Orthogonal activation of the reengineered A3 adenosine receptor (neoceptor) using tailored nucleoside agonists

Abstract

An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A(3) adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3': amino, aminomethyl, azido, guanidino, ureido; and at 5': uronamido, azidodeoxy. N(6)-Variations included 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N(6)-3-iodobenzyl-3'-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC(50) = 0.18 microM) or phospholipase D in chick primary cardiomyocytes, both mediated by a mutant (H272E), but not the wild-type, A(3)AR. The affinity enhancements for 10 and the corresponding 3'-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A(3)AR (EC(50) of 1.0 microM), but had no effect on the H272E mutant A(3)AR (100 microM). Compound 10 was inactive at human A(1), A(2A), and A(2B)ARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.

Fig. 1

Binding inhibition and functional activation in wild-type and H272E mutant A₃ARs. The receptors were expressed transiently in COS-7 cells as described in Experimental Procedures. The binding affinity (K_i) was determined by using the agonist radioligand [¹²⁵I]I-AB-MECA (0.5 nM). The structure of compound 10 is given in Table 1.

Fig. 2

Activation of the neoceptor by the neoagonist 10 protects heart cells from ischemia-induced injury. A) Activation of PLD by 10 in chick cardiomyocytes expressing the mutant human H272E A₃AR. B) Effect of 10 on anti-ischemic cardioprotection in neoceptor-transfected cardiac myocytes is shown. Cardiac ventricular myocytes were transfected with cDNA encoding the neoceptor H272E, and the percentage of cells killed was determined in the absence or the presence of 10 during the 90-min simulated ischemia, as described in Experimental Procedures. Data were plotted as the percentage of cells killed during the prolonged simulated ischemia. #P < 0.01 compared with control (ANOVA). ANOVA (all four groups), F = 15.6, P < 0.0001; all posttest comparisons were significant at P < 0.01 except for the percentage of cells killed at 300 nM 10 compared with 1,000 nM and that for 100 nM compared with 300 nM.

Fig. 3

Two energetically favorable binding modes of the N⁶-(3-I-benzyl)-3′-ureidoadenosine 10 in the binding site of the mutant H272E hA₃AR. The binding mode similar to the hA₃/Cl-IB-MECA complex (A) and another binding mode (B), which was energetically unfavorable in the hA₃/Cl-IB-MECA complex, are shown. All ligands are displayed as ball-and-stick models in the atom-by-atom color, and the side chains of the hA₃AR are shown as stick models. The H-bonding between each ligand and the mutant hA₃AR is displayed in yellow. The A₃AR is represented by a tube model with a different color for each TM (TM2 in orange, TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple).

Fig. 3

Two energetically favorable binding modes of the N⁶-(3-I-benzyl)-3′-ureidoadenosine 10 in the binding site of the mutant H272E hA₃AR. The binding mode similar to the hA₃/Cl-IB-MECA complex (A) and another binding mode (B), which was energetically unfavorable in the hA₃/Cl-IB-MECA complex, are shown. All ligands are displayed as ball-and-stick models in the atom-by-atom color, and the side chains of the hA₃AR are shown as stick models. The H-bonding between each ligand and the mutant hA₃AR is displayed in yellow. The A₃AR is represented by a tube model with a different color for each TM (TM2 in orange, TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple).

Scheme 1

Reagents and conditions: (a) Tf₂O, pyridine, 0 °C, 1 h; (b) NaN₃, DMF, rt, 48 h; (c) i) 75% AcOH, 55 °C, 1.5 h; ii) NaIO₄/H₂O, EtOH, 0 °C, 20 min then NaBH₄; (d) Ac₂O, pyridine, rt, 3 h; (e) i) 85% HCO₂H, 60 °C, 1.5 h; ii) Ac₂O, pyridine, rt, 16 h; (f) Silylated 6-chloropurine or 2,6-dichloropurine, TMSOTf, C₂H₄Cl₂, 0 °C to 60 °C, 2 h; (g) MeNH₂, 1,4-dioxane, rt, 4 h or 3-iodobenzylamine hydrochloride, Et₃N, EtOH, 50 °C, 18 h then NaOMe, MeOH, rt, 2 h; (h) TBSCl, imidazole, DMF, rt, 24 h; (i) Ph₃P, NH₄OH/H₂O, THF, rt, 18 h; (j) Chloroacetyl isocyanate, DMF, 0 °C, 3 h; (k) NaOMe, MeOH, rt, 18 h; (l) TBAF, THF, rt, 4 h. IB = 3-iodobenzyl.

Scheme 2

Reagents and Conditions: (a) i) 75% AcOH, 55 °C, 1.5 h; ii) NaIO₄, RuCl₃.H2O, CCl₄/CH₃CN/H₂O, rt, 4h; (b) i) (COCl)₂, DMF, CH₂Cl₂, rt, 16 h; ii) 2M NH₂CH₃, CH₂Cl₂, 0 °C, 3 h; (c) AcOH/Ac₂O/c-H₂SO₄, rt, 16 h; (d) Silylated 6-chloropurine, TMSOTf, C₂H₄Cl₂, 0 °C to 60 °C, 2 h; (e) MeNH₂, 1,4-dioxane, rt, 4 h or 3-iodobenzylamine hydrochloride, Et₃N, EtOH, 50 °C, 18 h then NaOMe, MeOH, rt, 2 h; (f) TBSCl, imidazole, DMF, rt, 24 h; (g) Ph₃P, NH₄OH/H₂O, THF, rt, 18 h; (h) Chloroacetyl isocyanate, DMF, 0 °C, 3 h; (i) NaOMe, MeOH, rt, 18 h; (j) TBAF, THF, rt, 4 h or Et₃N.3HF, THF, 50 °C, 16 h. IB = 3-iodobenzyl.