G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

Abstract

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A_2AAR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson’s disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.

Introduction

There are four subtypes of adenosine receptors (ARs, or alternately P1 receptors), i.e., A₁, A_2A, A_2B, and A₃, and eight subtypes of P2Y receptors (P2YRs), i.e., a family of G_q-coupled P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁Rs and a second family of G_i-coupled P2Y₁₂, P2Y₁₃, and P2Y₁₄Rs (Table 1) [1, 2]. The native agonists for these twelve receptors are clearly divided between purine nucleosides (ARs) and purine and pyrimidine nucleotides (P2YRs), although a high concentration of AMP (1 mM) activates the A₁AR, independent of P2YR activity [3]. The two A₂ subtypes are coupled to G_s protein to stimulate adenylate cyclase, and the other two AR subtypes inhibit adenylate cyclase through G_i protein. In some cells, the A_2BAR is dually coupled to G_s and G_q and consequently elevates phosphoinositides, mobilizes calcium and activates phospholipase C and MAPK [111–115]. This signaling pathway appears to be important in mast cells, in which A_2A and A_2BARs have opposing actions. ARs are the site of action of widely consumed alkylxanthines, which act as competitive antagonists. Knockout mice and selective ligands as pharmacological tools (Figs. 1, 2, and 3) are now available for all AR subtypes and for many of the P2Y subtypes (except for P2Y₁₁ that is absent in the mouse) [4]. Many ligands for these receptors are under consideration for pharmaceutical development.

Table 1.

Properties of ARs and P2Y receptors and key ligands (including radioligands) suitable for cell biological studies (Ado = adenosine)

Group	Subtype, gene symbol	Chromosome, human, (length, amino acids)	Native agonist (human, pEC50)	Selective agonist (pEC50)	Selective antagonist (pIC50)	G protein
ARs	A1, Adora1	1q32.1 (326)	Ado 1 (6.51)	S-ENBA 13 (9.47), CCPA 8 (9.08),dR-PIA 6 (8.82),d CPA 7 (8.65), CHA 11 (8.64),d GR79236 10 (8.51), SPA 5 (6.2)	PSB-36 38 (9.8), KW3902 37 (9.1), SLV320 39 (9.0), DPCPX 36 (8.5)d	Gi, Go
	A2A, Adora2a	22q11.23 (412)	Ado 1 (6.14)	ATL-146e 18 (9.30), UK-432097 19 (8.40), CGS21680 16 (7.64)d	ZM241385 44 (9.0),d SCH442416 47 (8.39), SCH58261 46 (8.3)	Gs, Golf
	A2B, Adora2b	17p12 (332)	Ado 1 (4.59)	Bay60-6583 21 (8.0)	PSB-603 57 (9.3),d MRS1754 52 (8.9),d MRE2029-F20 54 (8.8),d PSB-0788 58 (8.6),d MRS1706 53 (8.4), PSB-1115 56 (7.7), CVT-6883 55 (7.7), PSB-298 (7.2)d	Gs, Gq
	A3, Adora3	1p13.2 (isoform 1: 347)	Ado 1 (6.53), inosine (6.60)	IB-MECA 23 (8.85), Cl-IB-MECA 24 (8.74), I-AB-MECA (8.50),d CP532,903 25 (8.24)	MRE3008-F20 62 (9.1),d MRS1220 64 (8.8), MRS1334 66 (8.6), VUF5574 67 (8.4), PSB-11 61 (8),d MRS1523 63 (7.7), MRS1191 65 (7.5), MRS3777 69 (7.3)	Gi
P2Y1-like	P2Y1, P2RY1	3q24-25 (373)	ADP 70 (5.09)	MRS2365 76 (9.40)	MRS2500 95 (9.02),d MRS2279 94 (8.10),d MRS2179 93 (6.48),d	Gq
	P2Y2, P2RY2	11q13.5 (377)	UTP 79 (8.10), ATP 71 (7.07)	MRS2698 (8.10), MRS2768 90 (5.72)	PSB-716 (5.01)	Gq, Gi
	P2Y4, P2RY4	Xq13 (365)	UTP 79 (5.60)a	MRS4062 83 (7.64)	b	Gq, Gi
	P2Y6, P2RY6	11q13.5 (328)	UDP 78 (6.52)	5-iodo-UDP 85 (7.83), PSB-0474 84 (7.15)	MRS2578 109 (7.43) [noncompetitive]	Gq
	P2Y11, P2RY11	19p31 (374)	ATP 71 (4.77)	NF157 103 (7.35), NF546 104 (6.27)c	NF340 105 (7.14)	Gq, Gs
P2Y12-like	P2Y12, P2RY12	3q21-25 (342)	ADP 70 (7.22)d	b	PSB-0739 101 (9.8), AR-C69931MX 98 (9.40), AZ11931285,d PSB-0413 (8.3),d AZD6140 99 (7.90)	Gi
	P2Y13, P2RY13	3q24-25 (354)	ADP 70 (7.94)	b	MRS2211 108 (5.97)	Gi
	P2Y14, P2RY14	3q24-25 (338)	UDP 78 (6.80),dUDP-glucose 88 (6.45)	MRS2690 89 (7.31), MRS2802 (7.20)	Compound 116 (8.7)	Gi

^aATP acts as an antagonist at the human P2Y₄ receptor and as agonist at the rat and mouse P2Y₄ receptors

^bSelective ligands not yet available

^cNF546 activates the P2Y₁₁R, although it belongs to a structural class of antagonists

^dUsed as a radioligand, either in [³H], [³²P], [³³P] or [¹²⁵I] form, as appropriate. The selectivity of [¹²⁵I]I-AB-MECA (4-amino analogue of 23) for the A₃AR is low, and therefore binding to A₁AR is also seen. The listed A₃AR antagonist radioligands are suitable for use in primate but not rodent species. Other (nonselective) radioligands are: agonist [³H]NECA 3 for A_2AAR, A_2BAR or A₃AR; agonist [¹²⁵I]I-APNEA (3-iodo analogue of 4) for the A₃AR; agonist [³H] or [³³P]2-MeSADP 72 for P2Y₁R or P2Y₁₂R. [³³P]ADP 70 is also used for binding to the P2Y₁₂R. [³H]UDP 78 (K_d 10 nM) has been used for binding to the P2Y₁₄R. Chemical names: [³H]PSB-0413, 2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride; [³H]PSB-298, [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine]; [¹²⁵I]AZ11931285 (used at 125 pM), (1S,2R,3S,4R)-2,3-dihydroxy-4-[7-[[(2E)-3-iodoprop-2-en-1-yl]amino]-5-(propylthio)3H-[1–3]triazolo[4,5-d]pyrimidin-3-yl]cyclopentane-carboxylic acid

Fig. 1.

a Nonselective AR and A₁AR selective agonists (including nucleosides and a nonnucleoside derivative 15). b A_2AAR, A_2BAR, and A₃AR selective agonists (including nucleosides and a nonnucleoside derivative 21). AR affinites (Table 1) and selectivities of many of these ligands are available [1, 55]

