A novel non-natural nucleoside that influences P-glycoprotein activity and mediates drug resistance

Abstract

Multidrug resistance during cancer chemotherapy is commonly acquired by overexpression of the ATP binding cassette transporter, P-glycoprotein (P-gp). As such, inhibitors that target P-gp activity represent potential therapeutic agents against this form of drug resistance. This study evaluated the ability of various non-natural nucleosides that mimic the core structure of adenosine to modulate drug resistance by inhibiting the ATPase activity to P-gp. Of several analogues tested, only one novel non-natural nucleoside, 5-cyclohexylindolyl-2'-deoxyribose (5-CHInd), behaves as a P-gp inhibitor. Although 5-CHInd is an adenosine analogue that should block the binding of ATP, the non-natural nucleoside surprisingly stimulates the ATPase activity of P-gp in vitro. However, 5-CHInd is not an exportable substrate for P-gp as it is not transported across an MDCK-MDR1 monolayer. In addition, 5-CHInd differentially modulates MDR by decreasing or increasing the cytotoxicity of several chemotherapeutic agents. Although 5-CHInd displays variable activity in modulating the efflux of various drugs by P-gp, there is a correlation between changes observed in the drug-stimulated ATPase catalytic efficiency induced by 5-CHInd and its effect on drug efflux. The paradoxical behavior of 5-CHInd is rationalized within the context of contemporary models of P-gp function. In addition, the data are used to develop a predictive in vitro model for rapidly identifying potential drug-drug interactions with P-gp.

Figure 1

Structures of non-natural deoxyribose nucleosides used in this study.

Figure 2

Stimulation of P-gp ATPase activity by non-natural nucleosides. (a) Inside-out P-gp enriched membranes were used for ATPase measurements and the Michaelis-Menten kinetic parameters, K_m and V_max, were obtained by stimulating P-gp ATPase activity with increasing concentrations of 5-CHInd. ATP concentration was fixed at 1 mM and initial velocities were measured over 20 minutes. K_m = 60 ± 18 nM, V_max = 3.25 ± 0.37 μM min⁻¹. (b) Stimulation of P-gp ATPase activity in inverted P-gp enriched membranes by increasing concentrations of 5-CHInd (▼) 5-CEInd (▲) and 5-PhInd (■).

Figure 3

5-CHInd is a competitive inhibitor of P-gp. Double reciprocal plot of rate versus calcein-AM concentration at several fixed concentrations of 5-CHInd. 5-CHInd concentrations were set at the following: no inhibitor (▲), 10 nM 5-CHInd (▼), 20 nM 5-CHInd (■), and 30 nM 5-CHInd (◆). The K_i value of 6.5 ± 0.7 μM was determined through a fit of the data to the following equation: rate = V_max[S]/K_m(1+[I]/K_i) + [S].

Figure 4

Modulation of drug resistance by 5-CHInd in KB-V1 cells. (A) Effects of 10 μM 5-CHInd over 72 hours on the log (LD₅₀) values of vinblastine (VBL), doxorubicin (DOX), colchicine (COLC), and paclitaxel (TAX). (B) Potentiation effects of 50 μM 5-CHInd over 48 hours on the LD₅₀ of paclitaxel (TAX). No treatment (■) LD₅₀ = 4.0 ± 0.24 μM, 50 μM 5-CHInd (▲) LD₅₀ = 0.49 ± 0.03 μM.

Figure 5

(A) Correlation in ATPase V_max/K_m ratios with modulation in MDR phenotype. The following drugs were used in this study: vinblastine (VBL), doxorubicin (DOX), colchicine (COLC), and paclitaxel (TAX). The left axis (■) indicates changes in drug stimulated ATPase V_max/K_m ratios in the presence of 60 nM 5-CHInd. The right axis (▲) indicates changes in log (LD₅₀) for cytotoxic drugs in KB-V1 cells in the presence of 10 μM 5-CHInd over 72 hours. (B) Model correlating the effect of a compound such as 5-CHInd on the ATPase activity of P-gp with drug resistance in MDR-positive cells. In this model, the influence on drug resistance reflects enhanced export or retention of the drug of interest. V_max/K_m ratios greater than 1 indicate an increase in P-gp activity which generates an increase in drug resistance. Conversely, V_max/K_m ratios that are less than 1 reflect a decrease in P-gp activity that correlates with a decrease in drug resistance.