Engineering of A₃ Adenosine and P2Y Nucleotide Receptors and Their Ligands

Abstract

Modification of the ribose moiety of nucleotides and nucleosides has provided new insights into structural and conformational requirements for ligands at P2Y nucleotide receptors and at adenosine receptors (ARs). Methanocarba derivatives (containing a rigid bicyclic ring system in place of ribose) of adenosine, ATP, ADP, UTP, UDP, and other receptor agonist analogs were synthesized. Biological evaluation led to the conclusion that in general the Northern (N)-conformation was favored over the Southern (S)-conformation of the pseudoribose moiety at A₁ and A₃ ARs and at P2Y₁, P2Y₂, P2Y₄, or P2Y₁₁ receptors, but not P2Y₆ receptors. At the hA₃ AR a new full agonist, MRS1898, the (N)-methanocarba equivalent of CI-IB-MECA (2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine), had a K_i value of 1.9 nM in binding to the hA₃ AR expressed in CHO cells. Functional assays confirmed the selectivity of MRS1898, although CI-IB-MECA was even more functionally selective for human A₃ vs. hA₁ and hA_2A ARs. Thirty μM MRS1898 did not induce apoptosis in HL-60 cells, suggesting that some of the proapoptotic effects of CI-IB-MECA may be nonreceptor-mediated. Manipulation of the sequence of A₃ ARs through site-directed mutagenesis has led to pharmacologically unique constructs: constitutively active receptors and “neoceptors.” Such engineered receptors may later prove to have potential for cardioprotection through gene transfer. Effects of single amino acid replacement were interpreted using a rhodopsin-based model of ligand-A₃ receptor interactions, leading to the proposal that a movement of the conserved W243 in TM6 may be involved in AR activation.

INTRODUCTION

We have adopted an integrated approach to the study of drug-receptor interactions in adenosine receptors (ARs) and P2 nucleotide receptors [Fredholm et al., 2001; Jacobson et al., 2002]. The receptor structure may be studied indirectly through exploring ligand structure-activity relationships (SAR) and directly by using mutagenesis and molecular modeling, based on the structure of rhodopsin. Goals of this structural approach are to generate a testable hypothesis for location of the binding site and subsequently to enable the rational design of new agonists and antagonists. Specifically, this review will describe recent advances both in the design of ligands, in which specific conformations are locked, and the mutation of the receptors, which have resulted in unique constructs that display unique pharmacological properties, such as constitutive activity. Finally, this review will present the concept of neoceptors, which are engineered for activation only by structurally tailored small molecules (“neoligands”) that are selective for the neoceptor vs. the native receptor.

LIGAND DESIGN

Modification of the ribose moiety of nucleotides and nucleosides has provided new insights into structural and conformational requirements for ligands at P2Y nucleotide receptors and ARs.

P2Y Receptor Ligands

In collaboration with the laboratory of T.K. Harden of the University of North Carolina, we studied the medicinal chemistry of nucleotides as agonists, partial agonists, and antagonists at P2Y receptors [Nandanan et al., 1999, 2000; Kim et al., 2001]. Molecular modeling of the docking of adenine nucleotides in the P2Y₁ receptor [Moro et al., 1998] has helped us predict that there is no specific interaction of the ribose ring oxygen with the receptor. Consequently, we replaced the ribose of adenine nucleotides with a variety of ribose substitutes lacking this oxygen, i.e., carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics. Biological evaluation led to the conclusion that affinity at the P2Y₁ receptor could be preserved or even enhanced by such substitution.

The ribose ring of pentose derivatives, such as adenosine and ATP, can adopt a range of conformations as described by a “pseudorotational cycle” [Marquez et al., 1996]. One approach to “freezing” the ribose-like moiety of nucleoside/nucleotide receptor ligands in what may be a receptor-preferred conformation is to use a carbocyclic modification in which the cyclopentane ring is sterically constrained through the inclusion of a second, fused ring (Fig. 1) [Marquez et al., 1996]. The “methanocarba” ring system, in which a fused cyclopropane moiety at either of two positions, has two conformational variations, depending on the position of fusion: Northern (N) or Southern (S) envelope conformations.

