Modeling Blood‐Testis Barrier Characteristics with Novel CRISPR/Cas9 Functional ENT1 and ENT2 Knockout HeLa Cell Lines

Abstract

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood‐testis barrier. ENT1 is located on the basal membrane while ENT2 is located on the apical membrane of human Sertoli cells, creating a transepithelial transport pathway across the blood‐testis barrier. Improving drug disposition to the testis with drugs that use transepithlelial transport pathways may more effectively treat viral infections and cancer within the male genital tract. Here, we characterized uridine transport in two novel ENT cell lines and assessed the impact of nucleoside reverse transcriptase inhibitors (NRTIs) on uridine uptake to gain further insight on ENT function and substrate selectivity. ENT1 or ENT2 functional knockout HeLa cell lines were generated using CRISPR/Cas9 to determine the kinetics and selectivity of each ENT individually. We demonstrate the functional loss of each transporter through quantitation of [3H]uridine uptake in the presence of the ENT specific inhibitor S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR). At low concentrations (100 nM), NBMPR inhibits ENT1; conversely, at high concentrations (100 μM) NBMPR inhibits both ENT1 and ENT2. In ENT2 knockouts, >97% of [3H]uridine uptake was inhibited by 100nM NBMPR, indicating ENT1 function is predominant. In ENT1 knockouts, only 5% of [3H]uridine uptake was inhibited by 100 nM NBMPR and 90% was inhibited by 100 μM NBMPR, indicating ENT2 function is predominant. The Jmax for ENT1 is 23.4 pmol cm−2 min−1 and for ENT2 is 34 pmol cm−2 min−1. The Kt for ENT1 is 10 μM and for ENT2 is 110 μM. We next evaluated the impact of nine NRTIs (abacavir, entecavir, zidovudine, tenofovir, lamivudine, emtricitabine, zalcitabine, didanosine and stavudine) on [3H]uridine uptake, revealing abacavir as the most potent inhibitor of [3H]uridine uptake, with an IC50 of 72 μM for ENT1 and 350 μM for ENT2. Zidovudine had an IC50 of 1.4 mM for ENT1 and 1.2 mM for ENT2. Entecavir had an IC50 of 2.6 mM for ENT1 and 1.08 mM for ENT2. This suggests that abacavir has higher affinity for ENT1, and entecavir has a higher affinity for ENT2. Based on previous results using wild‐type HeLa cells and NBMPR, we believe that abacavir is an inhibitor of the ENTs, entecavir is a substrate, and zidovudine is a low‐affinity substrate. These functional knockout cell lines will allow further screening of therapeutic compounds to establish the substrate selectivity between ENT1 and ENT2, which can aid in the development of future compounds that are able to circumvent the blood‐testis barrier via the ENT1‐ENT2 transepithelial transport pathway.Support or Funding InformationNIGMS R01GM123643, 5P30ES006694, 2T32ES007091‐36A1