Pharmacology of P2X receptors - Wiley Online Library

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Pharmacology of P2X receptors Nawazish-i-Husain Syed1,2 and Charles Kennedy2∗ P2X receptors are ligand-gated cation channels that mediate many of the extracellular actions of adenosine 5 -triphosphate (ATP). The genes encoding seven different P2X subunits, P2X1–7, have been cloned and when expressed alone all P2X subunits form functional receptors, except P2X6, which usually only assembles in a heteromeric form. Subunits can also interact with each other and to date seven functional heteromultimers with pharmacological and/or biophysical properties that differ from the individual homomultimers have been identified: P2X2/3, P2X4/6, P2X1/5, P2X2/6, P2X1/4, P2X1/2, and possibly P2X4/7. These are distributed widely throughout the body and are involved in many physiological and pathophysiological processes. ATP is an agonist at all subtypes and few selective agonists and no useful antagonists were available when P2X receptors were first defined. Subsequently, nonselective antagonists, such as suramin and PPADS, were discovered and since then, and particularly in the past decade, numerous potent and subtype-selective antagonists have been developed. These include NF449 and RO-1 at P2X1 receptors; PSB-1011 at P2X2 receptors; A317491, compound A, RO-3, and AF-353 at P2X3 and P2X2/3 receptors; 5-BDBD at P2X4 receptors; and A740003, A438079, A804598, GSK314181A, AZ10606120, AZ11645373, AZD-9056, CE-224535, and EVT-401 at P2X7 receptors. The therapeutic usefulness of these compounds is currently being investigated in a variety of disorders, including thrombosis (P2X1), chronic neuropathic and inflammatory pain (P2X3, P2X2/3, P2X4, P2X7), dysfunctional urinary bladder (P2X1, P2X3, P2X2/3), rheumatoid arthritis and osteoarthritis (P2X7), and depression (P2X7).  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. How to cite this article:

WIREs Membr Transp Signal 2012, 1:16–30. doi: 10.1002/wmts.1

INTRODUCTION

T

he role of the endogenous nucleotide, adenosine 5 -triphosphate (ATP), as a major intracellular energy source is a well-established phenomenon. In addition, many studies have shown that ATP and related nucleotides have widespread extracellular actions via P2X receptors, which are ligand-gated cation channels, and P2Y receptors, which are G-protein-coupled receptors.1–3 These receptors are distributed widely throughout the body and are involved in many physiological and pathophysiological processes.4,5 Indeed, it is very likely that all cell types express at least one P2X and/or P2Y receptor subtype.6 Furthermore, the receptors ∗ Correspondence

to: [email protected]

1

University College of Pharmacy, University of the Punjab, Lahore, Pakistan

2 Strathclyde

Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom

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present in the central nervous system (CNS) and the peripheral nervous system, the cardiovascular system, cardiac, skeletal, and smooth muscle, and immune and inflammatory cells are of great therapeutic interest and are increasingly being targeted by pharmaceutical companies.7,8 The genes encoding seven different P2X subunits, P2X1–7 (based on the order of discovery), have been cloned.3 The P2X1–P2X6 subunits are 379–472 amino acids long, whereas the P2X7 receptor comprises 595 amino acids, due to a much longer COOH terminus. The COOH termini of the seven subtypes display substantial sequence divergence, but the rest of the sequence has 40–55% pairwise identity, with the P2X4 subunit being the most closely related to the others and the P2X7 subunit being the most dissimilar. Hydropathy analysis of the amino acid sequences of subunits indicated that each possesses two hydrophobic, transmembrane-spanning regions that are long enough to span the cell plasma

 2012 WIL EY-VCH Verlag GmbH & Co. K GaA, Wein h eim.

Vo lu me 1, Jan u ary/Febru ary 2012

WIREs Membrane Transport and Signaling

membrane. Furthermore, it predicted that the NH2 and COOH termini are intracellular and that the bulk of the protein, about 280–300 amino acids, forms an extracellular loop. In addition, data obtained using a variety of experimental techniques indicated that three subunits are required to form a functional P2X receptor and that three agonist molecules are required to bind to a single receptor to activate it.5 This structural model has recently been confirmed in a recent report of the three-dimensional crystal structure of the P2X4 receptor.9 When expressed alone all P2X subunits form functional receptors, except the P2X6, which usually only assembles in a heteromeric form. Subunits can also interact with each other10 and to date seven functional heteromultimers with pharmacological and/or biophysical properties that differ from the individual homomultimers have been identified: P2X2/3, P2X4/6, P2X1/5, P2X2/6, P2X1/4, P2X1/2, and possibly P2X4/711 (but see Ref 12). Regardless of the subunit composition, P2X receptors are permeable to Na+ , K+ , and Ca2+ and on activation cause depolarization and excite cells.5 Few agonists and no useful antagonists were available when P2X receptors were first defined in 1985. Since then, and particularly in the past decade, numerous potent and subtype-selective antagonists have been developed. In this review, we will discuss the pharmacological properties of each of the subtypes of P2X receptor, focusing where appropriate on recent developments. In addition, we will also highlight potential therapeutic uses of drugs active at these receptors and recent patents that relate to such compounds.