Fig. 1.

Structures of selected (N)-methanocarba analogs of nucleotides described in this review.

Methanocarba derivatives of ATP, UTP, UDP, and other nucleotide analogs (Fig. 1) were synthesized [Kim et al., 2002a; Ravi et al., 2002] by methods requiring some adaptation of standard procedures for nucleotide synthesis [Burgess and Cook, 2000]. The routine phosphorylation method using phosphorous oxychloride followed by a diphosphate (the “one pot” synthesis of nucleoside 5′-triphosphates) could not be used in the (N)-methanocarba series due to steric hindrance. When attempted, that method produced principally 3′,5′-cyclic monophosphate analogs [Kim et al., 2002a]. Instead, reaction of the 5′-hydroxyl group with a phosphoramidite reagent, followed by oxidation and deprotection, provided the 5′-monophosphates, which then were treated with 1,1′-carbonyldiimidazole for condensation with additional phosphate groups.

Professor H. Zimmermann and co-workers at the University of Frankfurt [Ravi et al., 2002] studied the stability of the methanocarba analogs of ATP and AMP toward the action of ectonucleotidases and concluded that in general cleavage occurs less readily with methanocarba nucleotides than with the native nucleotides. (N)- and (S)-methanocarba-ATP analogs were exposed to recombinant NTPDases 1 and 2 and methanocarba-AMP derivatives to recombinant 5′-nucleotidases. Phosphate cleavage by these ectonucleotidases occurring progressively closer to the 5′-position was increasingly impeded by the methanocarba modification (Table 1). The monophosphates were poor substrates for 5′-nucleotidase, suggesting that the activity of these compounds in pharmacological assays is less likely to result in receptor activation by their metabolites as compared to the unmodified, native nucleotides.

TABLE 1.

Rates of Hydrolysis of Nucleotide Analogs by Recombinant Ecto-Nucleotidases [Ravi et al., 2002]

Enzyme	NTPDase1	NTPDase2		5′-NT
ATP	100%	100%	AMP	100%
(N)-mcATP	52%	4%	(N)-mcAMP	0.14%
(S)-mcATP	57%	34%	(S)-mcAMP	0%
100%=	42.1	13.5		5.6
	nmol/min/106cells			nmol/min/μg prot

Harden and co-workers [Kim et al., 2002a] carried out functional assays of phospholipase C activated by the methanocarba nucleotide analogs through various P2Y receptors. There was a preference for the (N)- vs. the (S)-conformation of nucleotides acting at P2Y₁, P2Y₂, P2Y₄, and P2Y₁₁ receptors (Fig. 2), but not at P2Y₆ receptors. At recombinant human P2Y₁ and P2Y₂ receptors, (N)-methanocarba-ATP, MRS2340 (Figs. 1, 2A) was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist (note that the enantiomerically pure (S)-methanocarba-ATP was not prepared for comparison). MRS2340 activated P2Y₁₁ receptors with a potency similar to ATP. A (N)-methanocarba-2-methylthio-ADP analog, MRS2365 (Fig. 1), displayed an EC₅₀ at the hP2Y₁ receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate, i.e., 2-MeSADP. (N)-Methanocarba-UTP, MRS2341 (Figs. 1, 2B), was equipotent to UTP as an agonist at human P2Y₂ receptors and also activated P2Y₄ receptors with an EC₅₀ of 85 nM. (N)-Methanocarba-UDP was inactive at hP2Y₆ receptors.

Fig. 2.

Effects of ribose substitution of nucleoside 5′-triphosphates with (N) or (S)-methanocarba ring systems. Potency at turkey P2Y₁ and recombinant human P2Y receptors was determined [Kim et al., 2002a]. A: ATP and its analogs. B: UTP and its analog.

