Evaluation and optimization of compound solubilization and delivery methods in a two-tiered ion channel lead optimization triage.

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 Ca⁺⁺ 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.

Chemical genetics reveals an RGS/G-protein role in the action of a compound.

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.

BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine]: a putative potassium channel opener with bladder-relaxant properties.

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.

Fluorine substitution can block CYP3A4 metabolism-dependent inhibition: identification of (S)-N-[1-(4-fluoro-3- morpholin-4-ylphenyl)ethyl]-3- (4-fluorophenyl)acrylamide as an orally bioavailable KCNQ2 opener devoid of CYP3A4 metabolism-dependent inhibition.

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.

(S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]- 3-phenylacrylamide: an orally bioavailable KCNQ2 opener with significant activity in a cortical spreading depression model of migraine.

(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.

P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer's disease.

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.

Partially purified Grammostola spatulata venom inhibits stretch activated calcium signaling in bladder myocytes and improves bladder compliance in an in vitro rat whole bladder model.

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.

Dual regulation of calcium mobilization by inositol 1,4, 5-trisphosphate in a living cell.

Changes in cytosolic free calcium ([Ca(2+)](i)) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca(2+)](i) oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca(2+)](i) oscillates at a particular frequency or becomes a sustained response is poorly understood.

cGMP inhibits IP3-induced Ca2+ release in intact rat megakaryocytes via cGMP- and cAMP-dependent protein kinases.

1. Inhibition of inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by using a combination of single cell fluorescence microscopy to monitor intracellular free calcium ([Ca2+]i) and flash photolysis of caged second messengers. 2.

Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell.

Interaction of intracellular free calcium ([Ca2+]i) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca2+]i and flash photolysis of caged Ca2+, inositol 1,4, 5-trisphosphate (IP3), or cAMP to elevate rapidly the concentration of these compounds inside the cell. Photolysis of caged IP3 stimulated Ca2+ release from an IP3-sensitive store. The cAMP-elevating agent carbacyclin inhibited this IP3-induced rise in [Ca2+]i but did not affect the rate of Ca2+ removal from the cytoplasm after photolysis of caged Ca2+.

[Ca2+]i oscillations and [Ca2+]i waves in rat megakaryocytes.

ATP activated [Ca2+]i oscillations were measured in single rat megakaryocytes using fluorescence ratio microscopy. With increasing ATP concentration the duration of the [Ca2+]i oscillations increased, however, there was considerable variation from cell to cell in the absolute value of the peak [Ca2+]i and the frequency and duration of the oscillations. This variation depended, in part, on the level of Fura-2 loading suggesting that megakaryocytes are sensitive to buffering of [Ca2+]i by Fura-2.

[Structure-activity study of the basic toxic component of venom from the ant Ectatomma tuberculatum].

A toxic principle of the Ectatomma tuberculatum ant venom called ectatomin was isolated. Ectatomin is a protein with molecular weight 7928 Da. Its complete amino acid sequence and spatial structure in aqueous solution were determined by protein chemistry methods and NMR spectroscopy techniques.

[Immunochemical study of antigenic determinants of surface protein s of the tropical malaria pathogen Plasmodium falciparum using synthetic peptides].

To develop new approaches to diagnostics and therapy of malaria, we carried out immunochemical study of the surface proteins of the tropical malaria parasite Plasmodium falciparum with the use of synthetic peptides corresponding to the suggested antigenic determinants of the parasite proteins. Rabbit antisera raised against the synthetic peptides bound to parasite proteins as shown by ELISA and immunoblotting. Affinity purified anti-peptide antibodies inhibited, in some cases, the parasite growth in the human erythrocytes culture.

[Peptide synthesis--protein fragments from Plasmodium falciparum and their conjugates with protein and synthetic carriers].

To study coat proteins of Plasmodium falciparum, seven putative antigenic-determinants of PMMSA, SHARP, GBP and four known determinants of CSP, CRA and RESA were synthesized. Computerized methods for predicting protein antigenic determinants were employed to select the peptides. For immunochemical studies the peptides were conjugated to proteins and synthetic carriers by means of nonspecific and regiospecific heterobifunctional reagents.