Overexpression of human transient receptor potential M5 upregulates endogenous human transient receptor potential A1 in a stable HEK cell line.

Transient receptor potential M5 (TRPM5), a monovalent cation channel, is primarily activated by increases in intracellular calcium. However, we found unexpectedly that allyl isothiocyanate (AITC) and structural analogs triggered a membrane potential and calcium dye responses in TRPM5-HEK cells (AITC EC₅₀ = 9.0 ± 2.

Pharmacologic antagonism of the oral aversive taste-directed response to capsaicin in a mouse brief access taste aversion assay.

Chemosensory signaling by the tongue is a primary determinant of ingestive behavior and is mediated by specific interactions between tastant molecules and G protein-coupled and ion channel receptors. The functional relationship between tastant and receptor should be amenable to pharmacologic methods and manipulation. We have performed a pharmacologic characterization of the taste-directed licking of mice presented with solutions of capsaicin and other transient receptor potential vanilloid-1 (TRPV1) agonists using a brief access taste aversion assay.

Quantitative assessment of TRPM5-dependent oral aversiveness of pharmaceuticals using a mouse brief-access taste aversion assay.

Many orally administered pharmaceuticals are regarded by humans as aversive, most often described as 'bitter'. Taste aversiveness often leads to patient noncompliance and reduced treatment effectiveness. 'Bitter' taste is mediated by T2R G-protein coupled receptors through a peripheral signaling pathway critically dependent upon function of the TRPM5 ion channel.

Development of a fluorescence polarization AKT serine/threonine kinase assay using an immobilized metal ion affinity-based technology.

A bead-based FP assay methodology, termed IMAP trade mark, has been developed for the serine/threonine kinase, AKT, that allows for direct measurement of product formation. The assay design utilizes a fluoresceinated peptide substrate that, when phosphorylated by the kinase, binds to nanoparticles derivatized with trivalent metal cations through a metal-phospholigand interaction. The result of this bound fluoresceinated phosphorylated product is an increase in polarization signal caused by a decrease in the molecular mobility of the bound product.

Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts.

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts.

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase.

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied.