An Introduction to P2X and P2Y Receptors

The P2 receptors for extracellular nucleotides participate in the regulation of almost every physiological process and are widely distributed in the body.^1,2 Nucleotide receptors in the muscular, immune, cardiovascular, inflammatory, and central and peripheral nervous systems are of particular interest.

The ubiquitous signaling characteristics of extracellular nucleotides acting at two distinct families of P2 receptors – fast P2X ion channels and P2Y receptors (G-protein-coupled receptors) – are now well recognized. These extracellular nucleotides are generated as a result of cell damage and tissue stress and in the process of neurotransmitter release and channel formation. Their concentrations can differ massively depending on circumstances.

Consequently, the state of activation of these receptors can rely heavily on the disease states or stress conditions affecting a particular organ. The P2 receptors react to numerous extracellular mono- and dinucleotides (Table 1).

In agonist selectivity, the P2X receptors are more structurally restrictive than P2Y receptors. The P2Y receptors are activated by a group of five or more naturally occurring nucleotides, while the P2X receptors are activated principally by ATPs.

The P2X receptors are distributed throughout the nervous system (enteric, autonomic, and sensory neurons, cochlear, central, and retinal cells), vascular system (endothelium, cardiomyocytes, and smooth muscle), the pulmonary and digestive systems (epithelium and visceral smooth muscle), bone, skeletal muscle, and hematopoietic cells.

The P2X receptors are made up of trimeric ligand-gated ion channels. The subunits are numbered P2X₁ to P2X₇, and both homotrimers and heterotrimers occur. Activation of P2X receptors results in an influx of cations like calcium and sodium, which depolarize excitable cells and activate cytosolic enzymes respectively. Upon prolonged agonist exposure, the P2X₇ receptor also opens a large pore, which can pass dye molecules and organic cations.

The knowledge of P2X receptor structures was recently advanced with the X-ray crystallographic determination of the P2X₄ subunit.^3,4 Yet, this structure did not determine the specific ligand binding site within the protein. A huge challenge in designing new agonist and antagonist ligands for a specific P2X receptor subtype is that the heterotrimers and homotrimers could possess totally different structural requirements.

The correspondence of the P2Y receptor subtypes along with their native nucleotide ligands can be seen in Table 1. Because the assignment of numbers to certain putative P2Y receptors was later shown to be premature, the numbering of unique human P2Y receptors has some gaps, with some of the sequences which were previously assigned being P2Y species homologs and others being other varieties of receptors.

Each of the native nucleotides can activate a variety of P2Y receptor subtypes. The structures of representative adenine (Figure 1A, 1–10) and uracil (Figure 1B, 11–28) nucleotides that activate P2 receptors are conveyed. The adenine nucleotide ATP is a full agonist at two human P2Y subtypes (P2Y₂ and P2Y₁₁ receptors), and the corresponding diphosphate ADP activates three different subtypes (P2Y₁, P2Y₁₂, and P2Y₁₃ receptors).

The uracil nucleotide UTP activates two subtypes (P2Y₂ and P2Y₄ receptors), while UDP is now known to also activate P2Y₁₄ receptors along with the originally designated native agonist UDP-glucose, it was previously thought to activate only a single subtype (P2Y₆ receptors).⁵ Several P2 receptors are activated by the naturally occurring dinucleotide Ap₄A and its homologs.

The structure, signaling, and regulation of P2Y receptors have been investigated, and subfamilies of P2Y₁-like and P2Y₁₂-like receptors have been realized. These subfamilies are made up of two pharmacologically distinct groups of P2Y receptors, which also correspond with similarities in the function of key amino acid residues.⁶

The preferential coupling of the first subfamily of P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors is to G_q, leading to the activation of phospholipase Cβ (PLCβ), and the second subfamily of P2Y₁₂, P2Y₁₃, and P2Y₁₄ receptors couple to G_i leading to the inhibition of adenylyl cyclase. To stimulate adenylyl cyclase, P2Y₁₁ receptors also activate G_s.