Fig. 2.

a. Nonselective AR antagonists and A₁AR and A_2AAR selective antagonists (including xanthines and nonxanthine derivatives 44–51). b A_2BAR (xanthines 52–59) and A₃AR selective (nonxanthine derivatives 60–69) antagonists. Compound 68 is a truncated nucleoside derivative that displays A₃AR antagonist properties. AR affinites (Table 1) and selectivities of many of these ligands are available [1, 42, 55]

Fig. 3.

a Nonselective and selective P2YR agonists and related substances (including nucleotide derivatives). b Nonselective and selective P2YR antagonists (including nucleotide and nucleotide derivatives). Note that NF546 104 is a P2Y₁₁R agonist, although it belongs to a structural class of antagonists. Compound 110 is a P2Y₁R antagonist containing a novel chemotype, and 111–115 are P2Y₁₂R antagonists. Compound 116 is a P2Y₁₄R antagonist containing a novel chemotype. P2Y potencies of many of these ligands are available in Table 1 or reference [2]

Processing of ARs and P2YRs in the cell has been studied, including posttranslational modification and trafficking, intracellular localization, and the related phenomena of agonist-induced desensitization, internalization, and degradation [5–7]. The mechanisms of release, uptake, and degradation of extracellular nucleosides and nucleotides have also been explored [8, 9]. The ubiquitous presence of pharmacologically active endogenous ligands of ARs and P2YRs warrants careful consideration in experimental design.

Structure of adenosine and P2Y receptors

The ARs and the P2YRs share the overall topological structure typical of G protein-coupled receptors (GPCRs) belonging to family A: seven α-helical domains (TM) that cross the cell membrane and are connected by three extracellular (ELs) and three intracellular (IL) loops, the N terminus in the extracellular part and the C terminus in the intracellular part of the receptor. Nevertheless, from comprehensive sequence comparisons and phylogenetic analyses, it is clear that ARs and P2YRs belong to two different groups of the rhodopsin-like family of GPCRs [10], respectively, the rhodopsin α-group and γ-group of GPCRs [11].

Within the AR family, the average sequence identity between subtypes of the same species is about 47%, which increases to an average of ∼57% if only the TM domains are considered. The residues in the binding cavity involved in ligand recognition are mostly conserved among the AR subtypes and between species, with the A₃AR being the most divergent from the other subtypes, as shown in Table 2. Specific variable amino acids in the binding site are most likely involved in the ligand selectivity or the unique pharmacological behavior of each AR subtype.

Table 2.

List of the key residues in the binding pocket of the A_2AAR compared with the corresponding residues in other AR subtypes and in ARs of different species (h, human; m, mouse; r, rat)

Rec/Res	TM2 2.61	TM3 3.32	TM3 3.33	TM3 3.36	TM3 3.37	EL2	EL2	TM5 5.38	TM5 5.42	TM6 6.48	TM6 6.51	TM6 6.52	TM6 6.55	TM7 7.35	TM7 7.39	TM7 7.42	TM7 7.43
h_A1	A66	V87	L88	T91	Q92	F171	E172	M180	N184	W247	L250	H251	N254	T270	I274	T277	H278
m_A1	A66	V87	L88	T91	Q92	F171	E172	M180	N184	W247	L250	H251	N254	I270	I274	T277	H278
r_A1	A66	V87	L88	T91	Q92	F171	E172	M180	N184	W247	L250	H251	N254	I270	I274	T277	H278
h_A2A	A63	V84	L85	T88	Q89	F168	E169	M177	N181	W246	L249	H250	N253	M270	I274	S277	H278
m_A2A	A60	V81	L82	T85	Q86	F163	E164	M172	N176	W241	L244	H245	N248	M265	I269	S272	H273
r_A2A	A60	V81	L82	T85	Q86	F163	E164	M172	N176	W241	L244	H245	N248	M265	I269	S272	H273
h_A2B	A64	V85	L86	T89	Q90	F173	E174	M182	N186	W247	V250	H251	N254	M272	I276	S279	H280
m_A2B	A64	V85	L86	T89	Q90	F173	E174	M182	N186	W247	V250	H251	N254	M272	I276	S279	H280
r_A2B	A64	V85	L86	T89	Q90	F173	E174	M182	N186	W247	V250	H251	N254	M272	I276	S279	H280
h_A3	A69	L90	L91	T94	H95	F168	V169	M177	S181	W243	L246	S247	N250	L264	I268	S271	H272
m_A3	A70	L91	L92	T95	H96	F169	R170	M178	S182	W244	L247	S248	N251	M265	I269	S272	H273
r_A3	A71	L92	L93	T96	H97	F170	R171	M179	S183	W245	L248	S249	N252	M266	I270	S273	H274

Residues are denoted in the top row by a numbering convention as described [20]

Images displaying the role in ligand binding of amino acids in the binding site of various ARs are found in references [12–19, 62]

For the ARs, structural information has been available since 2008 with the high resolution X-ray structure of the human A_2AAR in complex with the antagonist 4-(2-[7-amino-2-(2-furyl)]1,2,4] triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol 44 (ZM241385) [12]. The crystal structure of the A_2AAR in its inactive conformation gave insight into the ligand recognition mechanism, showing the key residues involved in the ligand binding and the major interactions anchoring the antagonist to the binding site. Many of the site-directed mutagenesis data previously available for the ARs were structurally explained, and new mutational experiments were guided by the knowledge gained from the A_2AAR structure, helping to further define the ligand binding cavity of this AR [13].

The antagonist-bound A_2AAR structure has since improved widely the modeling approaches to the ARs, suggesting for example the possible binding modes of agonists to the A_2AAR [13, 14] or aiding the modeling of the other AR subtypes [15]. Before the release of the A_2AAR X-ray structure, other structural templates were used for the modeling of the ARs, as detailed in a recent review by Dal Ben et al. [16]. More recently, new crystal structures of a thermostabilized (by mutagenesis) A_2AAR in complex with the xanthine derivative 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-l,3-dipropylxanthine (XAC, structure not shown) and caffeine 30 were made available [17]. New structural insights into the activation mechanism and the conformational changes that occur upon agonist binding to the A_2AAR were revealed with the recent release of new crystal structures of the A_2AAR in complex with different agonists, the bulky substituted agonist 2-(3-[1-(pyridin-2-yl)piperidin-4-yl]ureido)ethyl-6-N-(2,2-diphenylethyl)-5′-N-ethylcarboxamidoadenosine-2-carboxamide 19 (UK432097) [18], the native agonist adenosine 1, and the non-selective adenosine-5′-N-ethyluronamide 3 (NECA) [19]. These new crystal structures lack a coupled G protein, but nevertheless, they are helpful in understanding the function of the ARs, and they will aid the drug design approaches for the AR family, thereby also improving the quality of models for other AR subtypes. The conformational changes upon activation of the A_2AAR resemble those of opsin, but there are other changes more specific to this receptor structure such as a see-saw movement of TM7 [18]. Homology models of A₁AR and A₃AR based on the agonist-bound structure of the A_2AAR, were recently proposed with docked agonists in the binding site [15].

