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 [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.

Fig. 1.

Reverse-transcription PCR showing mRNA expression of human ENT1 and ENT2 in HeLa cells. Analysis of PCR-amplified DNA products for ENT1 and ENT2 from HeLa S3 cells and positive control full-length plasmids by means of agarose gel electrophoresis and stained with ethidium bromide. Product bands were excised and sent for Sanger sequencing. L: DNA ladder. (1) ENT1 amplified from total HeLa S3 cDNA. (2) ENT1 amplified from a control ENT1 plasmid. (3) ENT2 amplified from total HeLa S3 cDNA. (4) ENT2 amplified from a control ENT2 plasmid.

Fig. 2.

Seven-minute time course of [³H]uridine uptake in HeLa cells. [³H]Uridine uptake is effectively linear through 7 minutes and approximately 80% blocked by 100 nM NBMPR and >95% blocked by 5 mM uridine and 100 µM NBMPR. The best fit line represents a one-phase association. All values are reported as mean ± S.D. (n = 3).

Fig. 3.

Inhibition of [³H]uridine uptake by increasing concentrations of NBMPR in HeLa cells. The experiments were conducted with approximately 20 nM [³H]uridine and terminated at 2 minutes. A best-fit line was fitted using a two-site inhibition model described in eq. 1. Data points are reported as mean ± S.D. of six replicate values (n = 3).

Fig. 4.

Michaelis-Menten kinetics of ENT-mediated uridine uptake in HeLa cells. Data were collected after 2 minutes. The experiments were conducted with approximately 20 nM [³H]uridine in the presence of increasing concentrations of uridine. Total uridine uptake (A), ENT1-specific uptake (B), and ENT2-specific uptake (C). ENT2-specific uptake was determined with the addition of 100 nM NBMPR, ENT1-specific uptake was determined by subtracting ENT2-specific uptake from total uptake. Best-fit lines were fitted to determine relevant values using GraphPad Prism 7. The red dashed lines in (A) represent predicted total uptake and nonspecific uptake determined using the kinetic parameters for ENT1- and ENT2-specific uptake. Data points in graphs are reported as mean ± S.D. of six replicates (n = 3).

Fig. 5.

Inhibition of [³H]uridine uptake in HeLa cells by endogenous nucleosides. IC₅₀ analysis of adenosine (A), guanosine (B), and thymidine (C), inosine (D), and cytidine (E). Total [³H]uridine uptake (black open circle), ENT1-mediated [³H]uridine uptake (blue diamond), and ENT2-mediated [³H]uridine uptake (red triangle) were measured in the presence of endogenous nucleosides. ENT2 mediated uptake was taken as that occurring in the presence of 100 nM NBMPR. ENT1 data points were determined by subtracting calculated ENT2 uptake from total uptake and fitting the data to a single component IC₅₀ equation. Best-fit lines were created to determine relevant values using GraphPad Prism 7. Increasing concentrations of each compound were incubated with approximately 20 nM [³H]uridine or 20 nM [³H]uridine and 100 nM NBMPR. Data points in the graph are reported as mean ± S.D. of triplicate values (n = 3).

Fig. 6.

Inhibition of ENT-mediated [³H]uridine uptake in HeLa cells by nine NRTIs. The experiments were terminated after 2 minutes. All graphs are represented by relative uptake as a percentage of control ([³H]uridine only). Three concentrations of abacavir (A), entecavir (B), zidovudine (C), didanosine (D), zalcitabine (E), emtricitabine (F), tenofovir disoproxil (G), stavudine (H), and lamivudine (I) were incubated with approximately 20 nM [³H]uridine or 20 nM [³H]uridine and 100 nM NBMPR as indicated on the x-axis. Significance between control without NBMPR and the NRTIs is indicated as *P < 0.05; ***P < 0.0001. Significance between control with 100 nM NMBPR and the NRTIs is indicated as ^†P < 0.05. Two-way analysis of variance with Bonferonni’s correction for multiple comparisons was used for statistical significance analysis using GraphPad Prism 7. Data in graphs are reported as mean ± S.D. of four replicate values (n = 3).

Fig. 7.

IC₅₀ analysis of abacavir (A), entecavir (B), and zidovudine (C). Total [³H]uridine uptake (black open circle), ENT1-mediated [³H]uridine uptake (blue diamond), and ENT2-mediated [³H]uridine uptake (red triangle) was measured in the presence of increasing concentrations of NRTIs. ENT2-mediated uptake was determined with the addition of 100 nM NBMPR. ENT1 data points were determined by subtracting ENT2 uptake from total uptake and fitting the data to a single component IC₅₀ equation. Best-fit lines were created to determine relevant values using GraphPad Prism 7. Increasing concentrations of each compound were incubated with approximately 20 nM [³H]uridine or 20 nM [³H]uridine and 100 nM NBMPR. Data points in the graph are reported as mean ± S.D. of six replicate values (n = 3).

Fig. 8.

Inhibition of uptake of uridine (A), abacavir (B), zidovudine (C), and entecavir (D) by NBMPR using radiolabeled substrates detected by a liquid scintillation counter and unlabeled substrates detected by LC-MS/MS. All data are reported as percentage of control. One-way ANOVA with Bonferonni’s multiple comparison correction was used to determine statistical significance. Data in graphs are reported as mean ± S.D. of duplicate values (n = 3).