The comparisons of the structural characteristics and functionally important amino acid residues within the family have been outlined. Specific cationic residues and other residues in the TM region (for example Phe in TM3), and on the extracellular loops possess conserved functions within the P2Y family. Molecular recognition in the P2Y₁, P2Y₂, P2Y₁₁, and P2Y₁₂ receptors has been explored extensively via mutagenesis.^7–10

P2Y receptor regulation has also been examined. The P2Y₁ receptor is desensitized more quickly than the P2Y₁₂ receptor in platelets, which express two ADP-responsive P2Y subtypes.¹¹ Primarily, the P2Y₁ receptor is desensitized through PKC-dependent processes, and the P2Y₁₂ receptor is a good substrate for the GPCR kinases (GRKs) which result in arrestin binding.

Residues on the cytosolic C-terminal domain related to the regulation of the P2Y₁ receptor have been inspected. The internalization of the P2Y₁₁ receptor depends on coexpression of the quickly desensitizing P2Y₁ receptor, proposing the occurrence of receptor dimers.¹²

A number of heterodimers of P2Y receptors with other P2Y and non P2Y GPCRs have been suggested. For example, a dimer of A₁ adenosine receptors and P2Y₁ receptors was characterized.¹³ Recurrent problems with the utilization of some P2 receptor ligands include cross-reactivity with numerous P2 receptors and low bioavailability because of polyanions, like sulfonates and phosphates which are present in the molecules.

Lability in biological systems is another issue of many of the currently used ligands. A big family of ectonucleotidase enzymes hydrolyzes the native nucleotides resulting in complications in the interpretation of biological results. In the final step by the action of CD73/5-nucleotidase on AMP, adenine nucleotides are progressively converted enzymatically, to create adenosine, which activates its own family of four receptors.

Selective inhibitors of ectonucleotidases that can act as modulators of receptor function are being investigated.¹⁴ Additionally, a lot of P2 receptor antagonists and agonists are known to inhibit ectonucleotidases at comparable concentrations. Known P2 antagonists frequently interact intracellularly with other signaling mediators, like G proteins.

Table 1. Subtypes of P2 receptors and their ligands (potency at the human homologs shown as pEC₅₀, unless noted r = rat)

One of the causes of the relatively slow progress in identifying competitive antagonists of the P2 receptors is that there are not many selective radioligands available for either the P2Y or P2X receptors. Formerly, numerous radioactive nucleotides have been proposed to bind to certain P2 receptors, but a number of these reports were questioned later, and presently just the P2Y₁, P2Y₁₂, P2X₁ and P2X₃ receptors have viable radioligands.^15–17

Hence, more versatile and improved affinity probes for the P2 receptors are still required. New selective antagonists and agonists have been identified recently for a few of the eight mammalian subtypes of P2Y receptors and for some of the seven mammalian subtypes of P2X receptors. Subtype-selective nucleotide agonists are a result of a cautious exploration of the structure-activity relationships (SARs) at relevant P2 receptors.

As a result of the conversion of agonists into antagonists, library screening, and the careful structural modification of known non-selective ligands, selective antagonist ligands for P2 receptors have been established. The structures of representative nucleotide (Figure 2A, 29–42) and non-nucleotide (Figure 2B, 43–73) antagonists of the P2 receptors are outlined.

Pharmacological Probes for P2X Receptors

The development of P2X receptor ligands for possible therapeutic applications has begun. Selective P2X receptor antagonists are of particular interest in diabetic retinopathy, urinary incontinence, pain control, inflammatory diseases such as rheumatoid arthritis, and some other conditions.

Non-Selective P2X Ligands

All subtypes of P2X receptors are activated by ATP, but at different concentrations varying from the low nanomolar through to the high micromolar.¹⁸ ADP and AMP, when purified, are inactive at P2X receptors. 2‑Methylthioadenosine 5′-triphosphate (2-MeSATP) is a potent agonist at multiple P2X receptors, for example, P2X₁ (EC₅₀ = 54 nM) and P2X₃ (EC₅₀ = 350 nM) receptors.

α,β‑Methyleneadenosine 5′-triphosphate (Figure 1A 6) activates and desensitizes the P2X₁ receptor and is inactive at the P2X₂ receptor. It acts as a radioligand of P2X₁ and P2X₃ receptors in tritiated form. Non-selective, older, and weak P2X antagonists (Figures 2A and 2B), for example, organic dyes 43 and 45, have been in use for decades.

