Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [³H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC₅₀ values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC₅₀-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC₅₀ values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells.

中文翻译：

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使用功能性敲除细胞系和贝叶斯模型预测药物与人类平衡核苷转运蛋白 1 和 2 的相互作用

平衡核苷转运蛋白 (ENT) 1 和 2 促进核苷转运穿过血睾丸屏障 (BTB)。使用使用内源性运输途径的药物改善药物进入睾丸可能会导致更有效的生殖道疾病治疗。在这项研究中，CRISPR/CRISPR 相关蛋白 9 被用于生成 HeLa 细胞系，其中 ENT 表达仅限于 ENT1 或 ENT2。我们对这些细胞系中的尿苷转运进行了表征，并生成了贝叶斯模型来预测与耳鼻喉科的相互作用。在 ENT 特异性抑制剂S存在下定量 [ ^{3 H] 尿苷摄取}-(4-nitrobenzyl)-6-thioinosine (NBMPR) 显示每个转运蛋白的功能丧失。评估了九种核苷逆转录酶抑制剂和 37 种核苷/杂环类似物，以确定耳鼻喉科的相互作用。21 种化合物抑制尿苷摄取，阿巴卡韦、奈韦拉平、替格瑞洛和三乙酸尿苷具有不同的 IC ₅₀ENT1 和 ENT2 的值。在有和没有 NBMPR 的情况下测量四种已识别抑制剂的总积累，以确定是否存在 ENT 介导的转运。氯法拉滨和克拉屈滨是 ENT1 和 ENT2 底物，而奈韦拉平和 lexibulin 是 ENT1 和 ENT2 非转运抑制剂。使用 Assay Central 机器学习软件生成的贝叶斯模型产生了相当高的内部验证性能（接收器算子特征 > 0.7）。基于ENT1 IC ₅₀的模型由 ChEMBL 生成；使用此训练数据集的子验证在按摄取百分比分析活性时正确预测了 58% 的抑制剂，在使用估计的 IC _{50时正确预测为 63%}价值观。确定与这些转运蛋白的药物相互作用可用于识别和预测作为 ENT1 和 ENT2 底物的化合物，从而可以通过支持细胞中的这种跨上皮转运途径绕过 BTB。