NONSELECTIVE P2X AGONISTS The P2X receptor was originally defined by the agonist potency profile of the nucleotides α,β-methyleneATP (α,β-meATP), 2-methylthioATP (2-meSATP), and ATP2 (Figure 1). Following the cloning of P2X subunits, ATP was found to be an agonist at all subtypes of P2X receptor, although with widely varying potency, and 2-meSATP is also active at all.3,13,14 In contrast, α,β-meATP was initially reported to be an agonist only at homomeric P2X1 and P2X3 receptors, as well as at heteromeric P2X4/6 receptors15 and P2X1/5 receptors.16 Subsequently, it was reported that α,β-meATP showed agonist activity at P2X517,18 and P2X6 receptors19 and also at P2X4 receptors, though in a species-dependent manner.20 An additional complicating factor in characterizing agonist activity is that ATP and 2-meSATP undergo degradation by surface-located ectonucleotidases, such Vo lu me 1, Jan u ary/Febru ary 2012

Pharmacology of P2X receptors

as ectonucleoside triphosphate diphosphohydrolases, while α,β-meATP does not.21–23 Thus, Burnstock and Kennedy2 originally defined the relative agonist potency profile of the P2X receptor as α,β-meATP 2-meSATP = ATP, but in the absence of degradation the true potency order was revealed to be 2-meSATP = ATP > α, β-meATP.24,25 2 ,3 -O-(4-benzoylbenzoyl)ATP (BzATP) (Figure 1) is the most potent P2X agonist currently available and acts at all P2X subtypes studied to date.14 It is also an agonist at P2Y11 receptors and an antagonist at P2Y1 and P2Y12 receptors.1 BzATP is commonly described as a selective agonist at P2X7 receptors, but in fact its affinity is highest for the P2X1 subtype. Diadenosine polyphosphates (APn A) (Figure 1) are naturally occurring molecules that are involved in numerous intracellular biochemical pathways. They may also be important extracellular signaling agents, as platelets, chromaffin cells, and some neurones release them in micromolar amounts.26 At these concentrations, they have widespread extracellular actions, for example, contraction of visceral and vascular smooth muscle, excitation of neurones, release of catecholamines from chromaffin cells, and inhibition of platelet aggregation. Initially, it was hoped that one or more of these compounds might display P2X subtype selectivity, but on the whole this has not proved to be the case, as AP6 A, AP5 A, and AP4 A are agonists at most P2X subtypes studied.13,26–28

NONSELECTIVE P2X RECEPTOR ANTAGONISTS When P2X receptors were first characterized in 1985 no antagonists were available. Shortly afterwards, suramin (Figure 2) and then PPADS (Figure 3) were shown to antagonize P2X receptors, but with low potency and little subtype selectivity. Suramin antagonizes most homomeric P2X subtypes at low micromolar concentrations, except for the P2X7 receptor, where hundreds of micromolar suramin are required, and the P2X4 receptor, which is insensitive.3,13,14 PPADS tends to have a similar potency or be slightly more potent than suramin, but again the P2X7 receptor is much less sensitive and the P2X4 receptor insensitive.3,13,14 The antagonistic actions of PPADS tend to develop and reverse slowly and antagonism is generally noncompetitive. Its ability to inhibit ATP breakdown by ectonucleotidases is less than other antagonists, such as suramin. Both suramin and PPADS are also antagonists at some of the P2Y receptor subtypes.1 Nonetheless, the introduction of these compounds was a major


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ATP

2-meSATP

a,b-meATP

b,g -meATP

BzATP

APnA

FIGURE 1 | Chemical structure of nonselective P2X receptor agonists.

breakthrough that allowed the physiological functions of P2X receptors to be studied in some detail.4,5 Subsequently, antagonists that are more potent and which have some degree of selectivity for individual P2X receptor subtypes have been developed and these will be discussed, where appropriate, in the sections below on individual P2X receptor subtypes.

P2X1 RECEPTORS The P2X1 receptor is widely expressed in smooth muscle and mediates the neurotransmitter actions of ATP released from sympathetic and parasympathetic nerves.4–6 It is also expressed in platelets and by mediating Ca2+ influx it appears to be involved in platelet shape change and aggregation.4 Thus, the platelet P2X1 receptor might be a target for novel antithrombotic agents. P2X1 receptors are also present in the smooth muscle of the urinary bladder, where they mediate the part of the contractile effects of parasympathetic nerve stimulation.29,30 Their expression is upregulated in the dysfunctional human urinary bladder, and therefore are a potential target for the treatment of disorders such as idiopathic detrusor instability and cystitis.

Agonists The P2X1 receptor corresponds to the P2X receptor initially characterized in pharmacological studies of native receptors in smooth muscle tissues by Burnstock and Kennedy,2 and when the recombinant 18

receptor is expressed in cell lines, it is activated by 2-meSATP = ATP > α, β-meATP.3,13,14 BzATP is the most potent P2X1 agonist available and depending on the assay system used, it is active at submicromolar concentrations. In addition, β, γ -methyleneATP (β, γ -meATP) (Figure 1), an agonist that was used extensively to study native P2X receptors,2 is a stable, partial agonist at P2X1 receptors, which is equipotent with α, β-meATP and acts in a nonstereo-selective manner.3,13,14 AP6 A is a full agonist at P2X1 receptors, whereas AP5 A and AP4 A are partial agonists.31 The activity of agonists at many classes of receptors is potentiated by positive allosteric modulators, but relatively few such compounds are known for P2X receptors. An exception is MRS2219 (Figure 4), which weakly potentiates agonist activity at P2X1 receptors.32

Antagonists Suramin-Based Compounds Suramin is a large polysulfonated compound and a number of antagonists with increased subtype selectivity and/or potency have been generated by modifying its structure13,33 (Figure 2). The first useful compound to be thus produced was NF023, which is a competitive and reversible antagonist at P2X1 receptors, albeit less than 10-fold more potent than suramin. It is also selective for P2X1 receptors over P2X2, P2X3, P2X4, and P2Y receptors and is a weak inhibitor of ATP metabolism by ectonucleotidases. NF110 is more potent than NF023 as a P2X1





FIGURE 2 | Chemical structure of suramin and its derivatives.