Polysulfonated Suramin, the antiparasitic drug, and the pyridoxal phosphate derivatives PPADS and positional isomer isoPPADS are relatively nonsubtype-selective P2X antagonists, which also block several P2Y subtypes.¹⁹ At the P2X₁ receptor, the PPADS analog MRS 2159 is more potent than PPADS and also antagonizes the P2X₃ receptor. The nucleotide derivative TNP-ATP is a potent P2X antagonist which is selective for numerous subtypes.²⁰

It antagonizes P2X₁, P2X₃ and heteromeric P2X_2/3 receptors with respective IC₅₀ values of 6, 0.9 and 7 nM, and exhibits 1000- fold selectivity for P2X₃ over P2X₂, P2X₄ and P2X₇ receptors. Evans Blue, the polysulfonated biphenyl derivative, behaves as a P2X receptor antagonist, but it also influences amino acid binding sites and other channels.

P2X₁ and P2X₂ Receptors

P2X₁ antagonists have been seen in many compound classes. The Suramin derivative NF 157 for example, is a P2X₁ antagonist which also inhibits the P2Y₁₁ receptor.²²

Some other Suramin derivatives which serve as selective P2X₁ antagonists include: NF 279, PPNDS, and the more highly selective P2X₁ antagonist NF 449.^23,24 The earlier-reported Suramin analog NF 023 is a competitive, moderately selective, P2X antagonist with IC₅₀ values of 0.21 and 28.9 μM at human P2X₁ and P2X₃ receptors respectively, and is inactive at P2X₂ and P2X₄ receptors.²⁵

The benzimidazole-2-carboxamide derivative Ro 0437626 was recently said to be a selective P2X₁ antagonist (IC₅₀ = 3 μM) that exhibits > 30-fold selectivity over P2X₂, P2X₃ and P2X_2/3 receptors (IC₅₀ > 100 μM) in a separate chemical series.²⁶ The dinucleotide Ip₅I was seen to antagonize the P2Y₁ receptor.⁴²

A weak potentiator of P2X₁-mediated responses is the pyridoxal phosphate derivative MRS 2219.²⁷ There are no selective ligands for the P2X₂ receptor. The nonselective antagonists RB-2, Suramin, isoPPADS, and TNP-ATP, have been utilized to examine this receptor.

P2X₃ Receptor

In combination with P2X₂ subunits, the P2X₃ receptor can act as a heterotrimer or a homotrimer. The Suramin derivative NF 110 is a high-affinity P2X₃ receptor antagonist (Ki values are 36, 82 and 4140 nM for P2X₃, P2X₁ and P2X₂ receptors respectively) that is inactive at P2Y₁, P2Y₂ and P2Y₁₁ receptors (IC₅₀ > 10 μM). The introduction of the competitive antagonist by Abbott Laboratories was a notable advance.

A 317491, which blocks P2X_2/3 (IC₅₀ = 92 nM), and P2X₃ (IC₅₀ = 22 nM) receptors and, in comparison to P2X₁ and P2X₂ receptors, is roughly three orders of magnitude selective for P2X₃.¹⁷ A 317491 is inactive at P2X₄ receptors and at all P2Y receptors. A 317491 is of limited bioavailability because of the presence of three carboxylic acid groups.

The pyrimidinediamine derivative RO-3 is another potent P2X₃ antagonist and is a selective antagonist of the heteromeric P2X_2/3 and homomeric P2X₃ receptors (pIC₅₀ values are 5.9 and 7.0 nM respectively). It is inactive at P2X₁, P2X₂, P2X₄, P2X₅ and P2X₇ receptors (IC₅₀ > 10 M).²⁹ The endogenous heptapeptide Spinorphin (LVVYPWT) was discovered to be an extremely potent P2X₃ antagonist (IC₅₀ = 8.3 pM).³⁰

P2X₄ Receptor

Not many of the known P2X antagonists act at the P2X₄ receptor. The benzofurodiazepinone derivative 5-BDBD is an antagonist of P2X₄-mediated currents (IC₅₀ = 0.50 μM).³¹ Ivermectin, the bacteria-derived broad-spectrum antiparasitic agent, which is a macrocyclic lactone, is a positive allosteric modulator for the P2X₄ receptor, but it influences some other ion channels too, for example, nicotinic acetylcholine receptors.³²