The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery by David Erlinge and co-workers (Univ. of Lund, Sweden) [Kim et al., 2002a]. MRS2341 was more potent than UTP in inducing a dilatory P2Y₄ response (pEC₅₀ = 6.1 ± 0.2). Consistent with its inactivity at recombinant P2Y₆ receptors, the corresponding diphosphate was inactive as either an agonist or an antagonist in a P2Y₆ receptor-mediated contractile response.

We have extensively explored the SAR of nucleotide bisphosphate analogs as P2Y₁ receptor antagonists [Nandanan et al., 2000], based on the initial discovery of receptor antagonism by adenosine-3′-phosphate-5′-phosphate (A3P5P) [Boyer et al., 1996]. The ability to constrain such analogs in a receptor-preferring conformation using the (N)-methanocarba ring system has aided the development of such antagonists as well as P2 receptor agonists. The (N)-methanocarba analog MRS2279 ((1R,2S,4S,5S)-1-[(phosphato)methyl]-4-(2-chloro-6-aminopurin-9-yl) bicyclo [3.1.0]-hexane-2-phosphate) proved to be a selective P2Y₁ receptor antagonist, with a K_B of 12 nM [Boyer et al., 2002]. The pharmacological profile of MRS2279 was characterized at recombinant P2Y receptors and in human platelets, in which it impedes the proaggregatory effects of ADP. As described by Harden and co-workers [Waldo et al., 2003], [³H]MRS2279 was synthesized and shown to be the first generally applicable radioligand for the P2Y₁ receptor.

In the antiviral field, acyclic analogs of nucleotides have proven exceptionally useful as inhibitors. We applied the acyclic approach to P2Y receptors. A series of acyclic nucleotide derivatives (all bisphosphates) were moderately potent P2Y₁ antagonists, demonstrating that the ribose moiety, or a cyclic substitute, was not required for antagonism of this receptor [Kim et al., 2001]. In fact, at least with respect to antagonists, the ribose appears to be nothing more than a spacer group, maintaining the preferred geometry of phosphate groups with respect to the adenine moiety. We evaluated the SAR of this series of acyclic antagonists in chain length and the terminal (phosphate or phosphate substitutes) groups. The optimal chain length occurred with MRS2298, which contains a single methylene group adjacent to the adenine moiety (Fig. 3A). MRS2298 displayed high selectivity for the P2Y₁ receptor [Brown et al., 2000]. The substitution of one of the phosphate groups with an uncharged ester group greatly reduced potency at the P2Y₁ receptor, indicating that both phosphates are required (Fig. 3B). An attempt to reduce the conformational degrees of freedom in the acyclic series by introducing a cyclopropyl ring in the alkyl chain failed to enhance potency [Kim et al., 2001].

Fig. 3.

Effects of (A) alkyl chain spacer length substitution and (B) terminal groups substitution on the antagonistic potency of bisphosphate P2Y₁ receptor antagonists. The optimal chain length antagonistic potency was found to be n = 1 (MRS 2298). plC₅₀ refers to inhibition of phospholipase C activated by 30 nM 2-MeSADP in turkey erythrocyte membranes [Kim et al., 2001].

AR Ligands

The analysis of ribose or ribose-like ring conformation also proved useful for studying preferred conformations at the ARs. As with certain P2Y receptors, ARs demonstrated a preference for the (N) conformation at A₁ and A₃ ARs. As demonstrated in binding assays, the affinity preserving or enhancing effects of the (N)-methocarba ring system were especially evident at the A₃ AR. (N)- and (S)-Methanocarba derivatives of adenosine and adenosine analogs substituted at the N⁶-, 2-, and/or 5′-positions were synthesized [Jacobson et al., 2000a; Lee et al., 2001].

Specific partial agonists selective at the A₃ AR receptor have been synthesized [Jacobson et al., 2000a] and characterized in CHO (Chinese hamster ovary) cells transfected with the hA₃ AR [Gao et al., 2002a]. A 2-H derivative (N)-Methanocarba N⁶-(3-iodobenzyl) adenosine, MRS1743 (Fig. 1) and its 2-chloro derivative, MRS1760, displayed K_i values of 9.2 and 1.9 nM, respectively, in binding to the hA₃ ARs and were highly selective partial agonists with high functional potency (EC₅₀ < 1 nM) but low efficacy (Table 2). These two partial agonists displayed cardioprotective properties (see below).