FIGURE 3 | Chemical structure of PPADS and its derivatives.

antagonist, with a Ki of 82 nM, but is even more potent at P2X3 receptors.34 NF279 is a more potent antagonist at P2X1 receptors than suramin or NF023, being active in the nanomolar range, and is selective for P2X1 receptors over P2X2, P2X3, P2X4, P2X7, and P2Y receptors. NF279 is also a weak inhibitor of ATP breakdown by ectonucleotidases. NF449 is the most potent of these suramin analogs that is commercially available and is currently the most Vo lu me 1, Jan u ary/Febru ary 2012

FIGURE 4 | Chemical structure of other drugs active at P2X1 receptors.

potent antagonist at rat and human P2X1 receptors known, being active at subnanomolar concentrations. It displays high selectivity for this site and at least four orders of magnitude higher concentrations are needed to inhibit P2X3, P2X7, P2Y1 , and P2Y2 receptors.


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Pyridoxal-Based Compounds PPADS is also a large compound and, like suramin, a number of antagonists with improved potency and selectivity have been generated by modifying its structure. PPNDS, a 6-napthylazo derivative of PPADS (Figure 3), inhibits P2X1 receptors in the nanomolar range and displays improved selectivity compared with the parent compound.33 More recently, PPNDS was shown to also antagonize P2X7 receptors, but at much higher concentrations and in a species-dependent manner (pIC50 ≈ 5 at mouse, 6 at rat, and 6.4 at human P2X7 receptors).35 MRS2159 is a derivative of pyridoxal-5 -phosphate (Figure 3), which also antagonizes P2X1 receptors at nanomolar concentrations. It is more than 15-fold less active at P2X3 receptors and over 1000-fold less potent at P2X2 receptors36 and, like PPNDS, antagonizes P2X7 receptors at much higher concentrations and in a species-dependent manner.35 MRS2220 is a cyclic phosphate analog of the PPADS isomer, isoPPADS (Figure 3), and the introduction of this modification appears to abolish the activity of the parent compound at P2Y receptors, but not P2X receptors. It is of moderate potency and displays several-fold selectivity for P2X1 over P2X3 receptors and is inactive at P2X2 and P2X4 receptors. MRS2220 is a surmountable and fully reversible antagonist at P2X1 receptors, with moderate potency and selectivity over P2X3 receptors.32 It has no effect at P2X2, P2X4, P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors or adenosine A1 , A2A , and A3 receptors.

Other Compounds

2 ,3 -O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) (Figure 4) was initially used to label ATP-binding sites, including P2 receptors, but was then discovered to be a potent antagonist of P2X1 receptors in the nanomolar range. Initial studies using recombinant receptors showed that TNP-ATP was a potent antagonist at P2X1, P2X3, and P2X2/3 receptors, with much less effect at P2X2, P2X4, and P2X7 receptors and at the P2X1/5 heteromer.3,13,14 . Note, however, that TNP-ATP appears to be rapidly degraded in intact tissues37 and so this limits its usefulness. Diinosine pentaphosphate (IP5 I) (Figure 4) is synthesized by deamination of AP5 A and is a potent antagonist at two types of nucleotide receptors. It was originally described as an antagonist of a specific dinucleotide receptor activated by diadenosine polyphosphates present in rat cerebral cortex synaptosomes.26 It is also an antagonist at native P2X receptors in these synaptosomes and in the guinea pig vas deferens. Subsequently, within 20

recombinant P2X receptors, IP5 I was reported to be a potent, nonsurmountable antagonist at rat P2X1 receptors and a moderately potent antagonist at rat P2X3 receptors. Moreover, it has no effect at P2X2 and P2X5 receptors and potentiates agonism at P2X4 receptors.28,38 All the antagonists discussed earlier are large polyanionic molecules or acidic nucleotides that are rapidly broken down. Consequently, they are poor starting points for the development of compounds that will be useful therapeutically. In an attempt to develop antagonists with improved potency and selectivity, other chemical structures have been investigated for P2X1 antagonism, including the benzimidazole2-carboxamide derivative, RO-139 (Figure 4), and 5-methyl-6,7-dihydro-5H-cyclopentapyrazine.8 These display some degree of P2X1 selectivity, but are less potent than NF449 at P2X1 receptors.

P2X2 RECEPTORS P2X2 receptors are widely expressed in central and peripheral neurones and have been implicated in mediating neurotransmission and sensory transduction, although their function in many cases is still unclear, as is their therapeutic potential.4–6 It is notable that they are expressed at high levels in nociceptive sensory neurones, and therefore might be targeted for the development of novel analgesics.5 It is likely, however, that targeting P2X3 receptors present in the same neurones will be more successful, as will be discussed later. P2X2 receptors are activated by micromolar amounts of BzATP, ATP, 2-meSATP, but not α, βmeATP or β, γ -meATP,3,13,14 or usually the diadenosine polyphosphates27 (but see Ref 40, where AP4A was reported to be a potent agonist) and are antagonized by micromolar suramin, PPADS, TNP-ATP, and NF279.3,13,14 Until recently, no ligands were available that display any degree of selectivity toward P2X2 receptors, but an anthraquinone derivative, PSB-1011, has been shown to be a potent and selective P2X2 antagonist.41 (Figure 5). It is active in the mid-nanomolar concentration range, displays more than fivefold selectivity over the P2X1 and P2X3 receptors, and is essentially inactive at the P2X4 and P2X7 receptors. 30 nM PSB-1011 shifted the ATP concentration–response curve to the right in a parallel manner, with no change in maximum response, but the right shift induced by 100 nM PSB-1011 was less than would be predicted for a true competitive antagonist, suggesting that the drug might in fact be a negative allosteric modulator.





order of magnitude, whereas ethanol had no effect on the maximum effect of ATP and increased its potency by a moderate amount.

