Rapid and accurate testing for urinary tract infection: new clothes for the emperor.

SUMMARYUrinary tract infection (UTI) is among the most common infections in clinical practice. In some cases, if left untreated, it can lead to pyelonephritis and urosepsis. In other cases, UTI resolves without treatment.

Quantification of TRPV1 protein levels in rat tissues to understand its physiological roles.

Transient receptor potential subfamily V, member 1 (TRPV1) is a nonselective cation channel expressed in both the peripheral and central nervous systems (CNS). TRPV1 protein levels in rat tissues were determined under normal and pain states using enzyme-linked immunosorbent assay. In naive rats, brain TRPV1 protein concentrations ranged from 1.

Discovery of TRPV1 antagonist ABT-116.

The synthesis and SAR of a series of indazole TRPV1 antagonists leading to the discovery of 21 (ABT-116) is described. Biological studies demonstrated potent in vitro and in vivo activity for 21, as well as suitable physicochemical and pharmacokinetic properties for advancement to clinical development for pain management.

Expression and purification of human TRPV1 in baculovirus-infected insect cells for structural studies.

TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes.

Characterization of A-425619 at native TRPV1 receptors: a comparison between dorsal root ganglia and trigeminal ganglia.

1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea (A-425619), a novel, potent, and selective transient receptor potential type V1 (TRPV1) antagonist, attenuates pain associated with inflammation and tissue injury in rats. The purpose of this study was to extend the in vitro characterization of A-425619 to native TRPV1 receptors and to compare the pharmacological properties of TRPV1 receptors in the dorsal root ganglion with trigeminal ganglion neurons. A robust increase in intracellular Ca(2+) was elicited by a variety of TRPV1 agonists with similar rank order of potency between both cultures: resiniferatoxin>tinyatoxin>capsaicin>N-arachidonoyl-dopamine (NADA).

Molecular determinants of species-specific activation or blockade of TRPA1 channels.

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g.

Transient receptor potential A1 mediates an osmotically activated ion channel.

Transient receptor potential (TRP)A1 channel has been implicated in various physiological processes, including thermosensation and pain. A recent study of TRPA1 knockout mice demonstrated deficits in sensing mechanical stimuli, suggesting a role for TRPA1 also in somatic mechanosensation. However, direct evidence of TRPA1 activation by mechanical forces has thus far been lacking.

Analgesic activity of metabotropic glutamate receptor 1 antagonists on spontaneous post-operative pain in rats.

Activation of metabotropic glutamate (mGlu) receptors has previously been shown to play a role in inflammatory or neuropathic pain states. However, the role of mGlu type 1 receptors in post-operative pain remains to be investigated. In the present study, effects of potent and selective mGlu1 receptor antagonists A-841720, A-794282, A-794278, and A-850002 were evaluated in a skin incision-induced post-operative pain model in rats.

[3H]A-778317 [1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea]: a novel, stereoselective, high-affinity antagonist is a useful radioligand for the human transient receptor potential vanilloid-1 (TRPV1) receptor.

1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778317) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pIC50 = 8.31 +/- 0.13).

Application of large-scale transiently transfected cells to functional assays of ion channels: different targets and assay formats.

Cell-based functional assays are increasingly being utilized for ion channels and other targets in drug discovery. However, development of functional assays is often hampered by problems related to stable expression of ion channels in host cell lines, such as variability in channel activity, cell line degeneration, toxicity associated with gene expression, and time and expense of maintaining the cells in culture. In a previous study, we showed that constitutive expression of the transient receptor potential ankyrin-1 (TRPA1) channel led to cellular toxicity and cell line degeneration.

Rapid hit to lead evaluation of pyrazolo[3,4-d]pyrimidin-4-one as selective and orally bioavailable mGluR1 antagonists.

Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties.

Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.

Capsaicin causes protein synthesis inhibition and microtubule disassembly through TRPV1 activities both on the plasma membrane and intracellular membranes.

TRPV1 is a non-selective cationic channel that is activated by capsaicin, acidic pH and thermal stimuli. Sustained TRPV1 channel activation causes severe cytotoxicity that leads to cell death. In this study, we investigated the mechanisms of capsaicin-induced cytotoxicity in HEK293 cells stably expressing TRPV1 with a focus on protein synthesis regulation and cytoskeleton reorganization.

Discovery of 3-methyl-N-(1-oxy-3',4',5',6'-tetrahydro-2'H-[2,4'-bipyridine]-1'-ylmethyl)benzamide (ABT-670), an orally bioavailable dopamine D4 agonist for the treatment of erectile dysfunction.