Figure 1A. Adenine derivatives that have been useful as antagonists in the study of P2 receptors. (Bold text denotes compounds available from Tocris at time of publication)

Figure 1B. Uracil derivatives that have been useful as antagonists in the study of P2 receptors. (Bold text denotes compounds available from Tocris at time of publication)

P2X₅ Receptor

No selective ligands exist for the P2X₅ and P2X₆ receptors. Yet, the dye Coomassie Brilliant Blue G (BBG) has been effectively utilized to inhibit P2X₅ receptor function (IC₅₀ = 0.5 μM),³³ but this dye also inhibits the P2X₄ receptors (IC₅₀ = 3 μM at human receptors) and P2X₇ receptors (IC₅₀ values are 10 nM and 267 nM at rat and human receptors respectively).

P2X₇ Receptor

2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) is a P2X₇ receptor agonist that displays an order of magnitude with higher potency than ATP. At P2X₁ (pEC₅₀ = 8.7) and P2Y₁ receptors, it is also a partial agonist.³⁴ The tyrosine and isoquinoline derivative KN-62 was one of the first antagonists of the P2X₇ receptor identified but it acts in a non-competitive way and is inactive at the rat P2X₇ homolog.

Additionally, KN-62 is an inhibitor of CaM kinase II.³⁵ Oxidized-ATP (o-ATP) has also been extensively utilized to antagonize P2X₇ receptors. It has been challenging to pinpoint antagonists which block the P2X₇ receptor in a species-independent manner.

A selective, potent, and competitive P2X₇ antagonist (pIC₅₀ = 6.9) is the tetrazolylmethylpyridine derivative A 438079.³⁶ The quinolinamino derivative A 740003 is a potent and selective competitive P2X₇ receptor antagonist (IC₅₀ values are 40 and 18 nM for human and rat P2X₇ receptors respectively). In comparison to various P2X and P2Y receptors, that is also highly selective.^36,37

Possessing KD values of 1.4 and 19 nM at human and rat P2X₇ receptors respectively, the adamantyl derivative from AstraZeneca AZ 10606120 antagonizes the P2X₇ receptor.³⁸ The biphenyl derivative AZ 11645373 potently antagonized the human P2X₇ receptor in a non-surmountable way with KB values between 5–20 nM and was inactive at the rat P2X₇ receptor and at every other P2X subtype.³⁹

The substituted glycyl anilide derivative GW 791343 is a positive allosteric modulator of the rat P2X7 receptor and a negative allosteric modulator of the human P2X₇ receptor (pIC₅₀ = 6.9–7.2).⁴⁰

Pharmacological Probes for P2Y Receptors

In the development of selective agonist and antagonist ligands for P2Y receptors for preclinical development progress has been made.² Until now, the antithrombotic P2Y₁₂ receptor antagonist Clopidogrel (Plavix) was the only P2Y receptor ligand in pharmaceutical use.⁴¹ Hence, there is a lot of activity to recognize new agents to act at the P2Y₁₂ receptor and at other P2Y receptors for pharmaceutical development.

The fast-growing progress in this field has already lead to new drug candidates for dry eye disease, pulmonary diseases, and thrombosis. Numerous selective ligand probes, both antagonists and agonists of the P2Y receptors, are now available. Yet, some subtypes, for example, the P2Y₄ receptor, are lacking such selective ligands entirely.

For P2Y₁ and P2Y₁₂ receptors, which both are proaggregatory in platelets, detailed SAR analyses have been created. Nucleotide agonists selective for P2Y₁, P2Y₂, P2Y₆, and P2Y₁₄ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors have been detailed. To avoid challenges of limited bioavailability and stability, selective non-nucleotide antagonists are now desired.

Such antagonists have been reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. The screening of chemically diverse compound libraries has lead to competitive P2Y₁₂ receptor antagonists which are being studied as possible antithrombotic agents.

