物的处置，以消除精液中可检测到的病毒的避难所。本研究的目的是研究耳鼻喉科药物与三种核苷类似物瑞德西韦、molnupiravir 和 molnupiravir 的活性代谢物β - d -N ^{4的相互作用}-羟基胞苷 (EIDD-1931)，以及四种非核苷分子，这些分子被重新用作 2019 年冠状病毒病 (COVID-19) 的抗病毒药物。该研究使用 ENT1 和 ENT2 底物和抑制剂的三维药效团以及贝叶斯机器学习模型来识别与这些转运蛋白的潜在相互作用。体外转运实验表明，瑞德西韦是最有效的 ENT 介导的 [ ³ H] 尿苷摄取抑制剂（ENT1 IC 50：39 μM；ENT2 IC 50：77 _μM），其次是 EIDD-1931（ENT1 IC _{50 ：} 259 μM ） ；ENT2 IC ₅₀：467 μM），而 molnupiravir 是一种适度抑制剂（ENT1 IC ₅₀：701 μM；ENT2 IC ₅₀: 851 微米)。其他提议的抗病毒药未能抑制 ENT 介导的 [ ³ H] 尿苷摄取低于 1 mM。在 6- S -[(4-硝基苯基)甲基]-6-硫代肌苷 (NBMPR)存在的情况下，瑞德西韦的积累在 ENT1 细胞中减少了 30% ( P = 0.0248)，在 ENT2 细胞中减少了 27% ( P = 0.0054)。EIDD-1931 积累在 NBMPR 存在的情况下在 ENT1 细胞中减少了 77% ( P = 0.0463)，在 ENT2 细胞中减少了 64% ( P= 0.0132），这支持了计算预测，即两者都是耳鼻喉底物，可能对对抗 COVID-19 的疗效很重要。NBMPR 未能降低 molnupiravir 的摄取，表明 ENT 相互作用可能是抑制性的。我们的综合计算和体外数据可用于识别额外的耳鼻喉药物相互作用，以提高我们对可以规避 BTB 的药物的理解。