The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability.

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources.

Acrylamide analog as a novel nitric oxide-independent soluble guanylyl cyclase activator.

Soluble guanylyl cyclase (sGC) is a target enzyme for endogenous nitric oxide (NO), and it converts GTP to cyclic GMP (guanosine 3',5'-cyclic monophosphate) as part of a cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, and inhibition of platelet aggregation. Here we examine a representative of the novel class sCG activators, A-778935 ((+/-)-cis-3-[2-(2,2-dimethyl-propylsulfanyl)-pyridin-3-yl]-N-(3-hydroxy-cyclohexyl)-acrylamide). A-778935 activated sGC synergistically with sodium nitroprusside (SNP) over a wide range of concentration, inducing up to 420-fold activation.

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells.

Transient receptor potential channel type V (TRPV) 1 is a non-selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes.

1-aryl-3-(4-pyridine-2-ylpiperazin-1-yl)propan-1-one oximes as potent dopamine D4 receptor agonists for the treatment of erectile dysfunction.

A new series of dopamine D4 receptor agonists, 1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes, was designed through the modification of known dopamine D4 receptor agonist PD 168077. Replacement of the amide group with a methylene-oxime moiety produced compounds with improved stability and efficacy. Structure-activity relationsips (SAR) of the aromatic ring linked to the N-4-piperazine ring confirmed the superiority of 2-pyridine as a core for D4 agonist activity.

Correlation between brain/plasma ratios and efficacy in neuropathic pain models of selective metabotropic glutamate receptor 1 antagonists.

We have discovered a novel, potent, and selective triazafluorenone series of metabotropic glutamate receptor 1 (mGluR1) antagonists with efficacy in various rat pain models. Pharmacokinetic and pharmacodynamic profiles of these triazafluorenone analogs revealed that brain/plasma ratios of these mGluR1 antagonists were important to achieve efficacy in neuropathic pain models. This correlation could be used to guide our in vivo SAR (structure-activity relationship) modification.

Modulation of human TRPV1 receptor activity by extracellular protons and host cell expression system.

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand-gated cation channel that can be activated by capsaicin, heat, protons and cytosolic lipids. We compared activation of recombinant human TRPV1 receptors stably expressed in human 293 cells, derived from kidney embryonic cells, and in human 1321N1 cells, derived from brain astrocytes. Cellular influx of calcium was measured in response to acid, endovanilloids (N-arachidonoyl-dopamine, N-oleoyl-dopamine and anandamide), capsaicin and other traditional vanilloid agonists under normal (pH 7.

Dopamine D4 receptor signaling in the rat paraventricular hypothalamic nucleus: Evidence of natural coupling involving immediate early gene induction and mitogen activated protein kinase phosphorylation.

The dopamine D4 receptor has been investigated for its potential role in several CNS disorders, notably schizophrenia and more recently, erectile dysfunction. Whereas studies have investigated dopamine D4 receptor-mediated signaling in vitro, there have been few, if any, attempts to identify dopamine D4 receptor signal transduction pathways in vivo. In the present studies, the selective dopamine D4 agonist PD168077 induces c-Fos expression and extracellular signal regulated kinase (ERK) phosphorylation in the hypothalamic paraventricular nucleus (PVN), a site known to regulate proerectile activity.

Structure-activity relationship of triazafluorenone derivatives as potent and selective mGluR1 antagonists.

SAR (structure-activity relationship) studies of triazafluorenone derivatives as potent mGluR1 antagonists are described. The triazafluorenone derivatives are non-amino acid derivatives and noncompetitive mGluR1 antagonists that bind at a putative allosteric recognition site located within the seven-transmembrane domain of the receptor. These triazafluorenone derivatives are potent, selective, and systemically active mGluR1 antagonists.

A-412997, a selective dopamine D4 agonist, improves cognitive performance in rats.

The recent development of a highly selective dopamine D4 receptor agonist, A-412997 (2-(3',4',5',6'-tetrahydro-2'H-[2,4'] bipyridinyl-1'-yl)-N-m-tolyl-acetamide), has provided a pharmacological tool with which to conduct systematic investigations into the putative role for dopamine D4 receptors in the central nervous system. These present studies evaluated the potential cognitive enhancing properties of A-412997 in rat models of ADHD (5-trial repeated acquisition inhibitory avoidance in Spontaneous Hypertensive Rat pups) and short-term memory (Social Recognition), in comparison with the less selective dopamine D4 receptor agonists PD168077 and CP226269. A-412997 showed significant dose-dependent efficacy in both models.