Antagonists

FIGURE 5 | Chemical structure of selective P2X2 and P2X4 receptor antagonists.

P2X3 AND P2X2/3 RECEPTORS The P2X3 subunit is expressed at high levels in sensory neurones, particularly the small-to-medium diameter nociceptive C and Aδ fibers, where it forms functional receptors on its own and in combination with the P2X2 subunit.42,43 Reports using a variety of technologies indicate that P2X3 and P2X2/3 receptors are likely to be involved in chronic inflammatory and neuropathic pain4,5,44 and so these represent important therapeutic targets that have generated much interest in the pharmaceutical industry. P2X3 receptors are also present in the peripheral terminals of sensory nerves present in the wall of the urinary bladder and in the urinary bladder epithelia, where they appear to contribute to transduction of sensory information relating to the filling state of the bladder.5,29,30 They are, therefore, also a potential target for the treatment of dysfunctional urinary bladder.

Agonists The P2X3 homomultimers and P2X2/3 heteromultimers have very similar pharmacological properties and hence will be discussed together.3,13,14 Indeed, it appears that in the heteromultimer the P2X2 subunit controls the receptor’s biophysical properties, whereas the P2X3 subunit defines its pharmacological properties. Like P2X1 receptors, they are activated by BzATP > 2-meSATP = ATP > α, β-meATP. β, γ -meATP is also a potent and stable, partial agonist at P2X3 receptors, but unlike at the P2X1 receptor, its action is stereo-selective, with only the d-isomer being active.45 Diadenosine polyphosphates are partial agonists, with AP6 A.27 The agonist action of ATP at the P2X3 homomer is potentiated by cibacron blue46 and ethanol.47 The former increased the maximum response evoked by ATP and shifted its concentration–response curve leftwards by almost an Vo lu me 1, Jan u ary/Febru ary 2012

Several compounds show a small degree of selectivity as antagonists at the P2X3 and P2X2/3 receptors over the P2X1 receptor. For example, TNP-ATP is a highly potent competitive antagonist at both forms of the P2X3 receptor, with IC50 values in the low nanomolar range, but has less than 10-fold selectivity over the P2X1 receptor.3,13,14 Similarly, the suramin derivative NF110, active in the mid-nanomolar range at P2X3 and P2X2/3 receptors, is about 2.5 times less potent at P2X1 receptors.34 A major breakthrough in this field was the development of A317491 by Abbott Laboratories (Figure 6), a tricarboxylic acid that is a highly selective, potent (nanomolar affinity), stable, and competitive P2X3 and P2X2/3 receptor antagonist, with more than 2 orders of magnitude selectivity over P2X1 and P2X2 receptors, and no effect at P2X4 and all P2Y receptors.14,48,49 A317491 inhibits chronic neuropathic and inflammatory pain in a range of animal models and tests,48–50 although its therapeutic usefulness is limited by its low oral bioavailability, high protein binding, and its inability to significantly cross the blood–brain barrier.50,51 In contrast, a drug developed by GlaxoSmithKline, compound A, effectively penetrates the CNS.50 More recently, Roche have developed RO-329 and AF-353 (formerly RO-4)52 (Figure 6), which are both derivatives of the diaminopyrimidine antibacterial agent, trimethoprim. RO-3 is active at P2X3 monomers and P2X2/3 heteromers at submicromolar concentrations and is highly selective for these over other homomeric P2X subtypes. It is also moderately stable and orally available (14%) and once absorbed has low plasma protein binding (49%), and a half-life of 0.41 h distributes widely and reaches the CNS. AF-353 has at least 300fold selectivity for the P2X3 and P2X2/3 receptors over other P2X receptors and a variety of non-P2X receptors, enzymes, and transporter proteins.52 In contrast to A-317491 and TNP-ATP, AF-353 acts in a noncompetitive manner. It also has good oral availability (%F = 32.9), a reasonable half-life (t 1/2 = 1.63 h), and in plasma is 98.2% protein-bound, and these favorable pharmacokinetic parameters suggest that AF-353 will be an excellent tool for use in in vivo studies. Indeed, AF-353 has already been found to be effective in a rat model of bone cancer pain.53 Another Roche compound, RO-85, is interesting as


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FIGURE 6 | Chemical structure of selective P2X3 receptor antagonists.

it shows selectivity for the P2X3 homomer over the P2X2/3 heteromer, and therefore may be useful in differentiating between their physiological roles.54 Finally, and intriguingly, spinorphin is an endogenous heptapeptide that is an extremely potent (IC50 = 8.3 pM), though noncompetitive, P2X3 antagonist.55 Several pharmaceutical companies have recently registered patents on a structurally diverse range of compounds that antagonize P2X3 and P2X2/3 receptors at nanomolar concentrations, including Roche: the diaminopyrimidines RO-3, RO-51, AF353 and benzamides containing piperazine, tetrazole, imidazole, triazole, and pyrazole; Renovis: 5,6,7,8tetrahydropyrido[4,3,d]-pyrimidines; AstraZeneca: pyrrolopyrimidin-7-one derivatives; Merck: biaryl benzamides; Shionogi: pyrrolinone derivatives; and Astrellas Pharma: minodronic acid (see Ref 8). This clearly indicates the great therapeutic potential of P2X3 and P2X2/3 antagonists.