The crystal structures of the A_2AAR revealed a peculiar orientation of the ligand in the binding site of the receptor when compared to the available structures of other GPCRs, e.g., bovine rhodopsin or the β-adrenergic receptors. The binding site for the ligands in the ARs is located near the extracellular portion of the domains TM3, TM5, TM6, and TM7, and the ligands are in a roughly vertical orientation with respect to the plane of the membrane. A crucial residue anchoring the aromatic core of agonists and antagonists in the binding pocket of A_2AAR is Asn6.55 (using the numbering convention of Ballesteros and Weinstein [20]), a residue conserved among the AR subtypes and also among different species. His6.52, Thr3.36, Ser7.42, and His7.43 play key roles in the binding of the hydrophilic ribose moiety of nucleoside agonists, while they are less critical in the binding of antagonists. Residues from the extracellular domain EL2 are also involved in anchoring ligands in the binding pocket. Phe168 in A_2AAR, a residue conserved among the ARs, interacts through a strong π–π stacking with the aromatic core of agonists and antagonists. Glu169, conserved in A₁AR and A_2BAR but substituted with a hydrophobic valine in human A₃AR, interacts with both agonist and antagonist H-bond donor groups, i.e., the exocyclic amino group of ZM241385, the exocyclic amino groups of NECA and adenosine, or the urea moiety at the C2 position of UK432097. Trp6.48, the so-called “toggle switch” of GPCR activation, is conserved among the ARs and was found in close proximity to the ligands in the A_2AAR complexes. While the residues surrounding the ligand core in the A_2AAR structure are mostly conserved in the binding pocket of the AR subtypes, other residues are less conserved, and possibly, they might be involved in the selectivity of the receptors for different substituted ligands. Those less conserved residues are located mainly in the most extracellular part of the binding cavity embedding the substitutuent groups projecting from the core of the ligands. For example, Leu267(7.32) in A_2AAR is substituted with a serine in A₁AR, with a lysine in A_2BAR, and a glutamine in A₃AR. Met270(7.35) in A_2AAR is a threonine in A₁AR and a leucine in A₃AR. Also in EL3, there are some nonconserved residues, such as His264 of A_2AAR, which is substituted with an asparagine in A_2BAR and a glutamate in A₃AR (Table 2).

Sequence alignments, phylogenetic analysis, and effector coupling of the P2YRs have distinguished two P2YR subfamilies [2, 21]. The P2Y₁-like family activates the phospholipase C signaling pathway through coupling with G_q protein. P2Y₁₁R also couples with G_s protein to activate adenylate cyclase. The other family of P2YRs is the P2Y₁₂-like family, which couples to G_i protein to inhibit the adenylate cyclase pathway [23, 24]. The sequence identity between the two subfamilies is quite low, with only 20% identity between P2Y₁R and P2Y₁₂R, while the sequence identity is higher between the members within the same subfamily, for example, with a 45% identity between P2Y₁₂R and P2Y₁₄R.

Unlike the ARs, no experimentally determined structural information is yet available for the P2YR family, and so far, the only structural characteristics of the P2YRs have come from structural modeling [21, 25–28]. Mostly, the modeling of P2YRs has focused on identifying the putative binding site and the analysis of the residues involved in the ligand binding and receptor specificity, with the aim to gain information on the ligand recognition mechanism. Site-directed mutagenesis and structure–activity relationship (SAR) analysis have been used to support and guide the modeling of the P2YRs [29–36], which has been used to identify new key residues important for the ligand binding and receptor activation [25, 37–39]. Several models based on different structural templates have been published for many of the P2YRs. The bovine rhodopsin crystal structure was used to build models for P2Y₁R [21, 39–41], P2Y₂R [31, 35, 42], P2Y₄R [42], P2Y₆R [43, 44], P2Y₁₁R [45], P2Y₁₂R [21], and P2Y₁₄R [46].

The putative binding pocket of the P2Y₁R, suggested by the modeling and supported by the many available mutagenesis data, is located near the extracellular region of TM3, TM6, and TM7. The positively charged residues of P2Y₁R Arg3.29, Lys6.55, and Arg7.39, conserved among the P2Y₁-like receptors (Table 3), appear to be involved in the coordination of the negatively charged phosphate groups of nucleotide ligands. Hydrophilic residues in the binding pocket were suggested to surround the ribose moiety of the nucleotides, while hydrophobic residues created a favorable environment for the aromatic nucleoside core of the nucleotides derivatives. Position 6.52 is a histidine residue that is conserved across the P2YRs, and mutagenesis studies on P2Y₁R, P2Y₂R, and P2Y₁₂R have implicated this residue in ligand recognition [28, 33, 35, 47]. Mutagenesis studies on the P2Y₁R also showed the crucial role of two disulfide bridges for the correct function of the receptor: a disulfide bridge between TM3 and EL2, conserved among family A GPCRs, and a second disulfide bridge between the N terminus and EL3 [32].

Table 3.

List of the key residues in the putative binding pocket of the human P2YRs

Rec/Res	TM3 3.29	TM3 3.32	TM3 3.33	TM3 3.37	TM5 5.47	TM6 6.48	TM6 6.51	TM6 6.52	TM6 6.55	TM6 6.58	TM7 7.35	TM7 7.36	TM7 7.39
P2Yb1	R128	F131	H132	Y136	F226	Y273	F276	H277	K280	N283	Y306	Q307	R310
P2Yb2	R110	F113	Y114	Y118	F207	F258	F261	H262	R265	Y268	Y288	K289	R292
P2Y4	R112	F115	Y116	Y120	F209	F258	F261	H262	R265	Y268	Y288	K289	R292
P2Y6	R103	F106	Y107	H111	F201	F252	F255	H256	K259	Y262	Y283	K284	R287
P2Y11	R106	F109	T110	L114	C214	Y261	Y264	H265	R268	N271	Y303	Q304	R307
P2Y12	S101	F104	Y105	Y109	F198	F249	F252	H253	R256	Y259	K280	E281	L284
P2Y13	S99	F102	Y103	Y107	F217	F247	F250	H251	R254	Y278	K278	E279	L282
P2Y14	A98	F101	Y102	Y106	F195	F246	F249	H250	R253	Y256	K277	E278	L281

Residues are denoted in the top row by a numbering convention as described [20]. Residues in bold have been shown using site directed mutagenesis to be important in ligand recognition

Images displaying the role in ligand binding of amino acids in the binding site of various P2YRs are found in references [25–27, 31, 33, 35, 38, 40, 42–47, 49, 50]

Residues in the ELs that affect ligand recognition include: D204, E209, and R287 (in P2Y₁R) [31]; R177, R180, and R272 (in P2Y₂R) [31]