P2Y₁ Receptor

Similar to ADP, 2-MeSADP activates the P2Y₁, P2Y₁₂ and P2Y₁₃ receptors. 2-MeSADP is a more potent agonist at the P2Y₁ receptor than 2-MeSATP. Consistent with a small hydrophobic pocket in the P2Y₁ receptor binding site surrounding the N⁶-position of adenine nucleotides, N⁶-methyl nucleotides are tolerated at the P2Y₁ receptor.

The favored ribose-ring conformation for all of the subtypes of the P2Y₁-like subfamily has been determined by utilizing conformationally-restricted (i.e. rigid) ribose equivalents, which also enhance the stability of the phosphate esters toward nucleotidases.

The methanocarba ring system made up of fused cyclopropane and cyclopentane has been particularly useful in looking at the biologically active conformations of nucleotide and nucleoside derivatives.

A high affinity, selective agonist of the P2Y₁ receptor is the North (N)- methanocarba analog of 2-MeSADP, MRS 2365 (EC₅₀ = 0.4 nM).¹¹ The introduction of a borano group within the phosphate moiety of P2Y receptor agonists is a way of further enhancing hydrolytic stability.⁴³

A number of nucleotide antagonists of the P2Y₁ receptor have been introduced. Typically, these are adenine nucleotides that contain bisphosphate groups, for example, a ribose 3′,5′-bisphosphate moiety.

An N⁶‑methyl 2′-deoxyadenosine bisphosphate derivative MRS 2179 (pK_B = 6.99) and its 2-chloro analog MRS 2216 are selective P2Y₁ antagonists.⁴⁴ The same (N)-conformational constraint of the ribose moiety which increases agonist action also favors the selectivity and potency in nucleotide antagonists.

The ring-constrained (N)-methanocarba nucleotide bisphosphates MRS 2279 (pK_B = 8.10) and MRS 2500 (pK_B = 9) for example are high affinity, selective antagonists of the P2Y₁ receptor.⁴⁵ The antithrombotic action of MRS 2500 (by selectively blocking the P2Y₁ receptor) has been shown in vivo in the mouse and also in some other species.^46,47

Antagonists of the P2Y₁ receptor of moderate affinity can also come from acyclic nucleotides, like the bisphosphates derivative MRS 2298.⁴⁶ Non-nucleotide antagonists of the P2Y₁ receptor have been found through the screening of structurally diverse chemical libraries.

The first of these compounds to be described was 63, which is an orally bioavailable and selective antagonist of the human P2Y₁ receptor from Pfizer of novel chemotype with a Ki value of 90 nM.⁴⁸ Some other structurally diverse antagonists of the P2Y₁ receptor have also been revealed. Pyridyl isatogen (PIT) is an allosteric modulator of the P2Y₁ receptor which exhibits mixed antagonism/potentiation.⁴⁹

Figure 2A. Nucleotide derivatives useful as antagonists in the study of the P2 receptors. (Bold text denotes compounds available from Tocris at time of publication)

P2Y₂ and P2Y₄ Receptors

Both P2Y₂ and P2Y₄ receptors are activated by UTP, and simple modifications increase the selectivity for the P2Y₂ receptor. A more stable and selective agonist of the P2Y₂ receptor than UTP is UTPγS.⁵⁰ Yet, it is subject to chemical oxidation. 2-ThioUTP is another selective agonist of the P2Y₂ receptor.⁵¹

In comparison to the P2Y₄ receptor, a combination of modifications of UTP in the selective P2Y₂ agonist MRS 2698 supplied an EC₅₀ of 8 nM and selectivity of 300-fold.⁵² Particular dinucleoside tetraphosphates activate the P2Y₂ and P2Y₄ receptors potently. The uracil dinucleotides which have been in clinical trials are Up₄dC (INS 37217, Denufosol, EC₅₀ = 0.22 μM) and Up₄U (INS 365, Diquafosol, EC₅₀ = 0.1 μM).⁵³

Recently, in Japan, diquafosol was approved for use in treating dry eye. They are more stable to enzymatic hydrolysis than nucleoside triphosphates, by being dinucleotides, but compared to the P2Y₄ receptor these agonists are non-selective. The 2′‑deoxycytidine (dC) moiety of 20 acts to increase the in vivo stability toward ectonucleotidases.

