Evidence for the Interaction of A3 Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1)

Abstract

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A₃ adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [¹²⁵I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound 3 as a stimulator and compound 8 as a partial inhibitor of P-gp ATPase activity). Compound 8 significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC₅₀ 2.4 ± 0.6 µM); however, the BODIPY-conjugated derivative of 8 (compound 24) was not transported by P-gp. In silico docking of compounds 3 and 8 was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A₃ adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp.

Graphical abstract

Fig. 1.

Synthesis of compounds 7 and 8. Reagents and conditions: (i) 3-azido-benzylamine Et₃N, MeOH; (ii) 40% methylamine, MeOH, rt.; (iii) arylethyne, Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, rt.; (iv) arylethyne, Pd(PPh₃)₂Cl₂, Et₃N, DMF, rt.; (v) 10% trifluoroacetic acid, H₂O, 70°C.

Fig. 2.

Synthesis of compound 24 by click chemistry coupling. Reagents and conditions: (i) 3-ethynyl-benzoic acid, HATU, DIPEA, DMF, rt.; (ii) sodium ascorbate, CuSO₄.5H₂O, DMF, H₂O, rt.

Fig. 3.

Effect of compounds 3 (A) and 8 (B) on the ATPase activity of P-gp. The total membranes of High-Five insect cells expressing human P-gp were used for the ATPase assay as described in the Materials and Methods section. The curves represent the mean ± S.D. values from three independent experiments performed in duplicate. The EC₅₀ = 0.45 ± 0.22 µM, shown in (A), and the IC₅₀ = 3.18 ± 0.017 µM, shown in (B), were calculated using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA). The chemical structures of compound 3 (C) and 8 (D) are shown.

Fig. 4.

Effect of compounds 3 and 8 on the photoaffinity labeling of P-gp by [¹²⁵I]iodoarylazidoprazosin. Total membranes of human P-gp–expressing High-Five insect cells (60 μg of protein per 100-μl volume) were incubated for 5 minutes, with increasing concentrations of compound 3 (A) 0–10 µM and 8 (B) 0–15 µM and later transferred to a 4°C water bath, followed by photo-crosslinking with ¹²⁵IAAP (3–4 nM) using 366 nm UV light for 10 minutes. The P-gp bands were separated using gel electrophoresis on 7% Tris-acetate gels. The gels were dried and exposed to X-ray films. Autoradiograms of IAAP-labeled P-gp bands using compounds 3 (A) and 8 (B) are shown. The curves were plotted using GraphPad Prism 7.0 with nonlinear one-phase exponential decay analysis. The values were obtained in three independent experiments carried out in duplicate. The value of IC₅₀ (compound concentration that produces 50% inhibition of IAAP incorporation into P-gp) is given as IC₅₀ = 3.65 ± 0.05 µM for compound 8 (B). IC₅₀ value was calculated using GraphPad Prism 7.0.

Fig. 5.

Compound 8 inhibits substrate transport mediated by P-gp. Bac-Mam P-gp baculovirus-transduced HeLa cells were assayed for BD-verapamil (0.5 µM) transport, as described previously (Vahedi et al., 2017). (A) Representative histogram showing the effect of compound 8 at indicated concentrations on reversal of BD-verapamil transport by P-gp. (B) Concentration-dependent inhibition of BD-verapamil efflux by compound 8. In the plot (B), transport of BD-verapamil in the absence of compound 8 was taken as 100%, and the percentage of transport in the presence of compound 8 was calculated with respect to it. Data points are plotted as the mean ± S.D. (n = 3). The value of IC₅₀ (compound concentration that produces 50% inhibition of BD-verapamil transport in HeLa cells expressing P-gp) is given as IC₅₀ = 2.4 ± 0.6 µM.

Fig. 6.

Docking of compounds 3 (A) and 8 (B) in the drug-binding pocket of human P-gp. In the docking studies using the structure of Taxol-bound human P-gp (pdb.6QEX), we obtained the 20 best, lowest-energy, Vina poses for each compound. Whereas only the best pose for each is presented here for clarity, superpositions of all 20 are shown in Supplemental Fig. 6 to assess structural variation. The color code is as follows: nitrogen, blue; oxygen, red; hydrogen, white; sulfur, yellow; fluorine, chartreuse; and chlorine, light green. The carbons of the ligands are colored magenta, those of the mutated residues, dark green; and gray for the rest of the residues. The residues selected for substitution are labeled.

Fig. 7.

Schematic representation of the modulatory effect of tested A3 adenosine receptor agonist nucleosides on the ATPase activity of human P-gp. See the Discussion section for details.