Dipyridamole analogs as pharmacological inhibitors of equilibrative nucleoside transporters. Identification of novel potent and selective inhibitors of the adenosine transporter function of human equilibrative nucleoside transporter 4 (hENT4)

Abstract

To identify needed human equilibrative nucleoside transporter 4 (hENT4) inhibitors, we cloned and stably expressed the recombinant protein in PK15NTD (nucleoside transporter deficient) cells, and, investigated its interaction with a series of dipyridamole analogs synthesized in our laboratory. Compounds were tested in this newly established hENT4 expressing system as well in previous stably expressed hENT1 and hENT2 expressing systems. Of the dipyridamole analogs evaluated, about one fourth of the compounds inhibited hENT4 with higher potencies than dipyridamole. The most potent of them, Compound 30 displayed an IC₅₀ of 74.4 nM, making it about 38 times more potent than dipyridamole (IC₅₀=2.8 μM), and selectivities of about 80-fold and 20-fold relative to ENT1 and ENT2, respectively. Structure-activity relationship showed nitrogen-containing monocyclic rings and noncyclic substituents at the 4- and 8-positions of the pyrimido[5,4-d]pyrimidine were important for the inhibitory activity against hENT4. The most potent and selective hENT4 inhibitors tended to have a 2,6-di(N-monohydroxyethyl) substitution on the pyrimidopyrimidine ring system. The inhibitors of hENT4 identified in this study are the most selective and potent inhibitors of hENT4 adenosine transporter function to date, and should serve as useful pharmacological/biochemical tools and/or potential leads for ENT4-based therapeutics. Also, the new hENT4-expressing PK15 cell line established will serve as a useful screening tool for the discovery and design of hENT4 ligands.

Keywords: Dipyridamole analogs; Equilibrative nucleoside transporters; Human equilibrative nucleoside transporter 4; Structure–activity relationships.

Fig. 1

Immunoblotting detection of recombinant hENT4 in PK15NTD cells. PK15NTD cells were transiently transfected with either pCMV-3tag-1A (empty vector) or pCMV-3tag-1A/hENT4. Cell lysates were analyzed by Western blotting using FLAG antibody (A1). The blot was reprobed with β-actin for normalization (A2). The quantification of the Western blots are shown in B datapoints are mean + S.EM. for two blots. * = (p< 0.05); ** = (p <01)

Fig. 2

Adenosine specificity of hENT4 and pH-dependence of transport activity of stably expressed recombinant hENT4 in PK15NTD cells. pH dependence was examined at variable pH by [³H]adenosine uptake compared with [³H]uridine uptake. Data points are the mean ±S.D. for 3 samples.

Fig. 3

Inhibition profiles of dipyridamole derivatives against hENT1-, hENT2- or hENT4-mediated nucleoside uptake. The uptake of [³H]-labeled nucleoside was determined after incubation with 10 μM test compounds for 15 min. Uptake values in the presence of compounds are given as the percentage of uptake values in their absence. Data points are the mean ±S.D. for 3 determinations.

Fig. 4

Concentration-dependent inhibition of hENT4 adenosine transport by the most potent dipyridamole analogues. Graded concentrations of test compounds were incubated with cells for 15 min prior to addition of [³H]adenosine for an uptake period of 2 min. Data are presented as percent inhibition relative to vehicle control (mean ± S.D.; n = 3). Compounds shown are the ones with IC₅₀s ≤ 200 nM for hENT4.

Fig. 5

Cell viability testing by MTT assay. PK15/hENT cells were seeded at 5 × 10³ cells were seeded in 96-well plates, treated with 10 μM test compounds or control and incubated at 37 °C in phenol-free MEM for 24 hours. Subsequently, 10 μl of 5 mg/ml methylthiazolyldiphenyl-tetrazolium bromide (MTT) in PBS was added to each well, and then incubated for 4 hrs at 37°C. Following incubation, 100 μl of 0.04N HCl in isopropanol was added for 3 hours at room temperature to solubilize the formazan produced. The absorbance was quantified at 570 nm using a microplate reader (SpectraMax M2, Molecular Devices). Data are mean ± S.E.M for 4 replicates. * = (p<0.05). Compared to the cytotoxic agent camptothecin, compound 30 was significantly non-cytotoxic at p < 0.02.