With moderate potency, the agonist MRS 2768 (uridine tetraphosphate δ-phenyl ester) is selective for the P2Y₂ receptor (EC₅₀ = 1.89 μM).⁵⁴ Definitive antagonists of the P2Y₂ receptor are unavailable. AR-C 126313 and its higher molecular weight analog AR-C 118925 were reported to selectively antagonize the P2Y₂ receptor, yet it appears that these compounds are only micromolar in affinity (Figure 2B).⁵⁵

The large polyanionic molecules Suramin and Reactive Blue 2 (RB2, an anthraquinone dye) are marginally selective antagonists of the P2Y₂ and P2Y₄ receptors, respectively. Yet, RB2 and Suramin also block numerous P2X receptors (Table 1). So, there are no truly selective ligands for the P2Y₄ receptor.

5-fold P2Y₄ selectivity was displayed by the agonist 2′-azido-2′-deoxy-UTP (Figure 1B 23).⁵¹ Hence, new antagonists and agonists are required to pharmacologically distinguish this subtype from the P2Y₂ receptor, which is also activated by UTP. ATP, the other native agonist of the P2Y₂ receptor, serves as an antagonist at the human, but not the rat, P2Y₄ receptor.

P2Y₆ Receptors

Both the P2Y₆ and P2Y₁₄ receptors are activated by UDP. However, it is worth noting that extracellular UDP can act as a substrate for the production of UTP through the action of nucleoside diphosphokinase (NDPK), which can complicate pharmacological examinations. The action of UPD at the P2Y₁₄ receptor has been controversial.

Initially, UDP was detailed as inactive at the newly cloned P2Y₁₄ receptor, but later study discovered an antagonist action of UDP at the human but not rat P2Y₁₄ receptor. Lastly, the antagonist action of UDP took place in cells expressing an unnatural, engineered chimeric G protein. Yet, UDP acts as a potent agonist in HEK293 and other cells in which endogenous G_i proteins mediate the functional response.⁵

Hence, if multiple P2Y subtypes are present, inaccurate results could be gathered by utilizing UDP alone in pharmacological studies. UDPβS (15) and Up₃U have been employed as more stable activators of the P2Y₆ receptor subtype than UDP,^50,53 although UDPβS activates the P2Y₁₄ receptor as well.

The SAR of nucleotide derivatives in activating the P2Y₆ receptor has been examined. Specific dinucleoside triphosphates have been looked at as P2Y₆ receptor ligands, for example, INS 48823 (EC₅₀ = 125 nM) activates the receptor potently.⁵⁶ Other UDP derivatives, e.g. 5-iodo-UDP (MRS 2693), and 3-phenacyl UDP (PSB 0474), are selective P2Y₆ agonists with respective EC₅₀ values of 15 and 70 nM.^57,58

Molecular modeling estimated that the South (S)-conformation of the ribose ring is the preferred conformation in receptor binding, which by synthesis of a conformationally constrained methanocarba analog of UDP, was then confirmed. The noncompetitive P2Y₆ receptor antagonist MRS 2578 is a diisothiocyanate derivative, which has low solubility and stability in liquid.⁵⁹ Competitive antagonists of the P2Y₆ receptor have not been reported as of yet.

P2Y₁₁ Receptors

ATPγS (Figure 1A) serves as a potent P2Y₁₁ receptor agonist. The P2Y₁₂ antagonist 2-propylthio-β,γ-dichloromethylene-ATP (AR-C 67085, Figure 2A) is also the most potent agonist of the P2Y₁₁ receptor (EC₅₀ = 8.9 μM) reported.⁶⁰ So, it must be employed cautiously in pharmacological studies in which both P2Y subtypes could be present.

Derived from nonselective P2 antagonist Suramin, a potent antagonist NF 157, has been said to be a selective antagonist of the P2Y₁₁ receptor (pK_i = 7.35).⁶¹ Yet, this compound also antagonizes the P2X₁, P2X₂, and P2X₃ receptors.

