P2X7 receptor-mediated release of microglial prostanoids and miRNAs correlates with reversal of neuropathic hypersensitivity in rats.

BACKGROUND: P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. METHODS: Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. RESULTS: Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1ß, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. CONCLUSIONS: We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. SIGNIFICANCE: Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.

Chemical biology of mGlu4 receptor activation: dogmas, challenges, strategies and opportunities.

Drug design necessitates a clear understanding of the phenotypic response to be elicited by a given ligandtarget interaction. This relationship is relatively well understood for classical biological targets of drug action, but for some novel targets, notably those amenable to allosteric modulation, developing such understanding may represent a more challenging task. In order to gain knowledge on the nature of the functional response derived from mGlu4 receptor activation, its molecular and cell biology are reviewed, including signalling pathways involved, receptor localization in central nervous system and beyond, and potential genetic links to disease. Broadly held views for both, orthosteric agonists as well as allosteric modulators, are compared with specific observations for the case of mGlu4 receptor activation via orthosteric and allosteric mechanisms. First, sub-type selectivity and brain penetration of amino acid mGlu4 receptor agonists are discussed, followed by the quantification of functional allosteric effects, the potential role of heterodimers in the functional response, and the observation of supra-physiological efficacy of mGlu4 receptor PAMs. We show that, in our analysis, these attributes differ from those that may be expected by extrapolating from broad knowledge. In addition, recent progress with mGlu4 receptor radioligands and PET ligands is summarized.

Fused thiazolyl alkynes as potent mGlu5 receptor positive allosteric modulators.

A new series of potent fused thiazole mGlu5 receptor positive allosteric modulators (PAMs) (10, 11 and 27-31) are disclosed and details of the SAR and optimization are described. Optimization of alkynyl thiazole 9 (Lu AF11205) led to the identification of potent fused thiazole analogs 10b, 27a, 28j and 31d. In general, substituted cycloalkyl, aryl and heteroaryl carboxamides, and carbamate analogs are mGlu5PAMs, whereas smaller alkyl carboxamide, sulfonamide and sulfamide analogs tend to be mGlu5 negative allosteric modulators (NAMs).

Discovery and structure-activity relationship of 1,3-cyclohexyl amide derivatives as novel mGluR5 negative allosteric modulators.

A novel series of trans-1,3-cyclohexyl diamides was discovered and characterized as mGluR5 negative allosteric modulators (NAMs) lacking an alkyne moiety. Conformational constraint of one of the amide bonds in the diamide template led to a spirooxazoline template. A representative compound (24d) showed good in vitro potency, high CNS penetration and, upon subcutaneous dosing, demonstrated efficacy in the mouse marble burying test, generally used as indicative of potential anxiolytic activity.

Azetidinyl oxadiazoles as potent mGluR5 positive allosteric modulators.

A novel series of aryl azetidinyl oxadiazoles are identified as mGluR5 positive allosteric modulators (PAMs) with improved physico-chemical properties. N-substituted cyclohexyl and exo-norbornyl carboxamides, and carbamate analogs of azetidines are moderate to potent mGluR5 PAMs. The aryl, lower alkyl carboxamides analogs and sulfonamide analogs of azetidines are moderate mGluR5 negative allosteric modulators (NAMs). In the aryl oxadiazole moiety, substituents such as fluoro, chloro and methyl are well tolerated at the meta position while para substituents led to either inactive compounds or NAMs. A tight pharmacophore and subtle 'PAM to NAM switching' with close analogs makes the optimization of the series extremely challenging.

N-Aryl pyrrolidinonyl oxadiazoles as potent mGluR5 positive allosteric modulators.

A novel series of N-aryl pyrrolidinonyl oxadiazoles were identified as mGluR5 positive allosteric modulators (PAMs). Optimization of the initial lead compound 6a led to the identification of the 12c (-) enantiomer as a potent compound with acceptable in vitro clearance, CYP, hERG and PK properties. Para substituted N-aryl pyrrolidinonyl oxadiazoles are mGluR5 PAMs while the meta and ortho substituted N-aryl pyrrolidinonyl oxadiazoles are negative allosteric modulators (NAMs). Para fluoro substitution on the N-aryl group and meta chloro or methyl substituents on the aryl oxadiazole moiety are optimal for mGluR5 PAM efficacy. The existence of an exquisitely sensitive 'PAM to NAM switch' within this chemotype making it challenging for simultaneous optimization of potency and drug-like properties.

Efficacy switching SAR of mGluR5 allosteric modulators: highly potent positive and negative modulators from one chemotype.

A series of metabotropic glutamate 5 receptor (mGluR5) allosteric ligands with positive, negative or no modulatory efficacy is described. The ability of this series to yield both mGluR5 PAMs and NAMs with single-digit nanomolar potency is unusual, and the underlying SAR is detailed.

Novel heterocyclic compounds as mGlu5 antagonists: WO2009015897.

Metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulators (NAMs) have previously been implicated in a number of pathophysiological conditions, based on preclinical, and to some extent clinical, proof of concept for migraine, gastroesophageal reflux disease, Parkinson's disease and anxiety. In the past, the potential use of known mGlu5 antagonists for the treatment of lower urinary tract disorders was disclosed. In the patent evaluated herein, novel derivatives of 4-(prop-2-ynylidene)piperidine are described and claimed by Recordati Ireland Ltd, Ireland, as NAMs at mGlu5 for the treatment of lower urinary tract disorders. Selected compounds reported in this application were efficacious in the cystometry model of bladder dysfunction in conscious rats, and mGlu5 NAMs are, therefore, suggested to have potential for the treatment of lower urinary tract disorders.

