Transport mechanisms of a novel antileukemic and antiviral compound 9-norbornyl-6-chloropurine

Figures & data

Figure 1. Structure of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP).

Figure 2. The time course of intracellular [³H]9-norbornyl-6-chloropurine ([³H]NCP) uptake. CCRF-CEM cells were incubated with 10 µM (0.2 µCi/ml) [³H]NCP for 0 to 60 min (•) or preincubated with 10 µM MK571 (MRP1 inhibitor, ▴) or 5 µM LY335979 (P-gp inhibitor, □) for 20 min prior to the addition of 10 µM (0.2 µCi/ml) [³H]NCP.

Figure 3. High-performance liquid chromatography analysis of extracellular [³H]9-norbornyl-6-chloropurine ([³H]NCP) metabolites. CCRF-CEM cells were incubated with 25 µM (10 µCi/ml) [³H]NCP (—) for 24 h. In a parallel experiment, medium without CCRF-CEM cells was incubated with 25 µM (10 µCi/ml) [³H]NCP (· · · ·) for 24 h. The identity of all peaks was confirmed using the authentic standards. NCP-GS, Glutathione conjugate.

Figure 4. Concentration dependence of [³H]9-norbornyl-6-chloropurine ([³H]NCP) uptake. CCRF-CEM cells were incubated at different concentrations of [³H]NCP (0.2 µCi/ml) for 2 min.

Table 1. The effect of ATP-depleting compounds, oligomycin and antimycin, and Na⁺ level on the transport of [³H]9-norbornyl-6-chloropurine ([³H]NCP).

	Intracellular [^3H]NCP (% of control)
Oligomycin A*
10 µM	92 ± 2
50 µM	72 ± 1
Antimycin A*
0.1 µM	81 ± 7
0.5 µM	74 ± 1
Na^+ depletion†	90 ± 2

*Incubation with 10 µM (0.2 µCi/ml) [³H]NCP for 2 min following a 20-min preincubation with the inhibitor; control sample (NCP only) = 0.8 ± 0.1 nmol.

†Incubation with 10 µM (0.2 µCi/ml) [³H]NCP for 2 min in the transport media w/o Na⁺; control sample (NCP only) = 1.2 ± 0.1 nmol.

Figure 5. The time course of intracellular [³H]9-norbornyl-6-chloropurine ([³H]NCP) levels following the CCRF-CEM cell treatment with ethacrynic acid and buthionine sulfoximine (BSO). CCRF-CEM cells were incubated with 10 µM (0.2 µCi/ml) [³H]NCP for 0 to 45 min (•) or preincubated with 50 µM ethacrynic acid (glutathione-S-transferase inhibitor, ▴) for 20 min prior to the addition of 10 µM (0.2 µCi/ml) [³H]NCP (A). In a parallel experiment, NCP uptake was evaluated after the cell treatment with 50 µM BSO (glutathione biosynthesis inhibitor, □) for 24 h before the addition 10 µM (0.2 µCi/ml) [³H]NCP (B).

Table 2. The effect of natural nucleosides on the uptake of [³H]9-norbornyl-6-chloropurine ([³H]NCP) after a 2-min incubation.

	Intracellular [^3H]NCP (% of control)
Competing nucleoside	400 µM*	1600 µM†
Guanosine	115 ± 4	N/A‡
Deoxyguanosine	107 ± 4	95 ± 22
Adenosine	120 ± 11	102 ± 10
Inosine	106 ± 5	107 ± 14
Uridine	80 ± 6	N/A‡
Thymidine	116 ± 7	133 ± 32
Cytidine	105 ± 2	138 ± 30

*[³H]NCP = 50 µM (0.2 µCi/ml); control sample (NCP only) = 9.9 ± 0.9 nmol.

†[³H]NCP = 400 µM (0.2 µCi/ml); control sample (NCP only) = 3.4 ± 0.6 nmol.

‡Nucleoside not soluble at the designated concentration.

Figure 6. The effect of the microtubule inhibitors on [³H]9-norbornyl-6-chloropurine ([³H]NCP) uptake. CCRF-CEM cells were incubated with 0 to 30 µM cytochalasin B (▪) and cytochalasin D (□, A) or with 0 to 20 mM colchicine (▪, B) for 20 min before the addition of 10 µM (0.2 µCi/ml) [³H]NCP. The intracellular NCP content was measured at t = 2 min. The absolute value of control sample (NCP only) = 1.4 ± 0.2 nmol. *p < 0.05 (one-way ANOVA with Tukey’s post hoc test).

Figure 7. The dependence of [³H]9-norbornyl-6-chloropurine ([³H]NCP) uptake on the clathrine-dependent endocytosis inhibitor, dynasore. CCRF-CEM cells were preincubated with 0 to 200 µM dynasore (□) for 20 min at 37°C after which 10 µM (0.2 µCi/ml) [³H]NCP was added and the intracellular level was measured following 2-min incubation. The absolute value of control sample (NCP only) = 0.9 ± 0.1 nmol.

Table 3. The cytotoxicity of 9-norbornyl-6-chloropurine (NCP) in CCRF-CEM cells in the presence of the inhibitors of drug efflux proteins.

Inhibitor	None	LY335979* (P-gp)	MK571* (MRP)
IC50 (µM)†	17.7 ± 1.8	14.1 ± 0.9	9.9 ± 1.0

*2 μM LY 335979 and 10 µM MK571, these concentrations did not to cause any intrinsic toxicity to the cells.

†The concentration causing 50% decrease in cells viability using XTT cytotoxicity test (calculated using the non-linear regression method by GraphPad Prism).