ABSTRACT
AIMS: To investigate the effects of two single nucleotide polymorphisms (SNPs) in the human P2X7 receptor gene (P2RX7)--1068G>A (A348T) and 1513A>C (E496A)--on P2X7 receptor function, using a specific receptor antagonist (GSK1370319A) and prospective genetic stratification. METHODS: Lipopolysaccharide- and ATP-stimulated interleukin-1ß production was determined in the presence or absence of GSK1370319A in blood culture from 32 prospectively genotyped subjects. RESULTS: There was approximately 6.7-fold difference (P < 0.0001) in IC50 for inhibition of ATP-stimulated interleukin-1ß release by GSK1370319A between individuals with the homozygous gain--(1068A) and loss-of-function (1513C) genotypes (expressing the 348T, 496E and 348A, 496A alleles, respectively). CONCLUSIONS: Leukocyte P2X7 receptors had significantly altered pharmacodynamic responses to a specific antagonist (GSK1370319A), directly related to SNP genotype.
Subject(s)
Leukocytes/drug effects , Polymorphism, Single Nucleotide , Purinergic P2X Receptor Antagonists/pharmacology , Pyrrolidines/pharmacology , Receptors, Purinergic P2X7/drug effects , Receptors, Purinergic P2X7/genetics , Adenosine Triphosphate/metabolism , Adult , Dose-Response Relationship, Drug , Female , Genotype , Heterozygote , Homozygote , Humans , Interleukin-1beta/pharmacology , Leukocytes/metabolism , Lipopolysaccharides/pharmacology , Male , Middle Aged , Phenotype , Prospective StudiesABSTRACT
A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.
Subject(s)
Imidazolines/pharmacology , Purinergic Antagonists/pharmacology , Receptors, Purinergic P2X7/drug effects , Administration, Oral , Animals , Biological Availability , Half-Life , Haplorhini , Imidazolines/administration & dosage , Imidazolines/chemistry , Imidazolines/pharmacokinetics , Purinergic Antagonists/administration & dosage , Purinergic Antagonists/chemistry , Purinergic Antagonists/pharmacokinetics , RatsABSTRACT
A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.
Subject(s)
Amides/pharmacology , Purinergic P2 Receptor Antagonists/pharmacology , Pyrrolidonecarboxylic Acid/chemistry , Receptors, Purinergic P2X7/drug effects , Amides/chemistry , Drug Discovery , Models, Molecular , Purinergic P2 Receptor Antagonists/chemistry , Structure-Activity RelationshipABSTRACT
A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X(7) receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X(7) antagonist with reduced in vitro metabolism and high solubility.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Imidazoles/chemistry , Purinergic P2 Receptor Antagonists , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Pyrazoles/chemistry , Rats , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7 , Structure-Activity RelationshipABSTRACT
Structure-activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund's adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res.1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain2006, 10, 537].
Subject(s)
Acetamides/chemistry , Purinergic P2X Receptor Antagonists , Pyrazoles/chemistry , Acetamides/chemical synthesis , Acetamides/therapeutic use , Administration, Oral , Animals , Disease Models, Animal , Humans , Pain/drug therapy , Pyrazoles/chemical synthesis , Rats , Receptors, Purinergic P2X7/metabolism , Structure-Activity RelationshipABSTRACT
High-throughput screening identified compound 1 as a potent P2X(7) receptor antagonist suitable for lead optimisation. Structure-activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were investigated and compound 32 was identified as a potent P2X(7) antagonist with enhanced potency and favourable physicochemical and pharmacokinetic properties.
Subject(s)
Acetamides/chemistry , Anti-Infective Agents/chemical synthesis , Purinergic P2 Receptor Antagonists , Pyrazoles/chemical synthesis , Acetamides/chemical synthesis , Acetamides/pharmacokinetics , Administration, Oral , Animals , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacokinetics , High-Throughput Screening Assays , Humans , Injections, Intravenous , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Rats , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7 , Structure-Activity RelationshipABSTRACT
BACKGROUND AND PURPOSE: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X(7) receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. EXPERIMENTAL APPROACH: Antagonist effects at recombinant and chimeric P2X(7) receptors were assessed by ethidium accumulation and radioligand-binding studies. KEY RESULTS: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X(7) receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X(7) receptors and, for each compound, their potency estimates at human and dog P2X(7) receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X(7) receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X(7) receptor. N(2)-(3,4-difluorophenyl)-N(1)-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373. CONCLUSIONS: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X(7) receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X(7) receptor at which antagonists can bind and affect receptor function.