The P2YR models have been improved and refined continually using updated information from studies of their structural biology and mutagenesis. Recent advances in the structural biology of GPCRs have provided alternative templates to the bovine rhodopsin structure as a basis for the homology modeling. For example, the sequence identity between rhodopsin and human P2Y₁₂R, the site of action of the active metabolite [37] of the blockbuster antithrombotic Clopidogrel [2], is only 16% overall and 19% for only the TM regions, while the sequence identity between human P2Y₁₂R and the chemokine receptor CXCR4 is 22% overall and 26% for the TM domains. The X-ray structures of the human CXCR4 in its inactive state in complex with a small antagonist and a long peptide were released in late 2010 [48]. A sequence comparison between P2Y₁₂R and CXCR4 suggested other structural features of CXCR4 that might be shared by P2Y₁₂R, making the crystal structure of CXCR4 a more suitable template than other available GPCR crystal structures for the modeling of P2Y₁₂ and other P2YRs [49, 50]. A model of P2Y₁₂R based on the CXCR4 crystal structure and guided by the mutagenesis data and SAR studies available on the P2Y₁₂R was recently published [49]. The homology models of P2Y₂R and P2Y₄R based on the CXCR4 crystal structure were used to explain the selectivity of agonists toward these two P2YR subtypes [50]. The modeling studies of P2Y₁₂R showed how key residues in the binding pocket in TM6 and TM7, Arg6.55, Lys7.35, and Tyr6.58, were involved in the anchoring of the negatively charged phosphate groups of the nucleotide ligands. Those residues are conserved across the P2Y₁₂-like subfamily of P2YRs, as shown in Table 3. Other hydrophobic or aromatic residues from TM1, TM3, TM6, and TM7 were suggested to form a suitable environment for the aromatic core of nucleotides derivatives, while the ribose moiety was surrounded by hydrophilic residues.

Key ligand tools for studying adenosine and P2Y receptors

AR agonists and antagonists

Selective agonist and antagonist ligands for each of the four AR subtypes are now available as pharmacological tools. The medicinal chemistry of the A_2BAR is the least developed of the four subtypes, with selective antagonists and a few selective agonists reported only since 2000 [51, 52]. The optimal binding features of AR ligands have also been predicted on the basis of quantitative SAR (QSAR) approaches, such as comparative molecular field analysis (CoMFA) [116–119], although the use of X-ray structural data is now able to provide greater insight than earlier approaches. The selective AR ligands now include compounds that are stable in vivo, high affinity radioligands for binding assays or in vivo imaging by positron emission tomography or single photon emission tomography [53], that bind irreversibly to the receptor affinity probes, fluorescent and other spectroscopic probes, and multivalent conjugates that retain high potency. An overview of SAR is provided below for AR agonists and antagonists.

1. Agonists: The SAR of adenosine derivatives as AR agonists has been well explored [1, 54], and selective agonists and antagonists for all four subtypes have been reported (Figs. 1 and 2 and affinities of selected compounds listed in Table 1). Data on selectivity of AR ligands have been collected [1, 55]. Typically, these agonists are nucleoside derivatives substituted at one or more of the following positions: ribose 5′, adenine C2, and adenine N⁶. Hydrophobic groups substituted at the adenine C2 position (linked by NH or S) often provide selectivity for the A_2AAR (e.g., 16, 18, 20), and hydrophobic groups substituted at the adenine N⁶ position often provide selectivity for the A₁AR (e.g., 5–14). An N⁶-(4-aminophenylethyl) derivative, APNEA 4, is a nonselective AR agonist with high affinity for both A₁ and A₃ARs, and has been used as a radioligand in its [¹²⁵I] 3-iodo form. In exceptional cases, this selectivity pattern may be altered to display A_2AAR selectivity, as in 17, or with combined modifications, as in 19. Nonnucleoside agonists of the A₁AR are also known, including the clinical candidate Capadenoson 15 [56].

   The most common ribose modification that enhances AR potency is a small N-alkyl-uronamide at the 5′ position, as in the potent nonselective agonist NECA 3, an N-ethyl-uronamide. The presence of an N-ethyl-uronamide is typical of A₂AR-selective agonists (A_2A: 16, 18, and 19; A_2B: 22), and a N-methyl-uronamide is typical of A₃AR-selective agonists (23–28). The ribose moiety of nucleoside ligands having high AR affinity could also be substituted with a limited set of other modifications [54, 55, 57–60], for example: carbocyclics, including a ring-constrained methanocarba (fused cyclopropyl and cyclopentyl rings as in A₃AR-selective 27 and 28), 4′-thio in place of oxo (68), 2′-methyl (9), 2′-methoxy (12), and 3′-amino-3′-deoxy (25). By comparing the AR binding affinities of isomeric bicyclic methanocarba adeonsine analogues that maintain either a North (N), as in 27 and 28, or a South (S) conformation, it was determined that there is a strong preference for the (N) conformation in binding to the A₃AR. This bicyclic modification of ribose often enhances the affinity, as well as selectivity, at the A₃AR [54]. The (N)-methanocarba modification is also preferred over the (S)-methanocarba modification at the A₁AR, but affinity enhancement was not observed. The preference of the (N) over (S) conformation of the ribose moiety was also determined using C methylation at the 2′ and 3′ positions [59].

   It is to be noted that some nucleoside derivatives act as full agonists at certain AR subtypes and antagonists or partial agonists at other subtypes. Typically, the efficacy at the A₃AR is particularly sensitive to structural modification of the nucleoside derivative. Thus, reducing the flexibility or H-bond donating ability of the ribose moiety, especially around the 5′-amide group, or introducing certain sterically bulky hydrophobic substituents at the N⁶ or C2 position tends to lower the relative efficacy at the A₃AR [61]. Various 8-cycloalkylamino adenosine derivatives or those modified at the ribose hydroxyl positions have reduced efficacy at the A₁AR or A_2AAR [60]. The introduction of bulky groups at the 5′ position has been shown to reduce efficacy at the A₁AR [56].

   Selective agonists for the A₁AR [54, 57] include: R-PIA 6, CPA 7 and its more selective 2-chloro analogue CCPA 8, and CHA 11 (all of which have been radiolabeled as tracers for binding experiments); SPA 5 (excluded from crossing the blood brain barrier). CPA, CCPA, and CHA are more selective for the A₁AR in mouse than in human, in comparison to the A₃AR. (S)-ENBA 13 displays high A₁AR selectivity (human, rat) in comparison to both A_2AAR and A₃AR, but also has reduced water solubility. A 4′-truncated (N)-methanocarba nucleoside containing an N⁶-dicyclopropylmethyl group (not shown) fully activated the A₁AR with moderate selectivity [15]. CGS21680 16 and DPMA 17 are A_2AAR selective in binding to the rat and mouse A_2AARs, but in the human, they bind with similar affinity to the A₃AR. The nonnucleoside 3,5-dicyanopyridine derivative 21 and the nucleoside derivative 22 are moderately A_2BAR selective. A₃AR-selective agonists typically have combined N⁶ and ribose 5′ modications (23–28). Introduction of certain bulky groups at the 5′ position of A₁AR reduced the efficacy in functional assays, to provide partial agonists [57]. N⁶-Benzyl substitution tends to provide greater between-species consistency in A₃AR binding affinity [61], while small N⁶-alkyl groups, such as methyl (26) are often more potent at the human A₃AR than at rat and mouse A₃ARs. However, N⁶-benzyladenosine derivatives are variable in their A₁AR binding affinity depending on the substitution pattern, which can reduce A₃AR selectivity. The product of enzymatic action of adenosine deaminase, inosine, also activates the A₃AR in the micromolar range [1].