P2X4 RECEPTORS

Agonists BzATP, ATP, and 2-meSATP stimulate the P2X4 receptor3,13,14 and α,β-meATP was originally reported to be inactive, but a subsequent report showed that its actions were species-dependent.20 Thus, α, βmeATP is a partial agonist at the human and mouse isoforms, but an antagonist at the rat P2X4 receptor. Diadenosine polyphosphates are partial agonists, with AP4 A > AP5 A > AP6 A.27 A distinguishing feature of this receptor is that currents evoked by ATP are potentiated by ivermectin,3 which is not the case with other subtypes of P2X receptors. Both the sensitivity to ATP and the maximum currents evoked are increased. The response to ATP is also potentiated by the dyes, cibacron blue57 and reactive blue 2.15

Antagonists

The P2X4 receptor, like the P2X2 receptor, is widely expressed in central and peripheral neurones, and also in various glandular tissues, but its function in these cells is mostly still unclear.4–6 Note that an added complication, however, is that, as described below, the P2X4 receptor pharmacological properties vary in a species-dependent manner. Currently, the most promising therapeutic target is the P2X4 receptors expressed in spinal microglial cells, as these appear to play a crucial role in the development of chronic neuropathic pain.56 Injury to spinal nerves greatly increased the expression of P2X4 receptors in the spinal microglia and downregulation of the receptors using specific antisense oligonucleotides depressed the associated tactile allodynia. Interestingly, TNP-ATP, which is a weak antagonist at P2X4 receptors,3,13,14 but not PPADS, also inhibited the tactile allodynia, 22

consistent with the involvement of P2X4 receptors. A potent and selective P2X4 antagonist might, therefore, be a useful, novel analgesic.

In contrast to other subunits, the rat P2X4 receptor subunit is relatively insensitive to conventional P2X receptor antagonists, such as suramin and PPADS;3,48 however, mouse and human orthologs of this receptor subunit display some, though reduced, sensitivity to PPADS and TNP-ATP.3,13,14,20 This scarcity of selective P2X4 receptor antagonists has greatly hindered the study of their physiological and pathophysiological roles, so the recent introduction of 5-BDBD, a benzofurodiazepine derivative that antagonizes P2X4 receptors at submicromolar concentrations, represents an interesting advance48 (Figure 5). This compound has yet to be studied widely, however, so its usefulness remains to be confirmed. Another interesting advance is the report that antidepressants, including the selective serotonin uptake inhibitors paroxetine and fluoxetine, and several tricyclic antidepressants,




including amitriptyline, also antagonize recombinant human and rat P2X4 receptors,58 and it was suggested that this might contribute to their ability to alleviate chronic pain. However, in a subsequent study, amitriptyline was found to have no effect at recombinant human P2X4 receptors and to act as a noncompetitive antagonist at recombinant rat and mouse P2X4 receptors.59 Interestingly, one group did find an inhibitory effect of tricyclic antidepressants and serotonin uptake inhibitors on native P2X4 receptors in mouse cerebellar microglia,60 but they concluded that this was due to inhibition of trafficking of the P2X4 receptors to the plasma membrane, rather than antagonism of the receptor per se. Given the involvement of P2X4 receptors in inflammatory and neuropathic pain, and so the potential usefulness of P2X4 antagonists as analgesics, it is not surprising that patents have been registered on numerous compounds, including benzofuro-1,4-diazepin-2-one derivatives (Bayer and Nippon Chemiphar); piperazine derivatives (Nippon Chemiphar); the tricyclic antidepressants imipramine, nortriptyline, amitriptyline, desipramine, and doxepin (Kyushu University); and the selective serotonin reuptake inhibitors paroxetine and fluoxetine (Kyushu University) (see Ref 8).

P2X5 AND P2X1/5 RECEPTORS The P2X5 receptor is expressed in various tissues, such as central neurones, the eye, and cardiac muscle, but it appears to be most prominent in tissues that are differentiating, including skeletal muscle, skin, and epithelia.4–6 On the whole, however, the physiological and pathophysiological functions of P2X5 receptors and the potential therapeutic usefulness of selective drugs are still unclear, particularly as a single nucleotide polymorphism at the 3 splice site of exon 10 of the P2X5 gene generates a nonfunctional protein lacking exon 10. Analysis of the expression of this splice variant in humans showed that white Americans, Chinese, and people from the Middle East predominantly express the nonfunctional form, whereas African-Americans are polymorphic.61 Thus, most humans in the populations studied do not appear to express functional P2X5 receptors. ATP and 2-meSATP are full agonists at the P2X5 receptor,3,13,14 but some species differences are apparent in the action of other agonists. BzATP is a full agonist of similar potency to ATP at the human isoform,17 whereas it is a less potent partial agonist at the rat.18 α, β-meATP is a full agonist, though less potent than ATP, at the human receptor17 and was initially reported to be inactive at the rat isoform.16,62 Vo lu me 1, Jan u ary/Febru ary 2012