P2Y₁₂ Receptors

The medicinal chemistry of the P2Y₁₂ receptor has been extensively explored. The thienopyridines, such as Clopidogrel (Figure 2B), act as liver-activated prodrugs that are irreversible inhibitors of the P2Y₁₂ receptor.⁶² This thienopyridine P2Y₁₂ receptor antagonist requires a two-step preactivation in vivo and therefore has a delayed onset of action and long reversal of the platelet effect after drug administration is stopped.

Prasugrel is another thienopyridine antagonist that has been present in clinical trials. It is a more potent P2Y₁₂ antagonist but exhibits a longer bleeding time. Prasugrel only needs one step of preactivation in vivo.⁶³ Directly-acting P2Y₁₂ receptor antagonists have also been reported.

The introduction of numerous 5′-triphosphate analogs as selective receptor probes and clinical candidates has been enabled because of the observation that ATP serves as an antagonist at this ADP-activated subtype. So, AstraZeneca’s antithrombotic nucleotide derivatives from AR-C 67085 (EC₅₀ = 30 μM) and ARC 69931MX (Cangrelor, EC₅₀ = 0.4 nM) have been clinically tested as antithrombotic agents.⁶⁴

A 5′-triphosphate group in adenine nucleotides is not a strict requirement for P2Y₁₂ receptor antagonism, similar to the instance of compound 38 and the potent antagonist and approved medicine ticagrelor (AZD 6140 pIC₅₀ = 7.9).^64,65

Nucleotide derivatives from Inspire Pharmaceuticals, INS 49266 (an ADP derivative with EC₅₀ of 52 nM) and INS 50589 (an AMP derivative with EC₅₀ of 11 nM) are some other nucleotide antagonists of the P2Y₁₂ receptor which have been discovered.⁶⁶ The identification of novel chemotypes that act as P2Y₁₂ receptor antagonists has been aided greatly by library screening, and many of these compounds are being developed by the pharmaceutical industry.

One very potent and selective competitive antagonist of the P2Y₁₂ receptor, which was reported recently, is PSB 0739, derived from RB2.

P2Y₁₃ Receptors

ADP is also the favored agonist ligand at the P2Y₁₃ receptor, and ATP is less potent. ADP is 3-5-fold more potent than 2-MeSADP in the rat. A selective P2Y₁₃ receptor antagonist, MRS 2211, a derivative of PPADS, has a pK_i of 6.0 at this receptor.⁶⁷

P2Y₁₄ Receptors

Recently, the SAR of analogs of UDP-glucose (EC₅₀ = 0.35 μM) and UDP at the P2Y₁₄ receptor was systematically examined.⁶⁸ Other naturally occurring UDP-sugars activate this receptor less potently. MRS 2690, the 2-thio analog of UDP-glucose, is a 6-fold more potent agonist for the P2Y₁₄ receptor and is inactive at the P2Y₂ receptor, unlike UDP-glucose.

Concerning alteration of the ribose, nucleobase, and phosphate moieties of agonist ligands, the P2Y₁₄ receptor is structurally restrictive. Yet, the glucose moiety may be deleted in UDP analogs, some of which are still extremely potent in receptor activation. For example, α,β‑difluoromethylene-UDP, MRS 2802, is inactive at the P2Y₆ receptor and fully activates the human P2Y₁₄ receptor with an EC₅₀ of 63 nM.

Conclusion

Novel ligands for the P2Y and P2X receptor families are now available for employment as tools in pharmacological studies. Although generally of low bioavailability and stability in vivo, selective nucleotide agonist ligands, have been designed. Recently, as a result of library screening, conversion of agonists into antagonist, and the careful structural modification of known non-selective ligands, selective antagonist ligands for P2 receptors have been reported.

Figure 2B. Non-nucleotides that have been useful antagonists in the study of P2 receptor. (Bold text denotes compounds available from Tocris at time of publication)

Special Mentions

This white paper was based on the original work of:

Kenneth A. Jacobson: Kenneth Jacobson serves as Chief of the Laboratory of Bioorganic Chemistry and the Molecular Recognition Section at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health in Bethesda, Maryland, USA. Dr. Jacobson is a medicinal chemist with interests in the structure and pharmacology of G-protein-coupled receptors, in particular receptors for adenosine and for purine and pyrimidine nucleotides.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA.

Email: [email protected]

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