2. Antagonists: The prototypical AR antagonists are theophylline 29, caffeine 30 and other naturally occurring xanthines, but these are of micromolar affinity and not subtype-selective antagonists. Both synthetic purine and nonpurine (e.g., nonselective 35, A₁AR-selective 39, A_2AAR-selective 44–49, A₃AR-selective 60–67) heterocycles have been extensively explored as subtype-selective AR antagonists [55]. Purine derivatives as selective AR antagonists include xanthines (e.g., high affinity 8-phenylxanthines 31–34 (including water-soluble and peripherally selective sulfophenyl derivatives 31 and 32), A₁AR-selective 8-cycloalkylxanthines 36–38, A_2AAR-selective 8-styrylxanthines 42 and 43, A_2BAR-selective 8-arylxanthines 52–59) and adenines (e.g., A₁AR-selective 39–41, A_2AAR-selective 50 and 51, A₃AR-selective 69). A₃AR-selective nucleoside 68 behaves as an antagonist in functional assays, which is related to the absence of the 5′-hydroxymethyl group that is associated with the conformational change needed to activate the A₃AR [15].

   The in silico screening of chemical libraries of diverse structure by docking to an X-ray structure or even a homology model is now an accepted method of discovering new chemotypes that bind to a given GPCR. Non-nucleotide antagonists of ARs and P2YRs have been discovered in this manner [62].

P2YRs

Progress in the development of selective agonist and antagonist ligands for P2YRs (Fig. 3, and potencies of selected compounds listed in Table 1) has accelerated in recent years. Detailed SAR analyses are available for activation by nucleotides of most of the P2YRs [2, 63]. One must keep in mind that extracellular nucleotides can be interconverted in situ to different phosphate forms or to the corresponding nucleoside, which may complicate pharmacological studies. In some cases, the addition of an inhibitor of ectonucleotidases or of other enzymes involved in this conversion, or even addition of a purified enzyme, aids in the interpretation of pharmacological data. A challenge is to design P2YR ligands that are stable in vivo. Nevertheless, there are now nucleotide agonists selective for P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₄Rs and nucleotide antagonists selective for P2Y₁ and P2Y₁₂Rs. The diastereoselectivity of binding of the phosphate groups of nucleotide agonists selective at the P2Y₁, P2Y₂, P2Y₄ and P2Y₁₁ Rs has been characterized [29, 41, 42]. Also, subtype-selective non-nucleotide antagonists have been introduced for P2Y₁, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄Rs. Isolated reports have suggested non-nucleotide antagonists of the P2Y₂R, but these so far are weakly binding. Chemically diverse library screening is now being applied to the problem of identifying new structural leads for receptor antagonists, e.g., the A_2AAR, P2Y₁₂R and P2Y₁₄R [49, 62, 64]. A general description of SAR is provided below for each of the P2Y subtypes.

P2Y₁R

One of the earliest potent agonists of the P2Y₁R identified was 2-MeSADP 72 (Fig. 3a). However, like the native agonist ADP 70, it also activates the P2Y₁₂R and P2Y₁₃R. There has been a question about the ability of 5′-triphosphate derivatives such as 2-MeSATP 73 to activate the P2Y₁R; some studies show it to be an agonist while others demonstrate low efficacy [2].

The introduction of conformationally restricted (i.e., rigid) ribose substitions has established the favored ribose-ring conformation for each of the subtypes of the P2Y₁-like subfamily [38, 43]. Principally, this approach has made use of the methanocarba ring system consisting of fused cyclopropane and cyclopentane, as applied earlier to the ARs, in exploring the biologically active conformations of nucleoside and nucleotide derivatives. Thus, the North (N)-methanocarba analog of 2-MeSADP, i.e., MRS 2365 76 is a selective, high affinity agonist of the P2Y₁R that does not appreciably activate the other ADP-preferring subtypes, i.e., P2Y₁₂ and P2Y₁₃Rs [65]. The (N)-methanocarba modification is also known to improve the stability of the phosphate esters toward nucleotidases, especially the 5′-monophosphate toward the hydrolytic action of the ectonucleotidase CD73. Borano analogues of the phosphate group have been found in some cases to preserve potency and to enhance selectivity of P2YR agonists, e.g., P2Y₁R agonists [66].

Many nucleotide antagonists of the P2Y₁R have been introduced. Usually, these are adenine nucleotides containing bisphosphate groups, for example, a ribose 3′,5′-bisphosphate moiety. N⁶-methyl 2′-deoxyadenosine bisphosphate derivatives MRS 2179 93 and its 2-chloro analogue MRS 2216 (not shown) are selective P2Y₁ antagonists [38]. In both agonist and antagonist series, only limited substitution of the N⁶ position of ADP and other nucleotides, i.e., methyl and ethyl, is tolerated at the P2Y₁R. The same (N)-conformational constraint of the ribose moiety that enhances P2Y₁R agonist action also favors potency and selectivity in nucleotide antagonists. For example, the ring-constrained (N)-methanocarba nucleotide bisphosphates MRS 2279 94 and MRS 2500 95 are selective, high affinity antagonists of the P2Y₁R [67]. Antagonists of the P2Y₁R of moderate affinity may also be derived from acyclic nucleotides, such as the bisphosphate derivative MRS 2298 (not shown) [67].

A representative antagonist of the P2Y₁R discovered through optimization of a high throughput screening hit is a substituted 1-phenyl-3-methyl pyrazol-5-one 110, which has a K_i of 90 nM and is orally bioavailable [68]. Other structurally diverse antagonists of the P2Y₁R have been reported.

P2Y₂ and P2Y₄Rs

UTP 79 is a native agonist of both P2Y₂R and P2Y₄R. Another native ligand, ATP 71, activates the P2Y₂R, but at the P2Y₄R its action is species-dependent, i.e., it acts as an antagonist at the human homologue and agonist at the rat P2Y₄R. Synthetic UTP analogues with selectivity for the P2Y₂R have been reported, e.g., UTPγS 77, 2-thioUTP 82 and MRS 2698 (not shown), which is 300-fold P2Y₂R-selective in comparison to the P2Y₄R [35]. Recently, an N⁴-alkoxyimino derivative of CTP, MRS 4062 83, was found to be a full agonist of the P2Y₄R with ∼30-fold selectivity in comparison to the P2Y₂R and P2Y₆R [50]. Molecular modeling and docking of N⁴-alkoxyimino derivatives of CTP defined a new subpocket facing the exterior of the P2Y₄R that could accommodate steric bulk.

