Irreversible Antagonists for the Adenosine A2B Receptor

Abstract

Blockade of the adenosine A2B receptor (A2BAR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A2BAR antagonists based on an 8-p-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα15 protein activation assays by performing ligand wash-out and kinetic experiments. p-(1-Propylxanthin-8-yl)benzene sulfonyl fluoride (6a, PSB-21500) was the most potent and selective irreversible A2BAR antagonist of the present series with an apparent Ki value of 10.6 nM at the human A2BAR and >38-fold selectivity versus the other AR subtypes. The corresponding 3-cyclopropyl-substituted xanthine derivative 6c (PSB-21502) was similarly potent, but was non-selective versus A1- and A2AARs. Attachment of a reactive sulfonyl fluoride group to an elongated xanthine 8-substituent (12, Ki 7.37 nM) resulted in a potent, selective, reversibly binding antagonist. Based on previous docking studies, the lysine residue K2697.32 was proposed to react with the covalent antagonists. However, the mutant K269L behaved similarly to the wildtype A2BAR, indicating that 6a and related irreversible A2BAR antagonists do not interact with K2697.32. The new irreversible A2BAR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs.

Keywords: BRET assay; G protein activation; G protein-coupled receptor; Gα15; adenosine; covalent binding; mutagenesis; radioligand binding studies; synthesis; xanthine.

Figure 1

Chemical structures of selected AR antagonists (h = human) [18,23].

Figure 2

Design of irreversible A_2BAR antagonists based on a published docking pose [18].

Scheme 1

Preparation of compounds 6a–c ¹. ¹ Reagents and conditions: (a) NaNO₂, AcOH (50% aq), 65 °C, 10 min, 81–94%; (b) Na₂S₂O₄, NH₃ (12.5% aq), 65 °C, 10 min, 68–80%; (c) EDC, DMF, room temperature (rt), 2–4 h, 66–73%; (d) PPSE, (1) 120 °C, 10 min; (2) 170 °C, 2 h, 52–68%.

Scheme 2

Preparation of compound 12 ¹. ¹ Reagents and conditions: (a) TEA, dioxane, rt, 36 h, 70%; (b) EDC, DMF, rt, 3 h, 68%; (c) PPSE, (1) 120 °C, 10 min; (2) 170 °C, 4 h, 60%; (d) HATU, DIPEA, DMF, rt, 18 h, 30%.

Figure 3

Assessment of reversibility of ligand binding. (a) Wash-out experiments after incubation of wildtype (wt) human A_2BAR with compounds 6a–c, 12 and IV at indicated concentrations. After an extensive washing procedure, specific binding of [³H]PSB-603 was determined. Data represents normalized mean values ± SEM from three independent experiments. (b) Competition association of [³H]PSB-603 alone (control) and in the presence of 6a or 12 in concentrations representing 3-fold of the respective apparent K_i values. Values represent means ± SEM from two independent experiments.

Figure 4

Functional assessment of A_2BAR inhibition by the new antagonists. (a) Assay principle of the TRUPATH BRET² assay. When the receptor is not activated, the G protein (Gα₁₅β₃γ₁₃) is in its inactive trimeric form, where Gα₁₅-RLuc8 and Gγ_13-GFP2 are in close proximity, resulting in a high BRET ratio. When the receptor is activated by an agonist (NECA). the trimer re-arranges, and the BRET ratio decreases due to a larger distance between the probes. (b–f) Concentration-response curves of (b) PSB-21500 (6a), (c) PSB-21501 (6b), (d) PSB-21502 (6c), (e) PSB-21503 (12), and (f) PSB-1115 (IV) showing the percent inhibition of NECA-induced Gα₁₅ activation without or after wash-out. Wash-out did not result in significant differences for 6a, 6b, and 6c. In contrast, significantly reduced inhibition of receptor activation was observed after wash-out of 12 and IV.

Figure 5

Suggested irreversible binding mode of PSB-21500 (6a) to the A_2BAR. (a) Docking pose of 6a in the previously published A_2BAR homology model [18]. The lysine residue in position 269^7.32 was chosen as an anchor for nucleophilic substitution. Covalent docking was performed using Maestro (Schroedinger). (b) Chemical structure of PSB-21500.

Figure 6

Binding and functional characterization of the A_2BAR K269^7.32L mutant. (a) Wash-out experiments after incubation of the A_2BAR mutant with compounds 6a and IV at 10-fold of the K_i value and at 100-fold of the K_i value, respectively. After an extensive washing procedure, the specific binding of [³H]PSB-603 was determined. Data represents normalized mean values ± SEM from three independent experiments. (b–f) Concentration-response curves for (b) PSB-21500 (6a), (c) PSB-21501 (6b), (d) PSB-21502 (6c), (e) PSB-21503 (12), and (f) PSB-1115 (IV) showing the percent inhibition of NECA-induced Gα₁₅ activation by the investigated antagonists at the A_2BAR K267^7.32L mutant.