TABLE 2.

Affinity and Efficacy of Adenosine Derivatives in CHO Cells Expressing hA₃AR

Derivative	Ki (nM)a	EC50(nM)a	Efficacymaxc
Riboside
CI-IB-MECA	2.8	1.9	100%
(N)-Methanocarba
MRS 1743	9.2	0.70b	13%b
MRS 1760	1.9	0.67b	3%b
MRS 1898	1.9	2.1	100%

^aK_i values refer to inhibition of the binding of [¹²⁵I]I-AB-MECA and EC₅₀ refers to inhibition of cyclic AMP production, unless noted.

^bIn a functional assay based on guanine nucleotide binding [Jacobson et al., 2000], the efficacy of MRS 1743 and MRS 1760 was determined to be 45% and 22%, respectively.

^ccAMP effects.

MRS1898, the (N)-methanocarba equivalent of Cl-IB-MECA (2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine), had a K_i value of 1.9 nM in binding to the hA₃ AR expressed in CHO cells and was a full agonist [Lee et al., 2001; Gao et al., 2002a]. Binding assays confirmed the selectivity of MRS1898 (Fig. 4), although Cl-IB-MECA was even more selective (Table 2) for the hA₃ AR vs. hA₁ and hA_2A ARs.

Fig. 4.

Binding displacement curves for Cl-IB-MECA (A) and MRS1898 (B) at human ARs expressed in CHO cells. A₁ (n), A_2A(s), and A₃ (I). K₁ values (Cl-IB-MECA; MRS 1989) at the following human ARs are (nM): A₁ (1,240 ± 320; 136 ± 22), A_2A(5,360 ± 2,470; 784 ± 97), and A₃ (1.4 ± 0.3; 1.9 ± 0.4).

Effects of A₃ AR Agonists on Apoptosis

The prototypical selective A₃ AR agonists, IB-MECA and Cl-IB-MECA, at high (≥20 μM) concentrations have been noted to induce apoptosis in various cell lines [Kim et al., 2002b; Gessi et al., 2002]. The apoptotic effect of IB-MECA is distinct from the noted anticancer (cytostatic) properties of the compound [Fishman et al., 2002], which occur at lower concentrations. The synthesis of this novel, selective A₃ agonist MRS1898 allowed the further examination of mechanism of this cell death phenomenon [Kim et al., 2002b]. First, the effects of Cl-IB-MECA on apoptosis in human leukemia cell lines, HL-60 and MOLT-4, were investigated. Cl-IB-MECA (30 μM) increased the apoptotic fractions as determined using FACS analysis and activated caspase 3 and poly-ADP-ribose-polymerase (PARP). Known messengers coupled to A₃ AR (phospholipase C and intracellular calcium) did not seem to play a role in the induction of apoptosis. Selective A₃ AR antagonists did not prevent the induction of apoptosis during a 48-h exposure of the cells to Cl-IB-MECA. Furthermore, micromolar concentrations of the A₃ AR antagonist MRS1220, which would also antagonize A₁ and A_2A receptors, were also ineffective in preventing apoptosis. Although Cl-IB-MECA has been shown in other systems [Shneyvais et al., 2000; Gessi et al., 2002] to cause apoptosis through an A₃ AR-mediated mechanism, in these cells it appeared to be an AR-independent effect that required prolonged incubation. Unlike its ribose analog, 30 μM MRS1898 did not induce apoptosis in HL-60 cells (Fig. 5), supporting the conclusion that at least under certain conditions the proapoptotic effects of Cl-IB-MECA may be nonreceptor-mediated.

Fig. 5.

Induction of apoptosis in HL-60 (human leukemia) cells upon 48-h exposure to nucleoside analogs selective for the A₃ AR [Kim et al., 2002b]. These included the full A₃ AR agonist Cl-IB-MECA, low efficacy A₃ AR partial agonist MRS1760, and full A₃ AR agonist MRS1898 (each 30 μM). Evidence indicates that in this system, CL-IB-MECA at this high concentration is not acting through an AR-mediated mechanism.

