TY - JOUR T1 - Nucleoside Reverse Transcriptase Inhibitor (NRTI) Interaction with Human Equilibrative Nucleoside Transporters 1 and 2 JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.120.090720 SP - dmd.120.090720 AU - Siennah R Miller AU - Raymond K Hau AU - Joseph L Jilek AU - Mark N Morales AU - Stephen H Wright AU - Nathan J Cherrington Y1 - 2020/01/01 UR - http://dmd.aspetjournals.org/content/early/2020/05/11/dmd.120.090720.abstract N2 - 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 Jmax (16.53 and 30.40 pmol•cm-2•min-1) and an eight-fold difference in Kt (13.6 and 108.9 μM). The resulting five-fold difference in intrinsic clearance (Jmax/Kt) 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 LC-MS/MS 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 NBMPR to differentiate between ENT1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse transcriptase inhibitors (NRTIs) 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. ER -