Interaction of A3 adenosine receptor ligands with the human multidrug transporter ABCG2

Abstract

Various adenosine receptor nucleoside-like ligands were found to modulate ATP hydrolysis by the multidrug transporter ABCG2. Both ribose-containing and rigidified (N)-methanocarba nucleosides (C2-, N⁶- and 5'-modified), as well as adenines (C2-, N⁶-, and deaza modified), were included. 57 compounds out of 63 tested either stimulated (50) or inhibited (7) basal ATPase activity. Structure-activity analysis showed a separation of adenosine receptor and ABCG2 activities. The 7-deaza modification had favorable effects in both (N)-methanocarba nucleosides and adenines. Adenine 37c (MRS7608) and (N)-methanocarba 7-deaza-5'-ethyl ester 60 (MRS7343) were found to be potent stimulators of ABCG2 ATPase activity with EC₅₀ values of 13.2 ± 1.7 and 13.2 ± 2.2 nM, respectively. Both had affinity in the micromolar range for A₃ adenosine receptor and lacked the 5'-amide agonist-enabling group (37c was reported as a weak A₃ antagonist, K_i 6.82 μM). Compound 60 significantly inhibited ABCG2 substrate transport (IC₅₀ 0.44 μM). Docking simulations predicted the interaction of 60 with 21 residues in the drug-binding pocket of ABCG2.

Keywords: A(3) adenosine receptor; ABC transporter; ABCG2; ATP hydrolysis; Drug transport; Multidrug resistance.

Fig. 1.

Modulation of ABCG2 ATPase activity by A₃ adenosine receptor ligands. Results for four representative ligands, the potent A₃ agonist 5 (A), triazole-extended 6-benzylamino derivative 17 (B), adenine (A₃ antagonist) derivative 37c (C), and (N)-methanocarba-7-deazaadenosine 5’-ester derivative 60 (D) are shown. Each experiment was performed at least three times in duplicate. The EC₅₀ and IC₅₀ values were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

Fig. 2.

Compound 60 inhibited mitoxantrone transport mediated by ABCG2. (A) Representative histogram showing the effect of compound 60 on mitoxantrone transport at indicated concentrations. Fluorescence intensity of pcDNA control cells is shown in red and R-5 cells expressing ABCG2 are shown in blue. The reversal effect of compound 60 for R-5 cells at 0.25, 0.5 and 1 μM are shown in orange, light green, and green, respectively. (B) Concentration-dependent inhibition of mitoxantrone efflux by compound 60. Mitoxantrone efflux by R-5 cells in the absence of compound 60 was taken as 100%, and the percentage of efflux at the indicated concentrations of compound 60 was calculated. Data points were plotted as the mean ± S.D. (n = 3). Compound concentration that produces 50% inhibition of mitoxantrone transport in R-5 cells is given as IC₅₀ value, calculated using GraphPad Prism.

Fig. 3.

Docking of (N)-methanocarba-7-deazaadenosine 5’-ester derivative 60 in the drug-binding pocket of human ABCG2. The cryo-EM structure of a human ABCG2 mutant bound to E₁S (PDBID: 6HCO) was used as a template for docking. On the left side, the structure of ABCG2 depicts the two monomers of (G2 and G2’) in cyan and yellow cartoons and the binding site for the compound 60 is boxed. The close-up in the right panel depicts the expanded region showing the lowest energy docking pose for compound 60 and the residues in the drug-binding pocket interacting with it. A total of 21 residues were identified to be within 4.5 Å of compound 60, and eleven of these (highlighted in yellow) were also within 4.5 Å of substrate E₁S.

Fig. 4.

Schematic representation of the modulatory effect of tested nucleoside adenines on the ATPase activity of human ABCG2.

Scheme 1.

Synthesis of (N)-methanocarba nucleoside derivatives 12, 16 and 18 – 23. A) Reagents and conditions: (i) phenylacetylene, sodium ascorbate, CuSO₄.5H₂O, t-BuOH-H₂O, rt, 87–90%. B) (i) 3-azidobenzylamine, DIPEA, 2-propanol, 87%, (ii) TBAF, THF, rt, 91%; (iii) 3,4-di-F-phenylacetylene, PdCl₂(Ph₃P)₂, CuI, Et₃N, DMF, 68%; (iv) 10% TFA, MeOH, 70°C, 89%; (v) phenylacetylene, sodium ascorbate, CuSO₄.5H₂O, t-BuOH-H₂O, rt, 87–88%.

Scheme 2.

Synthesis of 7-deaza (N)-methanocarba nucleoside 5’-esters 61 – 64. Reagents and conditions: (i) Ph(CH₂)₃NH₂, DIPEA, 2-propanol, 69–72%; (ii) arylalkyne, PdCl₂(Ph₃P)₂, CuI, Et₃N, DMF, rt, 67–72%; (iii) 10% TFA, MeOH, 70 °C, 88–91%.