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. SIGNIFICANCE STATEMENT This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [³H]uridine uptake by NBMPR was biphasic, with IC₅₀ values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [³H]uridine uptake revealed no difference in J_max (16.53 and 30.40 pmol cm⁻² min⁻¹) and an eightfold difference in K_t (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (J_max/K_t) for ENT1- and ENT2 transport accounted for observed inhibition of [³H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC₅₀ values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [³H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions (OAT3, OCT3, OCTN1, OATP3A1, OATP4A1, ENT1, and ENT2). Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [³H] uridine was linear over 10 min and inhibited by NBMPR with an IC₅₀ value of 1.35 ± 0.37 nM. These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.

Plasma protein binding (PPB) studies on the SARS-CoV-2 main protease inhibitor nirmatrelvir revealed considerable species differences primarily in dog and rabbit, which prompted further investigations into the biochemical basis for these differences. The unbound fraction (f_u) of nirmatrelvir in dog and rabbit plasma was concentration (2–200 µM)-dependent (dog f_u,p 0.024–0.69, rabbit f_u,p 0.010–0.82). Concentration (0.1–100 µM)-dependent binding in serum albumin (SA) (f_u,SA 0.040–0.82) and alpha-1-acid glycoprotein (AAG) (f_u,AAG 0.050–0.64) was observed from dogs. Nirmatrelvir showed minimal binding to rabbit SA (1–100 µM: f_u,SA 0.70–0.79), while binding to rabbit AAG was concentration-dependent (0.1–100 µM: f_u,AAG 0.024–0.66). In contrast, nirmatrelvir (2 µM) revealed minimal binding (f_u,AAG 0.79–0.88) to AAG from rat and monkeys. Nirmatrelvir showed minimal-to-moderate binding to SA (1–100 µM; f_u,SA 0.70–1.0) and AAG (0.1–100 µM; f_u,AAG 0.48–0.58) from humans across tested concentrations. Nirmatrelvir molecular docking studies using published crystal structures and homology models of human and preclinical species SA and AAG were used to rationalize the species differences to plasma proteins. This suggested that species differences in PPB are primarily driven by molecular differences in albumin and AAG resulting in differences in binding affinity.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5‐year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood–testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6‐nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6‐nitrobenzylthioinosine 5′‐monophosphate (NBMPR‐P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first‐line treatment to avoid testicular relapse should be considered.