Identification of positive modulators of TRPM5 channel from a high-throughput screen using a fluorescent membrane potential assay.

Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.

From High-Throughput Screening to Target Validation: Benzo[d]isothiazoles as Potent and Selective Agonists of Human Transient Receptor Potential Cation Channel Subfamily M Member 5 Possessing In Vivo Gastrointestinal Prokinetic Activity in Rodents.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

An isogenic cell panel identifies compounds that inhibit proliferation of mTOR-pathway addicted cells by different mechanisms.

The mTOR pathway is a critical integrator of nutrient and growth factor signaling. Once activated, mTOR promotes cell growth and proliferation. Several components of the mTOR pathway are frequently deregulated in tumors, leading to constitutive activation of the pathway and thus contribute to uncontrolled cell growth. We performed a high-throughput screen with an isogenic cell line system to identify compounds specifically inhibiting proliferation of PTEN/mTOR-pathway addicted cells. We show here the characterization and mode of action of two such compound classes. One compound class inhibits components of the PTEN/mTOR signaling pathway, such as S6 ribosomal protein phosphorylation, and leads to cyclin D3 downregulation. These compounds are not adenosine triphosphate competitive inhibitors for kinases in the pathway, nor do they require FKBP12 for activity like rapamycin. The other compound class turned out to be a farnesylation inhibitor, blocking the activity of GTPases, as well as an inducer of oxidative stress. Our results demonstrate that an isogenic cell system with few specific mutations in oncogenes and tumor suppressor genes can identify different classes of compounds selectively inhibiting proliferation of PTEN/mTOR pathway-addicted isogenic clones. The identified mechanisms are in line with the known cellular signaling networks activated by the altered oncogenes and suppressor genes in the isogenic system.

Essential role of protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development but not in glucose homeostasis.

Protein kinase B is implicated in many crucial cellular processes, such as metabolism, apoptosis and cell proliferation. In contrast to Pkb(alpha) and Pkb(beta)-deficient mice, Pkb(gamma)(-/-) mice are viable, show no growth retardation and display normal glucose metabolism. However, in adult Pkb(gamma)mutant mice, brain size and weight are dramatically reduced by about 25%. In vivo magnetic resonance imaging confirmed the reduction of Pkb(gamma)(-/-) brain volumes with a proportionally smaller ventricular system. Examination of the major brain structures revealed no anatomical malformations except for a pronounced thinning of white matter fibre connections in the corpus callosum. The reduction in brain weight of Pkb(gamma)(-/-) mice is caused, at least partially, by a significant reduction in both cell size and cell number. Our results provide novel insights into the physiological role of Pkb(gamma) and suggest a crucial role in postnatal brain development.

Protein kinase B alpha/Akt1 regulates placental development and fetal growth.

Protein kinase B alpha (PKB alpha/Akt1) is implicated in the regulation of metabolism, transcription, cell survival, angiogenesis, cell migration, growth, and tumorigenesis. Previously, it was reported that PKB alpha-deficient mice are small with increased neonatal mortality (Cho, H., Thorvaldsen, J. L., Chu, Q., Feng, F., and Birnbaum, M. J. (2001) J. Biol. Chem. 276, 38349-38352 and Chen, W. S., Xu, P. Z., Gottlob, K., Chen, M. L., Sokol, K., Shiyanova, T., Roninson, I., Wenig, W., Suzuki, R., Tobe, K., Kadowaki, T., and Hay, N. (2001) Genes Dev. 15, 2203-2208). Here we show that PKB alpha is widely expressed in placenta including all types of trophoblast and vascular endothelial cells. Pkb alpha-/- placentae display significant hypotrophy, with marked reduction of the decidual basalis and nearly complete loss of glycogen-containing cells in the spongiotrophoblast, and exhibit decreased vascularization. Pkb alpha-/- placentae also show significant reduction of phosphorylation of PKB and endothelial nitric-oxide synthase. These defects may cause placental insufficiency, fetal growth impairment, and neonatal mortality. These data represent the first evidence for the role of PKB alpha and endothelial nitricoxide synthase in regulating placental development and provide an animal model for intrauterine growth retardation.