Dinucleoside tetraphosphates, e.g., INS 365 (Diquafosol) 92 and Up₄-2′-deoxyC (structure not shown, INS 37217, Denufosol) are moderately potent agonists of both P2Y₂R and P2Y₄R. In general, dinucleotides are more stable to hydrolysis by nucleotidases than are nucleotides bearing a free terminal phosphate group [69]. MRS 2768 90 (uridine tetraphosphate δ-phenyl ester) is somewhat selective for the P2Y₂R but is less potent than other P2Y₂R agonists [70].

Several weak antagonists of the P2Y₂R that are uracil derivatives, e.g., AR-C126313 and AR-C 118925 (not shown), have been reported but full pharmacological characterization is still lacking [2]. For lack of better antagonists, the anti-infective drug suramin 102 and the large anthraquinone dye Reactive blue 2 100 (RB2), which is a mixture of isomers, are used as partially selective antagonists of the P2Y₂R and P2Y₄R, respectively. It should be noted that suramin and many other weak P2YR antagonists typically display other activities, such as inhibition of ectonucleotidases, which may complicate the interpretation of experiments [2].

P2Y₆R

UDP 78 is the native agonist of the P2Y₆R, but was recently found to also activate the P2Y₁₄R [71]. UDPβS 80, 3-phenacyl UDP (PSB 0474) 84, 5-iodo-UDP (MRS 2693) 85 and dinucleoside triphosphates, such as Up₃U 91 and INS 48823 (not shown) [2], have been used as moderately selective agonists of the P2Y₆R [44]. Probing the conformation of the ribose ring at the P2Y₆R by molecular modeling and chemical synthesis of ring-constrained analogues has clearly identified the South (S)-conformation as the receptor-preferred conformation at this subtype [43]. Thus, a rigid bicyclic (S)-methanocarba-UDP (not shown) was more potent than UDP, and the corresponding ring-constrained isomer with a (N)-conformation was inactive. The di-isothiocyanate derivative MRS 2578 109 is a non-competitive P2Y₆R antagonist that has limited stability in aqueous medium and presumably reacts irreversibly with the receptor.

P2Y₁₁R

ATPγS 75 is usually used as a potent but nonselective P2Y₁₁R agonist. Few P2Y₁₁R-selective agonists have been reported, but an atypical agonist NF546 104 of the suramin class of antagonists was reported to activate this receptor selectively [72]. However, several reported P2Y₁₂R antagonists, such as 2-propylthio-β,γ-dichloromethylene-ATP (AR-C 67085 97), also act as potent P2Y₁₁R agonists. The suramin derivative NF 157 103 is an antagonist of the P2Y₁₁R, but it is not selective with respect to the nucleotide-gated ion channels P2X₁R, P2X₂R, and P2X₃R. NF340 105 related to suramin is a selective P2Y₁₁R antagonist.

P2Y₁₂R

ADP 70 is the native agonist of the P2Y₁₂R, and another native ligand, ATP 71, acts as a competitive antagonist. Many nucleotide (96–98) and non-nucleotide (111–115) antagonists of the P2Y₁₂R have been reported, because of commercial interest. The thienopyridine Clopidgrel 113 is a blockbuster antithrombotic agent, which must be first activated in two steps by cytochrome P450 in the liver to subsequently irreversibly inhibit the P2Y₁₂R [2, 37, 70]. The recently approved antithrombotic Prasugrel 114 belongs to this thienopyridine family of P2Y₁₂ antagonists. Competitive P2Y₁₂R antagonists that do not require preactivation are also under development, for example, the antithrombotic nucleotide derivative AR-C 69931MX 98 (Cangrelor). An uncharged nucleoside derivative that binds potently to the P2Y₁₂R, AZD 6140 99 (Ticagrelor) was recently approved to reduce cardiovascular death and heart attack in cases of acute coronary syndrome. A major metabolite of 99 that is formed by oxidative loss of the hydroxyethyl side chain also acts as a potent P2Y₁₂R antagonist [22]. A sulfonate derivative related to RB2, PSB-0739 101, is a representative nonnucleotide antagonist of the P2Y₁₂R that displays high affinity and was used in the characterization of the effects of site directed mutagenesis of the receptor and in molecular modeling [34, 49].

P2Y₁₃R

ADP 70 is a native agonist of the P2Y₁₃R, while ATP 71 at high concentrations is at best a weak partial agonist. The pyridoxal phosphate derivative MRS 2211 108, is a selective antagonist of the P2Y₁₃R and related to the nonselective P2 antagonists PPADS 106 and iso-PPADS 107 [73]. However, MRS2211 and other pyridoxal phosphate derivatives also inhibit protein interactions of the 14-3-3 family of intracellular phosphoserine/threonine-recognition proteins [74].

P2Y₁₄R

UDP-glucose 88, other UDP-sugars and UDP 78 are native agonists of the P2Y₁₄R [71]. A synthetic 2-thio analog of UDP-glucose, i.e., MRS 2690 89, is a more potent and selective agonist at the P2Y₁₄R. α,β-difluoromethylene-UDP, MRS 2802 86, and the more potent α,β-methylene-2-thio analogue MRS2905 87 are inactive at the P2Y₆R and fully activate the human P2Y₁₄R.

Allosteric modulation of ARs and P2YRs

In addition to orthosteric agonists that bind at the same site on the receptor as the native agonist, allosteric modulators for ARs and P2YRs have been studied. The structure and action of positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) for ARs and P2YR have been recently reviewed [75]. This includes both heterocyclic derivatives and nucleotide analogues that resemble a native P2Y agonist. Some of the PAMs have no action of their own and require the presence of an agonist, either the endogenous ligand or a synthetic agonist, and other PAMs are allosteric agonists that act in the absence of orthosteric ligands. The SAR of PAMs of the A₁AR (e.g., the tetrahydrobenzothiophene derivative T-62) and A₃AR (e.g., the imidazoquinolinamine derivative LUF6000 and the quinoline derivative LUF6096, structures not shown) has been extensively explored. Recently, AEA061 was reported as a PAM of the A_2AAR [76].

Ligands in the clinic and in current clinical trials

The biological role of adenosine and P2YRs has been extensively explored, contributing to the entry of certain selective ligands on a clinical pathway [54, 70, 77, 78]. Table 4 lists those AR and P2YR ligands in the clinic for therapeutic and diagnostic applications, including those currently in clinical trials for chronic diseases, such as inflammatory, ischemic, and neurodegenerative diseases, and for other conditions.

Table 4.