Subject(s)
Adamantane/analogs & derivatives , Azabicyclo Compounds/pharmacology , Purinergic P2 Receptor Antagonists , Thiazoles/pharmacology , Adamantane/metabolism , Adamantane/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Azabicyclo Compounds/metabolism , Binding Sites , Cell Line, Tumor , Dogs , Dose-Response Relationship, Drug , Glycine/analogs & derivatives , Glycine/pharmacology , Guinea Pigs , Humans , Mice , Piperazines/pharmacology , Protein Conformation , Radioligand Assay , Rats , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7 , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Proteins/antagonists & inhibitors , Species Specificity , Structure-Activity Relationship , Temperature , Thiazoles/metabolism , Transduction, Genetic , Vanadates/pharmacologyABSTRACT
Chronic inflammation is known to occur in the brains of Alzheimer's Disease (AD) patients, including the presence of activated microglia close to amyloid plaques. We utilised real time autoradiography and immunohistochemistry to investigate microglial activation and the potential anti-inflammatory effects of PPARgamma agonists in the Thy-1 APP695swe/Thy-1 PS-1.M146V (TASTPM) overexpressing transgenic mouse model of AD. An age dependent increase in specific [3H](R)-PK11195 binding to peripheral benzodiazepine receptors (PBR)/translocator protein (18 kDa) (TSPO) was observed in the cortex of TASTPM mice compared to wild type mice, indicative of microglial activation. This was consistent with immunohistochemical data showing age-dependent increases in CD68 immunoreactivity co-localised with amyloid beta (Abeta) deposits. In 10 month old TASTPM mice, pioglitazone (20 mg/kg) and ciglitazone (50 mg/kg) significantly reduced [3H](R)-PK11195 and [3H]DPA-713 binding in cortex and hippocampus, indicative of reduced microglial activation. In AD brain, significant [3H](R)-PK11195 and [3H]DPA-713 binding was observed across all stages of the disease. These results support the use of PBR/TSPO autoradiography in TASTPM mice as a functional readout of microglial activation to assess anti-inflammatory drugs prior to evaluation in AD patients.
Subject(s)
Brain Mapping , Cerebral Cortex/drug effects , PPAR gamma/agonists , Receptors, GABA-A/metabolism , Receptors, GABA/metabolism , Acetamides/metabolism , Age Factors , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Autoradiography , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Female , Humans , Isoquinolines/pharmacology , Male , Mice , Mice, Transgenic , Neuroglia/drug effects , Neuroglia/metabolism , Peptide Fragments/metabolism , Pioglitazone , Presenilin-1/genetics , Protein Binding/drug effects , Protein Binding/physiology , Pyrazoles/metabolism , Pyrimidines/metabolism , Thiazolidinediones/pharmacology , Time FactorsABSTRACT
A novel series of oxadiazole EP1 receptor antagonists was identified by replacing the amide of a known glycine sulfonamide derivative with a 1,3,4-oxadiazole. Optimization of the substitution patterns on the three aromatic rings led to the identification of high affinity EP1 receptor antagonists. The derivative with highest affinity displayed a binding IC50 of 2.5 nM (pIC50 8.6).
Subject(s)
Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Receptors, Prostaglandin E/antagonists & inhibitors , Models, Molecular , Molecular Structure , Protein Binding , Receptors, Prostaglandin E, EP1 Subtype , Structure-Activity RelationshipABSTRACT
A high-throughput screen targeting the EP(1) receptor identified non-acidic glycine sulfonamide derivative 2a with a pK(i) of 6.2. Analogue synthesis allowed a thorough investigation of the structure-activity relationship (SAR) and led to a 100-fold increase in recombinant potency.