A subsequent study using elaborate ionic conditions found α, β-meATP to display partial agonism in the rat.18 β, γ -meATP is also a partial agonist with low potency in the rat and the diadenosine polyphosphates are partial agonists, with a potency order of AP4 A > AP5 A > AP6 A = AP3 A.18 Agonist action is blocked by the conventional and nonselective antagonists PPADS > suramin in both species, but TNP-ATP is more potent than suramin at the rat P2X5 receptor, but less active at the human receptor.17,18 The dye, Brilliant Blue G, is also a moderately potent antagonist at human P2X5 receptors.17 When rat P2X1 and P2X5 subunits are coexpressed, α, β-meATP evokes a novel maintained inward current, which is inhibited by TNP-ATP, PPADS, and suramin.16,63 Recently, evidence for functional P2X1/5 heteromers in vivo was obtained in mouse cortical astrocytes.64 In these cells, ATP and α, β-meATP evoked biphasic inward currents, which were inhibited by PPADS and TNP-ATP, but unaffected by ivermectin. Quantitative real-time polymerase chain reaction (PCR) showed strong expression of P2X1 and P2X5 mRNA and some expression of P2X2 mRNA, whereas the other subunits were absent. This genetic and pharmacological profile is most consistent with the expression of the P2X1/5 heteromer.

P2X6, P2X2/6, AND P2X4/6 RECEPTORS The P2X6 subunit is widely expressed in the CNS.62 There have been relatively few studies on properties of the homomeric P2X6 receptor, as it expresses extremely poorly on its own in Xenopus laevis oocytes and HEK293 cells15,62 and is usually unable to establish functional homomers.65 However, it was then shown that functional expression depends on increased N-glycosylation, which enables migration of P2X6 subunits from the endoplasmic reticulum to the plasma membrane.19,66 Under these conditions, ATP and α, β-meATP are full agonists, while TNP-ATP and PPADS, but not suramin, are antagonists.19 The distribution of the P2X6 subunit closely overlaps that of the P2X2 and P2X4 receptors, and therefore P2X2/667 and P2X4/6 heteromers15 may be the predominant native forms of the receptor. Their physiological functions are presently unclear. ATP and 2-meSATP are equipotent at the P2X2/6 receptor and more potent than BzATP and AP4 A, whereas α, β-meATP and β, γ -meATP are inactive.67 Suramin antagonized the actions of ATP. ATP and 2-meSATP are also equipotent at the P2X4/6 receptor, but only slightly more potent than α, β-meATP, and ATP was antagonized by suramin and PPADS.15


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P2X7 RECEPTORS The P2X7 receptor is mainly expressed in glial cells of the CNS and the peripheral nervous system, such as astrocytes, microglia, oligodendrocytes, and Schwann cells, and in cells of the immune system, including macrophages, monocytes, lymphocytes, dendritic cells, and mast cells.4–6 Prolonged activation leads not only to opening of the intrinsic cationic channel but also of a larger pore that enables substances of up to approximately 1 kDa molecular weight to enter cells. Although the P2X7 receptor is a ligand-gated cation channel, its activation leads to rapid changes in intracellular enzyme activity normally associated with the activation of G-proteincoupled receptors, such as activation of caspase-1 and the production of signaling molecules, especially the proinflammatory cytokines, interleukin-1β and interleukin-18. Thus, it is not surprising that the role of the P2X7 receptor in initiating inflammatory responses has been studied widely and recently it was shown to be a major stimulant of the NLRP3 inflammasome, a protein complex that initiates the production and release of interleukin-1β.68 Because of its crucial role in initiating the inflammatory signaling molecule cascade, the P2X7 receptor is currently under intense scrutiny as a target for the treatment not only of chronic inflammatory disorders, such as rheumatoid arthritis and osteoarthritis,4,7 but also of conditions which appear to involve inflammatory cytokine production, including chronic neuropathic pain,48 contact hypersensitivity,31 and affective disorders, including depression and anxiety.69 In addition, interleukin-1β release in the hippocampus is impaired in P2X7 receptor knockout mice and this is associated with a spatial memory deficit,70 so the P2X7 receptor could be a novel target for reducing memory loss. Thus, the selective targeting of P2X7 receptors clearly has great therapeutic potential.

Antagonists

Agonists In standard physiological bathing solutions containing millimolar amounts of divalent cations, ATP has a surprisingly low potency, with an EC50 value of around 100 µM, which is 1–2 orders of magnitude greater than for the other P2X subtypes.3,13,14 2-meSATP and BzATP, but not α, β-meATP, β, γ -meATP, or the diadenosine polyphosphates, are also agonists and since BzATP is the most potent, it tends to be the agonist of choice for studying P2X7 receptor function.3,13,14 Agonist potency is increased by removal of divalent cations and therefore experiments are often conducted in low [Ca2+ ] and [Mg2+ ] media. 24

