Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective,dual P2X3/P2X2/3 antagonist for the treatment of pain

The purinoceptor subtypes P2X₃ and P2X_2/3 have been shown to play a pivotal role in models of various pain conditions. Identification of a potent and selective dual P2X₃/P2X_2/3 diaminopyrimidine antagonist RO-4 prompted subsequent optimization of the template. This paper describes the SAR and optimization of the diaminopyrimidine ring and particularly the substitution of the 2-amino group. The discovery of the highly potent and drug-like dual P2X₃/P2X_2/3 antagonist RO-51 is presented.     

Identification and SAR of novel diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X3/P2X2/3 antagonist for the treatment of pain

P2X purinoceptors are ligand-gated ion channels whose endogenous ligand is ATP. Both the P2X₃ and P2X_2/3 receptor subtypes have been shown to play an important role in the regulation of sensory function and dual P2X₃/P2X_2/3 antagonists offer significant potential for the treatment of pain. A high-throughput screen of the Roche compound collection resulted in the identification of a novel series of diaminopyrimidines; subsequent optimization resulted in the discovery of RO-4, a potent, selective and drug-like dual P2X₃/P2X_2/3 antagonist.     

Selective P2X7 receptor antagonists for chronic inflammation and pain

ATP, acting on P2X₇ receptors, stimulates changes in intracellular calcium concentrations, maturation, and release of interleukin-1β (IL-1β), and following prolonged agonist exposure, cell death. The functional effects of P2X₇ receptor activation facilitate several proinflammatory processes associated with arthritis. Within the nervous system, these proinflammatory processes may also contribute to the development and maintenance of chronic pain. Emerging data from genetic knockout studies have indicated specific roles for P2X₇ receptors in inflammatory and neuropathic pain states. The discovery of multiple distinct chemical series of potent and highly selective P2X₇ receptor antagonists have enhanced our understanding of P2X₇ receptor pharmacology and the diverse array of P2X₇ receptor signaling mechanisms. These antagonists have provided mechanistic insight into the role(s) P2X₇ receptors play under pathophysiological conditions. In this review, we integrate the recent discoveries of novel P2X₇ receptor-selective antagonists with a brief update on P2X₇ receptor pharmacology and its therapeutic potential.     

Expression of P2X(2) and P2X (3) receptors in the rat carotid sinus, aortic arch, vena cava, and heart, as well as petrosal and nodose ganglia

With single- and double-labeling immunofluorescence techniques, the distribution patterns and morphological characteristics of P2X₂- and P2X₃-immunoreactive nerve fiber terminals and neuronal bodies have been studied in the main circulatory system baroreceptors and the nodose and petrosal ganglia of rats. A high density of P2X₂- and P2X₃-immunoreactive nerve fiber terminals was detected in the carotid sinus. P2X₂- and P2X₃-immunoreactive nerve fiber terminals were also distributed widely in the aortic arch, atrium, vena cava, and ventricles. Almost all the P2X₂-immunoreactive nerve fiber terminals were immunoreactive for P2X₃ receptors. P2X₂- and P2X₃-immunoreactive neuronal bodies were also detected in the nodose and petrosal ganglia, which are the sources of the P2X₂- and P2X₃-immunoreactive nerve terminals. P2X₂ and P2X₃ receptors were expressed in the same ganglionic neurons. These data indicate that extracellular ATP, via the homomeric P2X₂ and P2X₃ receptors, and heteromeric P2X_2/3 receptor in the sensory receptors of carotid sinus, aortic arch, atrium, and vena cava, may be involved in the regulation of systematic circulation blood pressure.     

Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo. The drug design is based on X-ray crystal analysis of a CaNmt complex with benzofuran derivative 4a. The optimization incorporates various biological investigations including a quasi in vivo assay and pharmacokinetic study. The computer aided drug design, synthesis, structure-activity relationships, and biological properties of RO-09-4879 are described in detail.     

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X_{1 –7}). P2X₃ homomultimer and P2X_2/3 heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X₅ receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X₅ receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X₅ receptors in cat. In mouse DRG, the P2X₅ receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X₅ receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X₅ receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X₅-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X₅-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X₅ receptors were coexpressed with NF-200 or with NOS. Only a few P2X₅-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X₅ receptors.     

New, potent P1/P2-morpholinone-based HIV-protease inhibitors

We have developed efficient synthesis of morpholinone-based cyclic mimetics of the P1/P2 portion of the HIV-1 protease inhibitor Amprenavir. This effort led to discovery of allyl- and spiro-cyclopropyl-P2-substituted inhibitors 17 and 31, both 500 times more potent than the parent inhibitor 1. These results support morpholinones as novel mimetics of the P1/P2 portion of Amprenavir and potentially of other HIV-protease inhibitors, and thus provide a novel medicinal chemistry template for optimization toward more potent and drug-like inhibitors.     

