Development of subtype-selective covalent ligands for the adenosine A2B receptor by tuning the reactive group

Abstract

Signalling through the adenosine receptors (ARs), in particular through the adenosine A_2B receptor (A_2BAR), has been shown to play a role in a variety of pathological conditions, ranging from immune disorders to cancer. Covalent ligands for the A_2BAR have the potential to irreversibly block the receptor, as well as inhibit all A_2BAR-induced signalling pathways. This will allow a thorough investigation of the pathophysiological role of the receptor. In this study, we synthesized and evaluated a set of potential covalent ligands for the A_2BAR. The ligands all contain a core scaffold consisting of a substituted xanthine, varying in type and orientation of electrophilic group (warhead). Here, we find that the right combination of these variables is necessary for a high affinity, irreversible mode of binding and selectivity towards the A_2BAR. Altogether, this is the case for sulfonyl fluoride 24 (LUF7982), a covalent ligand that allows for novel ways to interrogate the A_2BAR.

Scheme 1. Synthesis of potential covalent ligands for the A_2BAR. Reagents and conditions: (a) EDC·HCl, DIPEA, respective benzoic acid, dry DMF, rt, 2–20 h, 41–68%; (b) PPSE, 170 °C, 1–4 h, 10–53%; (c) 2 M NaOH, dioxane, 120 °C, 2–3 h, 60–84%; (d) (i) TFA, DCM, rt, 1 h; (ii) thiophosgene, 3 M HCl, rt, 2 h, 68–77%; (e) EDC·HCl, respective benzoic acid, dry DMF, rt, 1 h – 2 days, 13–54%; (f) PPSE, 150–170 °C, 2–7 h, 55–88%; (g) (i) piperidine, DMF, rt, 5 min; (ii) thiophosgene, 3 M HCl, rt, 2–4 h, 71–75%.

Fig. 1. Displacement of [³H]PSB-603 from the A_2BAR by (A) SO₂F-substituted LUF7982; (B) OSO₂F-substituted LUF7993 and (C) NCS-substituted LUF8002. Displacement measured after 0 or 4 h of pre-incubation of the respective ligand with CHO-spap membranes stably overexpressing the A_2BAR. Data represent the mean ± SEM of three individual experiments performed in duplicate.

Fig. 2. Wash-out assays on the adenosine A_2B receptor using the N¹-propyl xanthines with para-substituted warheads. CHO-spap cell membranes stably expressing the adenosine A_2B receptor were pre-incubated with buffer (vehicle) or 1 μM of ligand (10 μM in case of PSB-1115), followed by a four-cycle washing treatment (4× wash) or no washing at all (control) before being exposed to [³H]PSB-603. Data represent the mean ± SEM of three individual experiments performed in duplicate. Statistics were determined using unpaired student's t tests. ns: no significant difference; ****P < 0.0001.

Fig. 3. Predicted binding mode of LUF7982. Panel A: Overview of the key interactions of LUF7982 in the binding site, which include two hydrogen bonds (yellow dashed lines) with N254^6.55 and π–π stacking with F173^EL2, both are conserved interactions in adenosine receptor ligand recognition. The sulfonyl fluoride warhead points towards the extracellular vestibule. Panel B: Top view of the A_2BAR–LUF7982 binding pocket, showing potential lysine residues involved in covalent binding (K267^EL3 and K269^7.32).