ABSTRACT
Low-volume dispensing of neat dimethyl sulfoxide (DMSO) into plate-based assays conserves compound, assay reagents, and intermediate dilution plate cost and, as we demonstrate here, significantly improves structure-activity relationship resolution. Acoustic dispensing of DMSO solutions into standard volume 384W plates yielded inconsistent results in studies with 2 cell lines because of apparent effects on the integrity of the cell monolayer (increased intracellular Caâºâº levels as indicated by elevated basal dye fluorescence after acoustic transfer). PocketTip-mediated transfer was successful at increasing apparent potency on a more consistent basis. Notably, the correlation coefficient among fluorescence imaging plate reader (FLIPR):electrophysiology (EP) across a representative ~125 compound collection was increased ~5× via conversion to a PocketTip direct dispensation, indicating a triage assay more predictive of activity in the decisional patch-clamp assay. Very importantly, the EP-benchmarked false-negative rate as measured by compounds with FLIPR EC50 more than the highest concentration tested fell from >11% to 5% assay-wide, and the relative FLIPR:EP rank-order fidelity increased from 55% to 78%. Elimination of the aqueous intermediate step provided additional benefits, including reduced assay cost, decreased cycle time, and reduced wet compound consumption rate. Direct DMSO dispensing has broad applicability to cell-based functional assays of multiple varieties, especially in cases where limit solubility in assay buffer is a recognized impediment to maximizing interassay connectivity.
Subject(s)
High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Ion Channels/drug effects , Acoustics , Buffers , Calcium/analysis , Calcium/chemistry , Centrifugation , Dimethyl Sulfoxide/chemistry , False Negative Reactions , Fluorometry , HEK293 Cells , Humans , Image Processing, Computer-Assisted , Indicator Dilution Techniques , Patch-Clamp Techniques , Solubility , Solutions , WaterABSTRACT
We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , GTP-Binding Protein alpha Subunits/metabolism , RGS Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Triazoles/pharmacology , Animals , Caenorhabditis elegans/drug effects , Caenorhabditis elegans Proteins/metabolism , Calcium/metabolism , Cell Line , Drug Evaluation, Preclinical , Female , GTP-Binding Protein alpha Subunits/genetics , Humans , RGS Proteins/genetics , Radioligand Assay , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Urinary Bladder/drug effects , Urinary Bladder/metabolismABSTRACT
BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] produced a concentration-dependent membrane hyperpolarization of cultured human bladder myocytes, assessed as either a reduction in fluorescence of the voltage-sensitive dye bis-(1,2-dibutylbarbituric acid)trimethine oxonol (EC50 = 1.26 +/- 0.6 microM) or by direct electrophysiological measurement (EC50 = 1.49 +/- 0.08 microM). BL-1249 also produced a membrane hyperpolarization of acutely dissociated rat bladder myocytes. Voltage-clamp studies in human bladder cells revealed that BL-1249 activated an instantaneous, noninactivating current that reversed near E(K). The BL-1249-evoked outward K+ current was insensitive to blockade by glyburide, tetraethylammonium, iberiotoxin, 4-aminopyridine, apamin, or Mg2+. However, the current was inhibited by extracellular Ba2+ (10 mM). In in vitro organ bath experiments, BL-1249 produced a concentration-dependent relaxation of 30 mM KCl-induced contractions in rat bladder strips (EC50 = 1.12 +/- 0.37 microM), yet had no effect on aortic strips up to the highest concentration tested (10 microM). The bladder relaxation produced by BL-1249 was partially blocked by Ba2+ (1 and 10 mM) but not by apamin, iberiotoxin, 4-aminopyridine, glyburide, or tetraethylammonium. In an anesthetized rat model, BL-1249 (1 mg/kg i.v.) decreased the number of isovolumic contractions, without significantly affecting blood pressure. Thus, BL-1249 behaves as a potassium channel activator that exhibits bladder versus vascular selectivity both in vitro and in vivo. A survey of potassium channels exhibiting sensitivity to extracellular Ba2+ at millimolar concentration revealed that the expression of the K2P2.1 (TREK-1) channel was relatively high in human bladder cells versus human aortic cells, suggesting this channel as a possible candidate target for BL-1249.
Subject(s)
Muscle, Smooth/drug effects , Potassium Channels/agonists , Tetrahydronaphthalenes/pharmacology , Tetrazoles/pharmacology , Urinary Bladder/drug effects , Anesthesia , Animals , Barium/pharmacology , Blood Pressure/drug effects , Humans , Male , Membrane Potentials/drug effects , Myocytes, Cardiac/drug effects , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The formation of a reactive intermediate was found to be responsible for CYP3A4 metabolism-dependent inhibition (MDI) observed with (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenyl-acrylamide (1). Structure-3A4 MDI relationship studies culminated in the discovery of a difluoro analogue, (S)-N-[1-(4-fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluoro-phenyl)acrylamide (2), as an orally bioavailable KCNQ2 opener free of CYP3A4 MDI.