Ligands of ARs or P2Y receptors currently in clinical use or trials (previous clinical trials with selective adenosine and P2Y receptor ligands are listed in refs. [1, 2, 57, 70])

Ligand	Subtype action	Route	Application	Phase	Company
Adenosine 1 (Adenocard)	A1 agonist	iv	Paroxysmal supraventricular tachycardia	Approved	Astellas
INO-8875	A1 agonist	Topical	Glaucoma	I–II	Inotek
Capadenoson 15, Bay68-4986	A1 agonist	Oral	Atrial fibrillation	II	Bayer-Schering
Adenosine 1 (Adenoscan)	A2A agonist	iv	Myocardial perfusion imaging	Approved	Astellas
Apadenoson 18, ATL146e (Stedivaze)	A2A agonist	iv	Myocardial perfusion imaging	III	Forest Laboratories
Regadenoson 20, CV-3146 (Lexiscan)	A2A agonist	iv	Myocardial perfusion imaging	Approved	Astellas/Gilead
Regadenoson 20, CV-3146 (Lexiscan)	A2A agonist	iv	Sickle cell disease	I	Dana-Farber Cancer Institute
IB-MECA 23, CF101	A3 agonist	Oral	Rheumatoid arthritis, psoriasis, dry eye, glaucoma	II/III	Can-Fite
Cl-IB-MECA 24, CF102	A3 agonist	Oral	Hepatocellular carcinoma, chronic hepatitis C (genotype 1)	II	Can-Fite
Caffeine 30	AR antagonist	iv or oral	Sleep apnea, cancer pain, PD	II/III	Univ. of Texas, McMaster Univ., Nobelpharma, Korea Research, McGill University
Theophylline 29	AR antagonist	Oral	Asthma, COPD	Approved	–
Istradefylline 42, KW-6002	A2A antagonist	Oral	PD	III	Kyowa Hakko
KW-6356	A2A antagonist		PD		Kyowa Hakko (in Asia), Lundbeck (non-Asia)
Preladenant 46, SCH-420814	A2A antagonist	Oral	PD	III	Schering
Tozadenant 45, SYN-115	A2A antagonist	Oral	PD, cocaine dependence	IIB	Biotie, NIDA (Synosia Therapeutics)
ST-1535 51	A2A antagonist	Oral	PD	I	Sigma-Tau
V81444	A2A antagonist	Oral	PD	I	Vernalisa
DT1133	A2A antagonist	Oral	PD	Pre-clinical	Domain Therapeutics
[11C]-SCH442416 47	A2A antagonist	iv	PET imaging of PD	I	Institute for Neurodegenerative Disorders
[123I]MNI-420 49c	A2A antagonist	iv	SPECT imaging of PD, Huntington’s disease	I	Institute for Neurodegenerative Disorders
CVT-6883 55, GS 6201	A2B antagonist	Oral	Chronic pulmonary and inflammatory diseasesd	I	Gilead
Diquafosol 92 (Diquas)	P2Y2 agonist	Local	Dry eye disease	Approved (Japan)	Santen (Inspire)
Clopidogrel 113 (Plavix)	P2Y12 antagonist	Oral	Acute coronary syndrome, atherosclerosis	Approved	BMS/Sanofi
Prasugrel 114 (Effient)	P2Y12 antagonist	Oral	Acute coronary syndrome, angioplasty	Approved	Lilly/Daiichi Sankyo
Ticagrelor 99, AZD6140 (Brilinta)	P2Y12 antagonist	Oral	Acute coronary syndrome	Approved	AstraZeneca
Cangrelor 98, AR-C69931MX	P2Y12 antagonist	iv	Coronary artery bypassb	III	The Medicines Co.
Elinogrel 115, PRT-060128	P2Y12 antagonist	Oral or iv	Acute coronary syndrome	II	Portola/Novartis

PD Parkinson’s disease

^aClinical trials of another A_2AAR antagonist, vipadenant (V2006/BIIB014), for PD were recently halted by Vernalis and partner Biogen Idec

^bEffective at maintaining platelet inhibition in patients on thienopyridines who required bypass surgery

^cReference [110]

^dClinical trials discontinued or unsuccessful (see also references [1, 2, 57, 70])

AR ligands as clinical candidates and approved drugs

Adenosine may be released from intracellular sources or generated by the action of ectonucleootidases on ATP that is released under stress conditions. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs that can be modulated by exogenous agents. AR agonists are currently in clinical trials for various conditions, including cardiac arrhythmias, neuropathic pain, myocardial perfusion imaging, cardiac ischemia, autoimmune inflammatory diseases, and cancer [57, 79–84].

The first AR agonist to be approved was adenosine 1 itself (as Adenocard), used as a rapidly metabolized therapeutic treatment of cardiac arrhythmias, specifically paroxysmal supraventricular tachycardia (PSVT), by slowing atrioventricular (AV) nodal conduction, an A₁AR effect. Its short half-life upon intravenous infusion (seconds) avoids some side effects, although A_2AAR-related side effects still may occur. Other agonists of the A₁AR have been in clinical trials for pain and cardiac arrythmias, including atrial fibrillation, supraventricular arrythmias, paroxysmal supraventricular tachycardia, and atrial flutter [54, 57]. CVT-3619 (GS9667, not shown), a partial A₁AR agonist, has been in clinical trials for type 2 diabetes [54]. Side effects associated with A₁AR agonists applied to cardioprotective and cardiovascular regeneration may be overcome by using partial A₁AR agonists such as Capadenoson 15 [85].

Adenosine (as Adenoscan) is used as a pharmacological stress agent for cardiovascular imaging based on its A_2AAR-dependent vasodilatory effect in the coronary artery. Also, more advanced A_2AAR-selective agonists [79] are either already approved for this purpose (CVT-3146, 20) or in clinical trials (ATL-146e, Apadenson, 18). A_2AAR agonists also have anti-inflammatory and anti-ischemic effects, and have been in clinical trials for related conditions, including sickle cell disease by targeting iNKT cells [58, 84]. Selective agonists of A_2A, A_2B or A₃ARs have been shown to have anti-inflammatory effects due to inhibition of the release of pro-inflammatory cytokines and other mechanisms [80, 86, 87]. This also led to former clinical trials of A_2AAR agonists for the treatment of chronic and neuropathic pain and diabetic foot ulcers. However, an A_2AAR agonist was found ineffective for treating foot ulcers. A_2AAR agonists also show beneficial effects in wound healing, because A_2A and A_2BARs stimulate granulation tissue formation by inducing new matrix production and angiogenesis [88, 89]. A_2BAR agonists have been proposed for the treatment of hyperlipidemia and atherosclerosis [90].

A_2AAR antagonists (e.g. 42, 45, 48, and 51) are being developed for treatment of Parkinson’s disease (PD) and other disorders of the central nervous system including addiction [79, 91], and several clinical candidates have been radiolabeled for in vivo imaging [53]. In the striatum, a heterodimer of the A_2AAR and the D2 dopamine receptor is thought to establish the inverse action of dopamine and adenosine agonists; thus, an A_2AAR antagonist would have a net effect similar to a D2 agonist. A_2AAR antagonists could also be of interest in preventing fibrosis in the liver and elsewhere [88] or in the treatment of cancer [92]. A_2BAR antagonists are under consideration for treating inflammatory diseases, diabetes, and asthma [81, 82], although trials of CVT-6883 55 were unsuccessful.