Discovery of a potent and brain penetrant mGluR5 positive allosteric modulator.

This Letter describes the discovery of a novel series of mGluR5 positive allosteric modulators (PAMs). The lead compound, 11c, exhibits excellent potency (EC(50)=30 nM) in vitro, and reaches high brain levels in both rats and mice after oral administration.

Identification and characterization of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a novel, orally bioavailable C5a receptor inverse agonist.

The complement system represents an innate immune mechanism of host defense that has three effector arms, the C3a receptor, the C5a receptor (C5aR), and the membrane attack complex. Because of its inflammatory and immune-enhancing properties, the biological activity of C5a and its classical receptor have been widely studied. Because specific antagonism of the C5aR could have therapeutic benefit without affecting the protective immune response, the C5aR continues to be a promising target for pharmaceutical research. The lack of specific, potent and orally bioavailable small-molecule antagonists has limited the clinical investigation of the C5aR. We report the discovery of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a small-molecule, orally bioavailable, selective, and potent inverse agonist of the human C5aR. NDT 9513727 was discovered based on the integrated use of in vitro affinity and functional assays in conjunction with medicinal chemistry. NDT 9513727 inhibited C5a-stimulated responses, including guanosine 5'-3-O-(thio)triphosphate binding, Ca(2+) mobilization, oxidative burst, degranulation, cell surface CD11b expression and chemotaxis in various cell types with IC(50)s from 1.1 to 9.2 nM, respectively. In C5a competition radioligand binding experiments, NDT 9513727 exhibited an IC(50) of 11.6 nM. NDT 9513727 effectively inhibited C5a-induced neutropenia in gerbil and cynomolgus macaque in vivo. The findings suggest that NDT 9513727 may be a promising new entity for the treatment of human inflammatory diseases.

Aminopyrazine CB1 receptor inverse agonists.

A series of 5,6-diaryl-2-amino-pyrazines were prepared and found to have antagonist-like properties at the CB1 receptor. Subsequent SAR studies optimized both receptor potency and drug-like properties including solubility and Cytochrome-P450 inhibition potential. Optimized compounds were demonstrated to be inverse agonists and compared in vivo with rimonabant for their ability to inhibit food intake, to occupy central CB1 receptors and to influence hormonal markers associated with obesity.

Leukocyte effects of C5a-receptor blockade during simulated extracorporeal circulation.

BACKGROUND: Distinct pathways of leukocyte activation during simulated cardiopulmonary bypass are mediated by the complement C5a anaphylatoxin. We hypothesized that a human C5a receptor antagonist would specifically inhibit the inflammatory response of neutrophils to simulated extracorporeal circulation, while preserving the C5b-9 pathway for innate immunity. METHODS: An in vitro extracorporeal circuit recirculated fresh heparinized whole blood through a membrane oxygenator with and without addition of a small molecule human C5a receptor antagonist. Samples were periodically drawn over 90 minutes for complement and leukocyte activation studies. RESULTS: Addition of the C5a receptor antagonist to simulated extracorporeal circulation abrogated both neutrophil CD11b upregulation and interleukin 8 release (p < 0.01 for both), despite full generation of C3a and C5b-9; however, elastase release from neutrophils was unaffected. Although C5a receptor blockade only trended toward inhibiting monocyte CD11b upregulation (p = 0.09), circuit clearance of both monocytes (p = 0.04) and neutrophils (p = 0.01) was significantly decreased. In addition, the C5a receptor antagonist completely blocked both neutrophil-platelet and monocyte-platelet conjugate formation (p < 0.001 for both), without affecting platelet P-selectin expression. CONCLUSIONS: C5a receptor blockade during simulated extracorporeal circulation completely blocked neutrophil beta2 integrin upregulation and induction of plasma interleukin 8, suggesting an acute downregulatory effect on neutrophil chemotaxis-related pathways, while preserving terminal complement generation and neutrophil elastase release. Inhibition of leukocyte-platelet conjugate formation suggests a novel function for leukocyte adhesive receptors, possibly related to preservation of elastase generation.

Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction.

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.

C5L2, a nonsignaling C5A binding protein.

C5a anaphylatoxin, a potent inflammatory mediator, is known to act through a specific G protein coupled receptor. However, some of the complex effects of C5a in vivo may not be explained solely by the deletion of the known receptor. Here, we show that an orphan receptor, identified as C5L2, is a high affinity C5a binding protein. Unlike the previously described C5aR, C5L2 is obligately uncoupled from heterotrimeric G proteins, in part by virtue of an amino acid alteration in the so-called DRY sequence at the end of the third transmembrane segment. Both human and murine C5L2 bear a leucine for arginine replacement at this site. C5L2, when transfected into several cell types, is weakly phosphorylated in transfected cells following binding of C5a but does not induce significant activation of MAP kinases, mediate calcium flux, or stimulate chemotaxis. Bone marrow cells from wild type respond robustly to C5a with induction and suppression of a number of inflammation related genes. In contrast, C5a receptor deficient mice, which bear C5L2 alone, do not respond to C5a with changes in gene transcription by microarray analyses. Biophysical properties of the C5L2, including slow ligand on and off rates, absence of internalization, and relatively high affinity for the C5a des Arg metabolite, suggest that this receptor may serve to modulate C5a biological functions in vivo. Finally, in contrast to previous reports, we find absolutely no interaction of C5L2 with other anaphylatoxins C3a and C4a.