A complicating factor in characterizing the pharmacological properties of the P2X7 receptor is that the absolute and relative agonist potency varies in a species-dependent manner. For example, when studied under identical conditions, 2-meSATP had a pEC50 of 3.75 at the human receptor, but was inactive at the mouse and rat.35 In contrast, ATP was active at each, but with substantially different pEC50 values; 2.62 (mouse), 3.89 (rat), and 4.13 (human). Similarly, BzATP was active at each and the corresponding values were 3.99 (mouse), 5.01 (rat), and 5.33 (human). The rat and mouse receptors have 84% identity and their amino acid sequences differ in 88 positions only, most of which are in the ectodomain. Swapping two stretches (amino acids 115–136 and 282–288) of the rat and mouse receptors caused the latter to display the agonist sensitivities of the rat receptor.71 Point mutations within these regions identified asparagine 284 in the rat homolog as being responsible for the difference in ATP potency, whereas asparagine 284 and lysine 127 accounted for the difference in BzATP potency. Recent radioligand binding studies using [3 H]compound-17 (AZ10606120) have identified two allosteric modulators of the P2X7 receptor.72,73 Compound-17 itself is a negative allosteric modulator at human and rat P2X7 receptors, while GW791343 displays positive allosterism at the human isoform, but negative allosterism at the rat isoform. Further mutational analysis identified amino acid 95 (phenylalanine in human, leucine in rat) as the main determinant of the species-dependent effects of GW791343.74 More recently, the H1 -receptor antagonist, clemastine, was also identified as a positive allosteric modulator of the P2X7 receptor.75 Clemastine shifted the ATP concentration–response curve to the left and increased its slope, without changing the maximum response. In addition, the speed of pore formation was accelerated.

Similar to agonists, the potency of the standard antagonists is much lower at P2X7 receptors compared with the other subtypes. PPADS antagonizes human, rat, and mouse P2X7 receptors, though it is less potent at the latter.35 Suramin, on the other hand, has variable or no inhibitory effect in any species.3,13,14 In early studies, several other compounds were used as P2X7 antagonists, including the Ca2+ /calmodulin-dependent protein kinase II inhibitors, KN-62 (Figure 7) and calmidazolium, but their antagonist properties were found to be far from ideal, due to variable potency and species differences. In addition, KN-62 and calmidazolium are large





cationic compounds and therefore not good structures on which to base the development of therapeutically useful drugs. The irreversible P2X7 antagonist, oxidized ATP (Figure 7), does not appear to display prominent species dependence in its actions, but has low potency, many other pharmacological actions, and requires long preincubation times to be effective. Finally, Brilliant Blue G is active at nanomolar concentrations and selective for the P2X7 receptor over other subtypes, but again it is a large charged compound that would be difficult to modify structurally to produce a compound that is therapeutically useful.3,13,14

compounds inhibited the pore formation and secretion of interleukin-1β induced by BzATP activation of native P2X7 receptors in human THP-1 cells. The antagonists were then tested in commonly used in vivo animal models of chronic pain. When administered i.p., both reversed the mechanical allodynia induced by ligation of the L5–L6 spinal nerves (Chung model of neuropathic pain). In addition, A740003 depressed the mechanical allodynia associated with sciatic nerve ligatures (Bennett model of neuropathic pain) and the thermal hyperalgesia induced by injection of carrageenan or complete Freund’s adjuvant into the hind paw (inflammatory pain).

Selective Antagonists

A804598

A major breakthrough in this area was the discovery of a number of lead compounds by pharmaceutical companies using high-throughput screening, followed by extensive chemical modification, which led to the development of many potent and selective P2X7 antagonists based on novel structures (see Ref 7 for detailed review). The majority of these compounds are reported in patents and therefore information on their functional properties is limited. The properties of several compounds have been published in the scientific literature, however, and most of them are commercially available.

More recently, Abbot Laboratories have reported the properties of a third, structurally novel P2X7 antagonist, the aminoquinoline, A80459878 (Figure 7). This compound is equipotent at human, rat, and mouse P2X7 receptors, is more potent than A740003 and A438079 in each species, and acts in a competitive manner, with a pA2 of 7.7. It also inhibits pore formation and secretion of interleukin-1β induced by BzATP in human THP-1 cells78 and crosses the blood–brain barrier.79 Its ability to treat chronic neuropathic and inflammatory pain has not yet, however, been reported.

AZ11645373

GSK314181A

This cyclic imide was developed by AstraZeneca and antagonizes human P2X7 receptors at low- to midnanomolar concentrations10 (Figure 7). It is highly selective for the P2X7 subtype, having no effect at other P2X subtypes at 10 µM. Its action is, however, nonsurmountable, slowly reversible, and species-dependent, and it is much less effective at rat P2X7 receptors. Finally, it inhibits the ATP-induced release of interleukin-1β from the differentiated human macrophage phenotype, THP-1 cell line.

This adamantane derivative, also known as AACBA, was developed by GlaxoSmithKline and antagonizes the human P2X7 receptor in the mid-nanomolar range and the rat P2X7 receptor at higher concentrations80 (Figure 7). In in vivo models of acute pain, it inhibited interleukin-6 release induced by lipopolysaccharide and the edema and acute mechanical hypersensitivity induced by carrageenan, whereas in a model of chronic pain it depressed the noxious effects associated with collagen-induced arthritis. In contrast, it had no effect on the mechanical allodynia caused by spinal nerve ligation (neuropathic pain), which might be due to insufficient penetration of the blood–brain barrier by the drug.