Native adenosine acting at various AR subtypes has antiischemic activities in multiple organs, for example, a cardioprotective action, either as a preconditioning agent or during ischemia reperfusion. Adenosine and more selective AR agonists, e.g. A₃AR agonists such as CP532,903 25, have been considered for treating acute myocardial infarction [80]. One of the first actions discovered for A₃AR agonists administered in vivo was cerebroprotection. Also noted were paradoxical effects in which nM concentrations of A₃AR agonists prevented apoptosis and high μM concentrations induced apoptosis. The relative lack of cardiovascular side effects of A₃AR agonists in comparison to other AR agonists is considered an advantage in application to ischemia. The orally active A₃AR agonist CF101 (IB-MECA) 23 is in clinical trials for rheumatoid arthritis, psoriasis, keratoconjunctivitis sicca (dry eye syndrome), and glaucoma [80]. The closely related CF102 (Cl-IB-MECA) 24 is in clinical trials for advanced hepatocellular carcinoma and for patients with chronic hepatitis C genotype 1.

P2YR ligands as clinical candidates and approved drugs

Although the ARs are a mature field of medicinal chemistry, the P2YRs generally lag behind in the development of selective ligands, radioligands and other affinity probes, imaging agents, and clinical candidates. The most successful application in that area is the use of P2Y₁₂R antagonists as antithrombotics, but other disease areas are potentially amenable to treatment using selective P2YR agonists or antagonists [70]. Since some P2Y subtypes have a widespread distribution, there might be substantial side effects, such as those noted to occur in bone [4].

Nucleotides, such as ATP 71 and UTP 79, are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. One of the consequences of this release is a proinflammatory effect [2], for example from ATP that accummulates in asthmatic airways. Consistently, antagonists and other ligands of the P2YRs could serve as therapeutic targets for a variety of conditions, including cardiovascular diseases and inflammatory diseases such as asthma and neurodegeneration [70, 93]. It has been suggested that antagonists of P2Y₂R, P2Y₆R, or P2Y₁₁R might be beneficial in asthma and inflammatory bowel disease [70]. Beneficial effects of P2 receptor antagonists have been observed in a stroke model [94]. The effects of various P2YR ligands on apoptosis in cell culture and in the central nervous system have been explored [95–98], suggesting application to a variety of diseases, from cancer to diabetes to ischemia.

P2YRs are widespread in hematopoietic cells, and therefore the effects of extracellular nucleotides and their antagonists are being studied in the immune/inflammatory system. The platelet expresses two P2YRs, i.e., P2Y₁R and P2Y₁₂R, both of which have to be activated in order for ADP to have a prothrombotic effect [23]. Therefore, blocking either of these receptors produces an antithrombotic effect. P2Y₁₂R antagonists, three of which are already approved as agents for acute coronary syndrome and for prevention of secondary thrombotic events, have been described above. The antithrombotic action of MRS 2500 95 by selectively blocking the P2Y₁R is evident in vivo in the mouse and other species, suggesting this receptor subtype as a clinical target. Furthermore, genetic deletion of the P2Y₁R is associated with fewer atherosclerotic lesions in ApoE^−/− mice. Bone marrow reconstitution has demonstrated the involvement of non-hematopoietic-derived cells, probably the endothelial cells [99].

Several agonists of the P2Y₂R have been in clinical trials for cystic fibrosis and other pulmonary conditions. Activation of the P2Y₂R on epithelial cells in the airways and the eye promotes chloride secretion, independently of the genetically defective transporter in cystic fibrosis. However, the P2Y₂R agonist Up₄-2′-deoxyC (Denufosol) was denied approval for the treatment of cystic fibrosis due to the failure to reproduce the positive results of the TIGER-1 study in the longer duration TIGER-2 trial. A P2Y₂R agonist of low selectivity, Up₄U 92 (Diquafosol), has been approved in Japan but not the U.S. for the treatment of dry eye disease [100]. P2Y₂R activation has also been shown to protect rat fetal cardiomyocytes against ischemia [101]. P2Y₄R activation by UTP promotes chloride and water secretion by intestinal epithelial cells, suggesting the use of agonists of this subtype in treating chronic constipation [102].

Pancreatic islets express both the P2Y₁R and P2Y₆R, both of which are coupled to G_q and promote insulin release. The use of P2Y₁R agonists in diabetes has been proposed, and relatively stable nucleotide analogues that activate this subtype have been applied in vivo [69]. Furthermore, agonists of the P2Y₆R have been shown to have beneficial antiapoptotic effects on pancreatic islets cells in culture, suggesting their possible application to diabetes [95] Endogenous UDP activating the P2Y₆R is involved in the autocrine potentiation of insulin secretion [103]. However, there are significant side effects of activation of the P2Y₆R, such as a proinflammatory effect, atherosclerotic plaques, cardiac fibrosis and possibly a loss of bone mass [4, 70, 104].

P2Y₁₁R activation mediates ATP-induced semi-maturation of human monocyte-derived dendritic cells and increases the release of interleukin-8 from human monocyte-derived dendritic cells, suggesting use of ligands of this subtype in immune modulation [72, 105]. Semi-maturation of dendritic cells is characterized by an increased expression of co-stimulatory molecules with no stimulation of interleukin-12 secretion, leading to a Th2 response or tolerance.

The activation and migration of microglia in the brain are modulated by P2YRs [106, 107]. ADP activating the microglial P2Y₁₂R induces a “find-me” signal (to induce migration), and UDP activating the microglial P2Y₆R induces an “eat-me” signal (to induce phagocytosis). These findings suggest application of P2Y₁₂R or P2Y₆R ligands to neuropathic pain and neurodegenerative diseases. Indeed, intrathecal administration of P2Y₁₂R antagonist AR-C69931MX 98 prevented the development of tactile allodynia [106].

Activation of the P2Y₁₃R by ADP promotes reverse cholesterol transport in hepatocytes with the endocytosis of HDL particles [108]. Thus, activation of P2Y₁₃R might be a new target for treatment of dyslipidemia and atherosclerosis.

Modulation of the P2Y₁₄R has potential for the treatment of immune and inflammatory disorders, pain, asthma, gastric disorders, central nervous system diseases, and glaucoma. Non-nucleotide antagonists of the P2Y₁₄R, e.g., 116, and prodrug derivatives to increase their bioavailability have been proposed [64]. Intracellular UDP sugars, many of which would activate the cell-surface P2Y₁₄R, are substrates for protein glycosylation, and are released as the proteins are trafficked to the surface [71], where they may fulfill a cell signaling role. The role of P2Y receptors in stem cell differentiation has been explored; P2Y₄ and P2Y₁₄Rs appear to regulate the onset of mesenchymal differentiation, and the downregulation of P2Y₁ and P2Y₂Rs are markers for early osteogenic differentiation [109].

Conclusions

There have been significant recent advances in the structural biology of purine receptors and in the medicinal chemistry of selective ligands and their pharmacology. Potent purine and pyrimidine analogues have aided in the characterization of regulation of many physiological and pathophysiological processes. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. Thus, this field has provided fertile ground for pharmaceutical development.

Glossary

AR

Adenosine receptor

EL

Extracellular loop

GPCR

G protein-coupled receptor

IL

Intracellular loop

TM

Transmembrane helix

NECA

Adenosine-5′-N-ethyluronamide

PD

Parkinson’s disease

PET

Positron emission tomography

SAR

Structure–activity relationship

SPECT

Single photon emission tomography

UDPG

Uridine-5′-diphosphoglucose