Nevertheless, the induction of apoptosis by Cl-IB-MECA in leukemic cells displayed unique mechanistic features, e.g., dependence on the expression of Fas, a death receptor, which appeared not to depend on p53 [Kim et al., 2002b]. Cl-IB-MECA-induced apoptosis in HL-60 cells was augmented by the immunological agonism of Fas and was suppressed by antagonism of Fas. Therefore, 30 μM Cl-IB-MECA induced apoptosis in HL-60 and MOLT-4 cells via a novel, p53-independent up regulation of Fas.

SAR of Efficacy of Nucleoside Derivatives at A₃ ARs

In the typical pharmacological characterization of a series of receptor agonists, the binding affinity or potency are the criteria which give rise to a pattern of SAR [Müller, 2000]. The effects of structural modification of adenosine derivatives on the efficacy SAR at the A₃ AR have been studied recently in a systematic fashion by our and other laboratories [van Tilburg et al., 2002; Gao and Jacobsen, 2002; Gao et al., 2002a]. In general, the efficacy of adenosine derivatives at the A₃ AR appears to be readily reduced by certain adenine or ribose modifications, such as 2-chloro or N⁶-iodobenzyl substituents. Therefore, agonist derivatives that are used widely as pharmacological tools at other AR subtypes, such as A₁ ARs (see below), must not be assumed automatically to be agonists (albeit weaker) at the other subtypes.

The functional effects of the potent adenosine A₁ receptor agonists N⁶-cyclopentyladenosine (CPA) and 2-chloro-N⁶-cyclopentyladenosine (CCPA) were compared [Gao and Jacobsen, 2002]. In CHO cells expressing the hA₃ AR, CPA, but not CCPA, induced phosphoinositide turnover, indicating greatly reduced efficacy of CCPA at this subtype. CPA inhibited forskolin-stimulated cyclic AMP production (EC₅₀ value of 242 ± 47 nM). CCPA competitively antagonized the effects of the agonist Cl-IB-MECA with a K_B value of 5.0 nM. CPA competition curves vs. the selective A₃ AR antagonist radioligand [³H]PSB-11(8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one) [Müller et al., 2002] were right-shifted 4-fold by 100 μM GTP, whereas the competition by CCPA or the A₃ AR antagonist MRS1220 (N-[9-chloro-2-(2-furanyl) [1,2,4]triazolo[1,5-c] quinazolin-5-yl]benzene-acetamide) was not affected by GTP. Thus, CCPA proved to be a moderately potent antagonist (K_i = 38 nM) of the hA₃ AR.

The 2-chloro substitution of adenosine derivatives, in combination with certain N⁶-substitution (such as 3-iodobenzyl), caused a selective decrease in efficacy at both human and rat A₃ ARs [Gao et al., 2002a]. In addition to the 2-chloro group, other structural factors influencing the degree of efficacy at the A₃ AR were flexibility of the ribose ring and the presence of a uronamide group (such as in NECA and IB-MECA) at the 5′-position. Rigidity of the ribose-like moiety (such as in MRS1743) reduced efficacy; however, the addition of a 5′-uronamide group restored full efficacy in the cases examined. The efficacy-enhancement by the uronamide dominated all other modifications that were found to reduce efficacy.

A₃ RECEPTOR STRUCTURAL CHARACTERIZATION AND MANIPULATION

Molecular Modeling of ARs

We are exploring the steric and electronic requirements of the A₃ AR binding site using molecular modeling [Gao et al., 2002a,b]. The docking of several ligands into the putative binding site of the A₃ AR model was accomplished.