A740003, A438079 In 2006, Abbot Laboratories described two new selective and competitive P2X7 antagonists, the disubstituted cyanoguanidine derivative, A74000376 , and the disubstituted tetrazolylmethylpyridine, A43807977 (Figure 7). A438079 is effective in the mid to high nanomolar range and approximately three times more potent at the human than rat homolog, with little or no activity at other P2X subtypes tested. A740003 is more potent than A438079 in both species, by approximately 10 times at the rat and 3 times at the human P2X7 receptors. A recent study reported that both are also active, though at slightly higher concentrations, at the mouse P2X7 receptor.35 Both Vo lu me 1, Jan u ary/Febru ary 2012

Patented Compounds Many pharmaceutical companies have registered patents on a range of structurally diverse P2X7 antagonists.7,8 These include quinoline carboxamides with cyclohexanes and cycloheptanes, such as AZD-9056 (AstraZeneca), oxoisoquinoline carboxamides with adamantanes or substituted phenyl rings, such as EVT-401 (Evotec), pyrazolo-[1,5,a]pyridine carboxamides with adamantane or without adamantine derivatives (Neurogen), carboxamides


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FIGURE 7 | Chemical structure of selective P2X7 receptor antagonists.

with oxoisoindoles or piperidinones or oxazolidines or morpholines or imidazolidine or pyrazoles (GlaxoSmithKline), carboxamides such as pyrrolo-[2,3,b]pyridine carboxamides and indole carboxamides (Lundbeck), polycyclic guanine derivatives (Schering), pyridazinone derivatives (Nissan), quinoline and isoquinoline carboxamides (Janssen), and indole-3carboxamides or azaindole-3-carboxamides (Affectis Pharmaceuticals). Several of these are currently in phase II clinical trials, including CE-224535 (Pfizer), AZD-9056 (AstraZeneca), and EVT-401 (Evotec).8

CONCLUSION It is now 26 years since the P2X receptor was first identified and named and 16 years since the first P2X subunit was cloned and functionally expressed. In that time, our knowledge and understanding of the physiological and pathophysiological roles of P2X receptors have expanded immeasurably. It was a surprise that the genes encoding seven different P2X subunits were cloned, as there was little indication of such heterogeneity in the published literature at the time. Together, these subunits form at least 12 different types of functional P2X receptor, which are distributed widely throughout the body. Initially, the pharmacological tools available to study the actions 26

mediated by these receptors and to identify individual native P2X subtypes expressed in tissues were very limited. ATP is an agonist at all subtypes and its structural analogs, α,β-meATP and 2-meSATP, have limited subtype selectivity of action. Similarly, the diadenosine polyphosphate agonists, AP6 A, AP5 A, and AP4 A, were of limited use in characterizing the subtype of native receptors. Receptors are more easily identified using selective antagonists, but to begin with no such compounds were available. Suramin and PPADS, already known for their other actions, were found to also antagonize P2X receptors, but with low potency and little subtype selectivity. Subsequently, modifying their structure generated a number of antagonists with increased subtype selectivity and/or potency. A major advance with P2X antagonists came, however, when pharmaceutical companies used high-throughput screening to identify lead compounds, followed by extensive chemical modification, which led to the development of many potent and selective P2X antagonists that are structurally novel. Currently, selective and potent antagonists include NF449 and RO-1 at P2X1 receptors; PSB-1011 at P2X2 receptors; A317491, compound A, RO-3, and AF-353 at P2X3 and P2X2/3 receptors; 5-BDBD at P2X4 receptors; and A740003, A438079, A804598, GSK314181A, AZ10606120,




AZ11645373, AZD-9056, CE-224535, and EVT-401 at P2X7 receptors. The introduction of P2X antagonists enabled the physiological roles of P2X receptors to be characterized. The P2X1 receptor is widely expressed in smooth muscle and mediates the neurotransmitter actions of ATP released from sympathetic and parasympathetic nerves. It is also expressed in platelets and by mediating Ca2+ influx it appears to be involved in platelet shape change and aggregation. P2X2 receptors are widely expressed in central and peripheral neurones and have been implicated in mediating neurotransmission and sensory transduction, although their function in many cases is still unclear. The P2X3 subunit is expressed at high levels in sensory neurones, particularly nociceptive C and Aδ fibers, where it forms functional receptors on its own and in combination with the P2X2 subunit. P2X3 and P2X2/3 receptors are likely to be involved in chronic inflammatory and neuropathic pain. P2X3 receptors are also present in the peripheral terminals of sensory nerves present in the wall of the urinary bladder and in the urinary bladder epithelia, where they appear to contribute to transduction of sensory information relating to the filling state of the bladder. The P2X4 receptor is widely expressed in central and peripheral neurones and in various glandular tissues, but its function in these cells is mostly still unclear. The P2X5 receptor is expressed in various tissues, such as central neurones, the eye, and cardiac muscle, but it appears to be most prominent in tissues that are


differentiating. Intriguingly, due to a single nucleotide polymorphism in the P2X5 gene, most humans in the populations studied to date do not appear to express functional P2X5 receptors. The distribution of the P2X6 subunit closely overlaps that of the P2X2 and P2X4 receptors and P2X2/6 and P2X4/6 heteromers may be the predominant native forms of the receptor, but their functions are presently unclear. Finally, the P2X7 receptor is mainly expressed in glial cells of the CNS and the peripheral nervous system and in cells of the immune system. In the latter, they play a crucial role in initiating inflammatory responses. The development of potent and selective antagonists has greatly increased our understanding of the physiological and pathophysiological functions of P2X receptors. Consequently, the therapeutic usefulness of these compounds is currently being investigated in a variety of disorders, including thrombosis (P2X1), chronic neuropathic and inflammatory pain (P2X3, P2X2/3, P2X4, P2X7), bone cancer pain (P2X3, P2X2/3), dysfunctional urinary bladder (P2X1, P2X3, P2X2/3), rheumatoid arthritis and osteoarthritis (P2X7), and depression (P2X7). With the continuing development of novel antagonists, for example, the very recent report described earlier on the first select P2X2 antagonist, it is very likely that our understanding of the roles of P2X receptors will continue to improve and further therapeutic applications will be discovered and developed.

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