Nucleoside Reverse Transcriptase Inhibitor Interaction with Human Equilibrative Nucleoside Transporters 1 and 2

Abstract

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 [ 3 H] uridine uptake by NBMPR was biphasic, with IC 50 values of 11.3 nM for ENT1 and 9.6 mM 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 [ 3 H]uridine uptake revealed no difference in J max (16.53 and 30.40 pmol cm 22 min 21 ) and an eightfold difference in K t (13.6 and 108.9 mM). The resulting fivefold difference in intrinsic clearance (J max /K t ) for ENT1-and ENT2 transport accounted for observed inhibition of [ 3 H]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 IC 50 values of ∼200 mM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [ 3 H] 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 STATEMENTThis 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.

Generation of a hTERT-Immortalized Human Sertoli Cell Model to Study Transporter Dynamics at the Blood-Testis Barrier

“…Although the maximum capacity for [ 3 H] uridine uptake was 4- to 5-fold lower compared to HeLa S3 cells after 7 min, the linear uptake and response to NBMPR inhibition was consistent with previous studies [ 38 ]. In addition, the apparent IC 50 value of 1.35 ± 0.37 nM for NBMPR of ENT1 was well within the range of low nanomolar values reported in previous studies using a variety of cell models [ 22 , 38 , 53 , 54 , 55 , 56 ]. These data suggest that ENT1 kinetics for nucleoside analogs and other exogenous substrates can be adequately modeled with hT-SerCs with the intent to evaluate xenobiotic transport across the BTB.…”

“…Protein expression of the CNTs in SCs is limited [ 13 ], thus requiring the ENTs to mediate the majority of nucleoside transport into SCs. The expression of ENT1 and ENT2 in SCs has been reported with extensive kinetic parameters being evaluated [ 13 , 14 , 38 ]. Therefore, the mRNA expression of these transporters was evaluated in the primary human SCs and hT-SerCs.…”

“…The expression of the ENTs in SCs has been well described and provides a route for endogenous nucleosides and nucleoside analog drugs to cross the BTB [ 13 , 14 ]. To validate the presence and function of the ENTs in hT-SerCs, the cellular uptake of a model substrate, uridine [ 13 , 14 , 22 , 38 , 53 , 54 , 55 , 56 ], was measured by transport assays. Total accumulation of ~25 nM [ 3 H] uridine into primary human SCs at passages 4–7 and post-passage 20 hT-SerCs after 10 min was measured in the presence and absence of unlabeled uridine or two concentrations of NBMPR.…”

“…This is consistent with the dominant expression of ENT1 in these cells. The apparent IC 50 of NBMPR on ENT1-mediated [ 3 H] uridine uptake was determined using a modified Equation (1) [ 38 , 57 ]: J represents total uridine transport, J app-max is the apparent J max for uridine times the ratio of IC 50 to the K m of for uridine transport (i.e., J app = J max (IC 50 /K m ); (fmol/cm 2 ·min)), [T] is the [ 3 H] uridine concentration, and [S] is the NBMPR concentration. Results were normalized to percent of the control.…”

“…Transport experiments were performed as previously described with minor modifications [ 38 , 39 , 40 ]. All transport buffers were made with Waymouth’s Buffer (WB; 2.5 mM CaCl·2H 2 O, 28 mM D-glucose, 13 mM HEPES, 135 mM NaCl, 1.2 mM MgCl 2 , 0.8 mM MgSO 4 ·7H 2 O, pH 7.4) and 1 μCi/mL [ 3 H] uridine (~25 nM) and 5 mM unlabeled uridine, 100 nM NBMPR, or 100 μM NBMPR.…”