Mutagenesis of ARs

To provide new insights into ligand–receptor interactions, site-directed mutagenesis of the A₃ AR was carried out [Gao et al., 2002b]. While mutagenesis of A₁ and A_2a ARs has been performed [Tucker et al., 1994; IJzerman et al., 1996; Rivkees et al., 1999], only recently were single amino acids replaced in the A₃ AR. A number of amino acid residues in the transmembrane (TM) domains III, VI, and the second extracellular loop were individually replaced with Ala and other amino acids. These residues are homologous to those that have been predicted in previous molecular modeling studies of ARs [IJzerman et al., 1996, and references therein] to be involved in the ligand recognition, including: His95, Trp243, Ser247, Asn250, and Lys152. The N250A mutant receptor lost the ability to bind both radiolabeled agonist ([¹²⁵I]I-AB-MECA) and antagonist ([³H]PSB-11). The H95A (3.37) mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity approximately 3–38-fold, suggesting that these residues were required for the high affinity of A₃ AR antagonists. Activation of phospholipase C by wild-type (WT) and mutant receptors was measured. The A₃ agonist Cl-IB-MECA stimulated phosphoinositide turnover in the WT, but at concentrations as high as 100 μM failed to evoke a response in cells expressing W243A and W243F mutant receptors. Curiously, the binding affinity of Cl-IB-MECA (K_i = 2–3 nM) was unchanged in this functionally impaired W243 mutant. Thus, the binding modes of nucleoside agonists and nonnucleoside antagonists at the hA₃ AR are different. Residues needed for antagonist but not agonist binding include Trp243 and Lys152. The residues proposed to be in proximity to the bound selective agonist Cl-IB-MECA and to the bound nonselective antagonist CGS15943 are shown (Fig. 6). Residues that are needed for both antagonist and agonist binding include His95 (3.37), His272 (7.43), and Asn250 (6.55). Furthermore, it was possible to separate the structural bases for binding and activation processes. Trp243 was required for activation of the receptor but not for binding of agonists.

Fig. 6.

Summary of the results of human A₃ AR docking of a selective A₃ AR agonist, CI-IB-MECA (A), a nonselective, nonpurine AR antagonist, CGS15943 (B). Residues surrounding the putative binding site are shown [Gao et al., 2002a]. The residues in the double-squared box indicate identity among four subtypes of ARs and those in the single-squared box are conserved amino acids similarity among ARs. lntermolecular H-bonds are shown with arrows.

Hypothesis for Conformational Changes Involved in Activation of ARs

The results were interpreted using a rhodopsin-based model of ligand-A₃ AR interactions. We have proposed that a movement of the conserved W243 may be involved in A₃ AR activation [Gao et al., 2002a,b]. In the docked A₃ AR complexes, a major difference between agonist and antagonist binding was that agonist binding required a rotation of the indole sidechain of W6.48, which was important for constraining the intramolecular TM network through both hydrophobic and H-bonding interactions. This rearrangement upon agonist binding suggested that the destabilization of the ground-state structure through the disruption of the intramolecular TM network facilitated the conformational change from inactive to active form. The conserved P6.50 above W6.48 may act as a flexible hinge for the straightening of TM6 and subsequently TM7 to rearrange IL3 and helix 8 important for signaling to the G-protein, as proposed for the activation of rhodopsin [Ballesteros et al., 2001].

Cardioprotective Effects of AR Activation Suggest Gene Therapy Using Engineered A₃ Receptors

Full activation of A₃ ARs or co-activation of A₁ and A₃ ARs can induce potent anti-ischemic effects. Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent protective effects in heart muscle cells [Strickier et al., 1996]. Thus, a synthetic AR agonist, selective for either the A₁ or A₃ subtype, might be beneficial to the survival of the ischemic heart [Liang and Jacobson, 1998]. The activation of both receptors exerted a cardioprotective effect greater than the activation of either receptor alone.

We have characterized the cardioprotective effects of novel ligands, including: binary drugs and amino acid conjugates that bind to both A₁ and A₃ ARs (and less potently to A_2A ARs), and selective, partial A₃ agonists. The binary agonist MRS1741, consisting of covalently fused moieties derived from the A₃ agonist IB-MECA and the A₁ agonist ADAC, coactivated A₁ and A₃ ARs, and full cardioprotection (EC₅₀ ~0.1 nM) was dependent on expression of both subtypes [Jacobson et al., 2000b].

