The neoceptor approach was validated for ARs, which respond to stress-elevated levels of extracellular adenosine and have diverse protective roles against ischemia and tissue damage [20
]. Much experience has been gained to tailor ligands for the native ARs, and several selective agonists are in advanced clinical trials for inflammation, cancer, arthritis, and cardiac arrhythmias and imaging [20
]. Because of the widespread distribution of native ARs, agonist therapy has been impeded by side effects. The A2A
ARs have been developed as test cases of the neoceptor approach [2
] to address the issue of inherent nonselectivity of agonist-based therapies.
In general, hypotheses for receptor docking of nucleosides and the proposed conformational changes of GPCRs that are associated with activation have guided the design of neoligands. Modeling of the putative ligand-binding site of the A3
AR receptor [8
] led to the identification of a conserved site for mutagenesis, i.e. a His residue (7.43) that has been implicated in agonist recognition [8
]. Modeling of A1
ARs places His(7.43) within a hydrophilic ribose-binding region of the putative agonist binding site. This residue corresponds to the Lys of rhodopsin which forms the Schiff base with retinal, and in the AR has been proposed to be H-bonded to the 3′-hydroxyl group of adenosine [2
]. Mutagenesis of A2A
ARs indicates His(7.43) is associated with agonist binding and less important for antagonist binding.
Initially the A3
AR was converted into a neoceptor that can recognize uniquely modified nucleosides that are inactive at the native ARs [2
]. This concept is also dependent on the retention of the ability of the neoceptor to activate signaling pathways known to be beneficial in cardiac myocyte cultures for antiischemic protection, such as A3
AR-activated phospholipase D [4
His7.43 of the A3
AR receptor was mutated to Glu. A complementary functional group was incorporated in a synthetic neoligand, i.e. 3′-amino-3′-deoxyadenosine (MRS1960, ). A novel electrostatic pair forming between the neoceptor and neoligand was intended for selective recognition of MRS1960 by the carboxylate-modified receptor [2
]. The consequences of H272E mutation are: 1) Adenosine (100 μM) no longer binds to the receptor. However, the affinity of a standard agonist radioligand (125
I-I-AB-MECA) is fortuitously only 2-fold decreased. Thus, an important synthetic agonist tool may still be used to characterize the mutant receptor. MRS1960 and the mutant H272E A3
AR form a suitable association to achieve a 6-fold enhancement of binding affinity. A novel electrostatic pair would account for the enhanced recognition and subsequent activation of second messenger systems by the tailored ligand.
Figure 2 Modifications of the structure of adenosine leading to neoligands for A2A and A3ARs. Known SAR of adenosine derivatives have identified sites of modification for achieving receptor subtype selectivity and in some cases for tuning the efficacy. Cumulative (more ...)
Since the ratio of selective enhancement of MRS1960 was only modest, we improved the interaction based on prediction from molecular modeling [4
]. A 3′-aminomethyl neoligand (MRS3176 [26
]) achieved a 20-fold enhancement of affinity at the H272E mutant A3
AR. The impetus for this structural change was the prediction from molecular modeling that the 3′-amino group may be at an excessive distance from the His imidazole group to form a direct H bond. This modified nucleoside also contained a substituted N6
-benzyl group, which tends to enhance affinity at the wild type and mutant A3
Thus, ligands may be tailored with strategically-positioned amino groups for recognition by carboxylate mutants of the native receptor. However, a more dramatic demonstration of selective affinity enhancement and orthogonality was desired. The substituent at the 3′-position of ribose was varied in charge, size, and H bonding ability, and each analogue was examined for binding affinity at several neoceptor variations. The optimal affinity enhancement and orthogonality followed the incorporation of a urea group in place of the 3′-hydroxyl group of adenosine analogues, as in MRS3481 (). The urea group is capable of forming multiple H bonds with the neoceptor and appears to preclude binding at the native ARs for steric reasons [27
]. A model of this analogue docked in the H272E receptor showed a bidentate coordination of between the carboxylate group and the urea moiety ().
Figure 3 Docking of agonist Cl-IB-MECA in the native human A3AR (left panel) and a neoligand, MRS3481, in the H272E neoceptor (right panel). The homology models were derived from human A3AR model based on rhodopsin and resembling the meta I state .
It was necessary to probe the coupling pattern of the neoceptor [4
]. Although the coupling specificity in GPCRs is principally a function of the second and third intracellular loops [28
], and ligand specificity is governed by functionality within the upper third of the TM regions, the preservation of the typical A3
AR second messengers could not be assumed. Coupling to one known effector pathway of the A3
AR, i.e. stimulation of phospholipase C through the Gβ,γ-subunits, is preserved upon activation of the H272E mutant receptor by MRS3481. Thus, at least part of the downstream signaling of this cytoprotective receptor is maintained. The stimulation of phospholipase C by MRS3481 occurred with an EC50
value of ~100 nM at the neoceptor, while it was inactive at 100 μM at the WT receptor similarly expressed in COS-7 cells. For comparison, the EC50
for adenosine at the WT receptor was ~1 μM and >100,000 at the neoceptor. The effects of MRS3481 acting through the H272E neoceptor on other signaling systems (e.g., cyclic AMP, ion channels, and arrestin) remain to be determined.
In a chick cardiac myocyte culture, which is an established model for cardioprotection [29
], the neoligand MRS3481 induced a potent antiischemic protection in cells expressing the H272E neoceptor [4
]. Also, this protection correlated with the activation of PLD, as occurs with the native A3
AR transfected in the same cell system. Thus, a neoceptor-neoligand pair has been demonstrated to be beneficial in inducing stages of a response to stress in a tissue known to respond similarly when the native parent receptor is present.
The anti-inflammatory A2A
AR was also converted into a neoceptor [3
]. The same approach of mutation of the conserved His278 in TM7 could not be used, because mutation of the corresponding His residue to Asp or Glu did not lower the potency of native adenosine. However, a hydrophilic residue, i.e. T88 in TM3, on the other side of the putative subdomain for ribose binding to the ARs was selected for mutation. This residue in the A2A
AR is exclusively associated with agonist binding [3
]. The T88D mutant receptor was unaffected in the ability to bind the nonselective AR antagonist CGS15943, however it failed to bind the nonselective AR agonist NECA, even at a concentration of 100 μM. The T88D mutant A2A
AR recognized a strategically 5′-modified amino derivative (MRS3366, ). Moreover, the precise position and spacer length of the amino group was critical to achieving a selective affinity enhancement at the neoceptor. Other mutant A2A
ARs displayed even greater degrees of enhancement for neoligands. For example, MRS3417 was functionally enhanced at the N181D mutant receptor by 110-fold, however, this combination was not truly orthogonal since this modified receptor was still capable of being activated by known AR agonists.
In addition to mutating the ligand binding pocket, it is also possible to mutate sites involved in phosphorylation and desensitization to retard these processes in neoceptors. This is particularly important with regard to A3 receptors since these are known to undergo exceptionally rapid desensitization. It should also be possible to mutate promoter regions of the neoceptor transcript. This could be used to enable induction of the neoreceptor mRNA - thus adding an additional layer of control in the response to the neoligand.