Nav 1.8-null mice show stimulus-dependent deficits in spinal neuronal activity

Extracellular ATP is known to mediate synaptic transmission as a neurotransmitter or a neuromodulator via ionotropic P2X and metabotropic P2Y receptors. Several lines of evidence have suggested that ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors, in which the P2X₃ subtype is considered as an important candidate for the effect. Conversely, we previously found that the activation of supraspinal P2X receptors evoked antinociception. However, the subtypes responsible for the antinociception via supraspinal P2X receptors remain unclear. In the present study, we showed that intracerebroventricular (i.c.v.) pretreatment with A-317491 (1 nmol), the novel non-nucleotide antagonist selective for P2X₃ and P2X_2/3 receptors, attenuated the antinociceptive effect produced by i.c.v. administered α,β-methylene-ATP (10 nmol), the P2X receptor agonist, in rats. Similarly, the abolishment of the P2X₃ receptor mRNA in the brainstem by repeated i.c.v. pretreatments with antisense oligodeoxynucleotide for P2X₃ gene once a day for 5 consecutive days diminished the antinociceptive effect of α,β-methylene-ATP. Furthermore, i.c.v. administration of A-317491 (1 and 10 nmol) significantly enhanced the inflammatory nociceptive behaviors induced by the intraplantar injection of formalin and intraperitoneal injection of acetic acid. Taken together, these results suggest that supraspinal P2X₃/P2X_2/3 receptors play an inhibitory role in pain transmission.     

Discovery and synthesis of a novel and selective drug-like P2X(1) antagonist

Although there is extensive literature to indicate that many different types of P2 purinoceptors are present in the lower urinary tract, the physiological role of these receptors in micturition is still uncertain. In part, this uncertainty has been caused by a lack of P2 subtype selective ligands. In this paper we report the discovery, gram scale synthesis, and binding results for 1, the first potent, drug-like, selective P2X(1) receptor antagonist described. Compound 1 was shown to be more than 30-fold selective over other purinergic receptor subtypes.     

Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X₇ receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X₇ receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X₇ receptors. Functional inhibition of P2X₇ receptors by P2X₇ receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X₇ receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were decreased in either RA- or oATP-differentiated or P2X₇ receptor knockdown N2a cells. Simply cultured N2a cells in low Ca²⁺ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X₇ receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X₇ receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X₇ receptor is important in maintaining cell survival of N2a neuroblastoma cells.     

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X_{1–5, 7}) was detected in all sampled equine tissues, whereas P2X₆ receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X_{1–3, 7} antibodies, and these were used in immunohistochemistry studies. P2X_{1–3, 7} receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X₃ subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X₂ and P2X₃ receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X_1–3 receptor subunits, whereas glial cells were positive for P2X₇ and P2X_{1 and 2} receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.     

Roles of P2X7 Receptor in Glial and Neuroblastoma Cells: The Therapeutic Potential of P2X7 Receptor Antagonists

Recently, one of the P2 purinergic receptors, the P2X₇ receptor, has been extensively studied in nervous system and important functions have been revealed in both astrocytes and microglia. Stimulation of the receptors induces a sustained and nondesensitized increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). In astrocytes purinergic receptors primarily regulate neurotransmission by inducing gliotransmitters release whereas in microglia the receptors stimulate the processing and release of proinflammation cytokines such as interleukin-1 and are thereby involved in inflammation and neurodegeneration. Thus, P2X₇ receptors are considered not only to exert physiological functions but also mediate cell death. P2X₇ receptors have also been identified in various cancer cells and in neuroblastoma cells. In these cells, the P2X₇ receptor-mediated sustained Ca²⁺ signal is important in maintaining cellular viability and growth. Accordingly, these findings not only lead to a better understanding of roles of the receptor but also prompt the development of more potent, selective and safer P2X₇ selective antagonists. These emerging antagonists bring new hope in the treatment of inflammatory-induced neurodegenerative diseases as well as neuroblastoma.     

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT₃ receptors, respectively. We have investigated the changes in P2X and 5-HT₃-mediated signalling in pelvic afferent neurons in mice deficient in P2X₂ and/or P2X₃ subunits by whole-cell recording of L₆–S₂ dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X₃ ^−/− neurons, whereas sustained currents were absent in P2X₂ ^−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X₂/P2X₃ ^Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT₃ receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X ₂ ^−/−, P2X₃ ^−/− and P2X₂/P2X₃ ^Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X₂/P2X₃ ^Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X₂/P2X₃ ^Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X₂/P2X₃ ^Dbl−/− preparations. These data suggest that P2X receptors containing the P2X₂ and/or P2X₃ subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X₂ or P2X₃ receptor genes. This effect is more pronounced when both subunits are absent.     

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X₇ receptor (P2X₇R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X₇R activation induced Ca²⁺-dependent vesicular glutamate release and significant Ca²⁺-independent glutamate efflux through the P2X₇R itself. In the present study, we investigated the effect of the new selective P2X₇R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca²⁺]_i signals and glutamate release, and evaluated whether P2X₇R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X₇R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X₇R-mediated [Ca²⁺]_i signals. These findings provide compelling evidence of functional presynaptic P2X₇R in cortical nerve terminals.     

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca²⁺]_i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X₃ subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg₂₊-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X₇ receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca²⁺]_i measurements in single synaptic terminals. Additionally, the P2X₇ receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.     

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulfide cross-linking between neighbouring P2X₁ subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X₂ (K69C, F289C), P2X₃ (K63C, F280C), and P2X₄ (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X₂K69C and P2X₂F289C mutants. Likewise, co-expression of P2X₁K68C and P2X₂F289C, but not P2X₁F291C and P2X₂K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X_1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X₃ or P2X₄ mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X₁ are preserved in P2X₂ receptors and at one of two possible interfaces in heteromeric P2X_1/2 receptors but appears to be redundant for P2X₃ and P2X₄ receptor function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.