Subject(s)
Cinnamates/chemical synthesis , Cytochrome P-450 Enzyme Inhibitors , Fluorine/chemistry , Morpholines/chemical synthesis , Potassium Channels/drug effects , Administration, Oral , Animals , Biological Availability , Cell Line , Cinnamates/metabolism , Cinnamates/pharmacology , Cytochrome P-450 CYP3A , Disease Models, Animal , Injections, Intravenous , Ion Channel Gating , KCNQ2 Potassium Channel , Male , Membrane Potentials , Migraine Disorders/drug therapy , Migraine Disorders/physiopathology , Morpholines/metabolism , Morpholines/pharmacology , Parietal Lobe/drug effects , Parietal Lobe/physiopathology , Patch-Clamp Techniques , Potassium Channels/physiology , Potassium Channels, Voltage-Gated , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity RelationshipABSTRACT
(S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide (2) was synthesized as an orally bioavailable KCNQ2 potassium channel opener. In a rat model of migraine, 2 demonstrated significant oral activity in reducing the total number of cortical spreading depressions induced by potassium chloride.
Subject(s)
Acrylamides/chemical synthesis , Cerebral Cortex/drug effects , Migraine Disorders/physiopathology , Morpholines/chemical synthesis , Potassium Channels/drug effects , Acrylamides/chemistry , Acrylamides/pharmacology , Administration, Oral , Animals , Biological Availability , Cell Line , Cerebral Cortex/physiopathology , Disease Models, Animal , Dogs , Humans , Ion Channel Gating , KCNQ2 Potassium Channel , Migraine Disorders/metabolism , Morpholines/chemistry , Morpholines/pharmacology , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Potassium Channels/physiology , Potassium Channels, Voltage-Gated , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship , Xenopus laevisABSTRACT
Primary rat microglia stimulated with either ATP or 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O(2)(-)*). ATP and BzATP stimulate O(2)(-)* production through purinergic receptors, primarily the P2X(7) receptor. O(2)(-)* is produced through the activation of the NADPH oxidase. Although both p42/44 MAPK and p38 MAPK were activated rapidly in cells stimulated with BzATP, only pharmacological inhibition of p38 MAPK attenuated O(2)(-)* production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O(2)(-)* production to a greater extent than an inhibitor of p38 MAPK. Both ATP and BzATP stimulated microglia-induced cortical cell death indicating this pathway may contribute to neurodegeneration. Consistent with this hypothesis, P2X(7) receptor was specifically up-regulated around beta-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576).
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Alzheimer Disease/genetics , Microglia/physiology , Receptors, Purinergic P2/physiology , Superoxides/metabolism , Adenosine Triphosphate/pharmacology , Animals , Animals, Newborn , Calcium/physiology , Cerebral Cortex/physiology , Chromones/pharmacology , Disease Models, Animal , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Female , Humans , Kinetics , MAP Kinase Signaling System/physiology , Mice , Mice, Transgenic , Microglia/drug effects , Morpholines/pharmacology , NADPH Oxidases/metabolism , Neutrophils/physiology , Phosphoinositide-3 Kinase Inhibitors , Pregnancy , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2X7 , Up-RegulationABSTRACT
PURPOSE: Stretch activated nonselective cationic channels (SACs) are present in urinary bladder myocytes and thought to be activated during bladder filling. We investigated the relationship of stretch induced calcium signaling inhibition in bladder myocytes and bladder compliance modulation in an in vitro whole bladder model. MATERIALS AND METHODS: Grammostola spatulata venom (SpiderPharm, Yarnell, Arizona) was purified by preparative high performance liquid chromatography. The resulting fractions were examined for their ability to inhibit the swelling activated intracellular free Ca2+ signal in cultured bladder myocytes. An in vitro rat whole bladder model was used to examine the effect of venom fractions on compliance, emptying and spontaneous contractions during bladder filling. RESULTS: The gadolinium ion, a SAC inhibitor, and venom fractions caused concentration dependent inhibition of the swelling activated intracellular free Ca2+ signal in bladder myocytes. When tested in a rat isolated whole bladder model, 0.1 and 0.2 mg./ml. partially purified venom produced a significant improvement in compliance (p <0.05), caused significant inhibition of the frequency of spontaneous bladder contractions (mean +/- SEM 35.8% +/- 3.7% and 62.3% +/- 4.4%, respectively, p