Partial A₃ agonists bearing the (N)-methanocarba ring system, e.g., MRS1760 (Fig. 1) and its 2-H analog, MRS1743, were further characterized at the level of native A₃ ARs in isolated chick cardiac myocytes. Both agonists were able to induce a marked protective effect against ischemia. However, the maximal protective effect of MRS1760 was less than the maximal effect caused by Cl-IB-MECA (P < 0.05, t-test). Consistent with the property of partial agonism, both MRS1760 and MRS1743 behaved as antagonists in the presence of Cl-IB-MECA by inhibiting the protection induced by a full agonist (Cl-IB-MECA). The underlying signaling mechanism for this protection was via phospholipase D, identical to that of the full A₃ agonist Cl-IB-MECA. The cardioprotective effect of MRS1760 was also demonstrated in isolated mouse hearts.

Manipulation of the sequence of A₃ ARs through site-directed mutagenesis has led to pharmacologically unique constructs that may later prove to have potential for cardioprotection through gene transfer.

Constitutively active mutant human A₃ ARs were created using single amino acid replacements, based on findings from other G-protein-coupled receptors [Chen et al., 2001]. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in functional assays of phospholipase C and adenylyl cyclase following expression in COS-7 cells (Fig. 7A).

Fig. 7.

Functional effects of constitutively active mutant human A₃ ARs [Chen et al 2001] (A) and enhancement of binding affinity of a human neoceptor derived from the A₃ AR, upon of a complementary functional group in the neoligand [Jacobson et al., 2001] (B).

A general concept of engineered “neoceptors” has been demonstrated for the hA₃ AR [Jacobson et al., 2001]. Such mutant receptors are designed to be activated only by “neoligands”, e.g., structurally modified adenosine derivatives of reduced potency at wild-type ARs. Molecular modeling suggested His272 in TM7 as a site for A₃ AR mutation, leading to selective activation of a receptor mutant (H272E) by adenosine derivatives that were modified in a chemically complementary fashion (Fig. 7B).

CONCLUSIONS

The design of novel ligands for purine and pyrimidine receptors, in some cases based on a rational, structural approach, has resulted in selective receptor probes that promise to be useful in pharmacological studies. These probes display enhanced potency and/or selective, and in some cases stability, over the previously used probes. The ribose ring has been a recent focus of this medicinal chemical effort in terms of effects of modification on both potency and efficacy of the nucleoside/nucleotide derivatives. In general the (N)-conformation was favored over the (S)-conformation for achieving selectivity for P2Y₁, P2Y₂, P2Y₄, or P2Y₁₁ receptors, but not P2Y₆ receptors, leading to the new P2Y₁ agonist MRS 2365. Also, the P2Y_2,4 agonist MRS 2341 and the P2Y₁ antagonists MRS 2297 and MRS 2298 have been characterized. At A₃ ARs, the new, potent agonist MRS 1898, containing a rigid (N)-methanocarba ring system that is locked in a receptor-preferred conformation, has been introduced. MRS 1898 has been shown to be free of the apoptosis-inducing effects previously observed with Cl-IB-MECA in human leukemic cells. Also, partial agonists of the A₃ AR MRS 1743 and MRS 1760 and A₁/A₃ AR binary agonists MRS 1740 and MRS 1741 have been reported and shown to be cardioprotective.

Advances in the general methods of molecular modeling and the resolution of the template structures of rhodopsin have brought this integrated perspective to a stage of practicality as a medicinal chemical approach to studying GPCRs in general and purine/pyrimidine receptors, specifically. Molecular models of the A₃ AR and P2Y₁ receptors have provided insights into the putative binding sites and recognition elements. Identification of microscopic complementarity between residues of the putative receptor binding site and the docked high-affinity ligands, i.e., energetically stabilizing elements in ligand recognition, has aided in the design process. The design of mutant A₃ ARs having unique pharmacological features has been accomplished, with the envisioned application in gene therapy. Constitutively active A₃ ARs and a neoceptor, which is activated selectively by tailored synthetic neoligands, have been designed.