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Glycine transporter-1 (GlyT1) inhibition has been extensively studied both in pharmaceutical companies and academic institutions primarily as a potential new approach to treat schizophrenia, a severe and chronic mental illness. More recently, preclinical results have suggested that this approach could also have therapeutic potential for CNS disorders beyond schizophrenia as well as for non-CNS indications. Over the past 17 years, Roche has been a key player in the GlyT1 field with the discovery and development of bitopertin, the most advanced GlyT1 inhibitor to date and the only one which completed Phase III clinical studies for schizophrenia. In this article, we relate the eventful journey of the discovery and development of bitopertin, from project initiation in 2001 to its evaluation today in patients suffering from beta-thalassemia, a monogenic hereditary haematological disorder.

Imaged-based screening has been developing extremely quickly in the past 10 years. Academic institutes quickly realized that the discovery capacity of this technology was huge, allowing the automatic detection and quantification of complex cell phenotypes. Associated with chemical or genetic perturbations, high content screening is the method of choice for a deep system biology analysis. The evolution of high-content screening is mainly due to the recent progress in the development of fast and high quality automated imagers and of a plethora of new very bright fluorescent markers, so that almost any cellular element can be seen and imaged. In this paper we review and summarize the major steps in the development of an image-based screening project.

A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by evanescent electromagnetic fields. The presented microplate-TIRF-reader combines two different screening procedures related to high throughput screening (HTS) and high content screening (HCS). HTS allows a rapid measurement of large sample numbers based on fluorescence intensity, whereas by HCS a high content of information from a single sample based on the parameters fluorescence lifetime (fluorescence lifetime screening) and fluorescence anisotropy (fluorescence lifetime polarization screening) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e.g. NBD-cholesterol, rhodamine 123) as well as stably transfected cells expressing a fluorescent membrane-associating protein (e.g. EYFP-mem, ECFP-mem). In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved by caspase-3, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times.

This account highlights some of our recent activities focused on developing microfluidic technologies for application in high-throughput and high-information content chemical and biological analysis. Specifically, we discuss the use of continuous and segmented flow microfluidics for artificial membrane formation, the analysis of single cells and organisms, nanomaterial synthesis and DNA amplification via the polymerase chain reaction. In addition, we report on recent developments in small-volume detection technology that allow access to the vast amounts of chemical and biological information afforded by microfluidic systems.

Vibrio cholerae ist ein Gram-negativer humanpathogener Erreger, der die Durchfallerkrankung Cholera verursacht. Bei Cholera handelt es sich um eine akute intestinale Infektion, die zu starken plötzlich auftretenden Durchfällen, und somit innerhalb kürzester Zeit zu einem hohen Wasser- und Elektrolytverlust führt. Die Übertragung von V. cholerae erfolgt hauptsächlich über die Zufuhr von kontaminiertem Wasser, seltener über die Aufnahme von kontaminierter Nahrung wie Fisch und Meeresfrüchte. Besonders Entwicklungsländer ohne adäquate Trinkwasseraufarbeitung oder Trennung von Abwässern und Frischwasser leiden unter Cholera-Epidemien. Aufgrund der steigenden Resistenzentwicklung gegen die bisher eingesetzten Antibiotika zur Behandlung von V. cholerae Infektionen, besteht ein dringender Bedarf an der Entwicklung neuer spezifischer Therapeutika. Für die Suche nach neuen Wirkstoffen wurden im Rahmen dieser Arbeit vier unterschiedliche Mikrotiterplatten-basierte Hochdurchsatz-Screening¬verfahren entwickelt und etabliert. Es handelt sich dabei um Verfahren zur Bestimmung von 1. der Wachstumsrate, 2. der Choleratoxin-Expression, 3. der Protoporphyrinogen IX Oxidase-Aktivität und 4. der bakteriellen Adhäsion an Epithel¬zellen. Die Verfahren 1-3 wurden für ein Screening nach aktiven Substanzen genutzt. Im Rahmen eines umfangreichen Screening wurden drei neuen Substanzen mit antibakteriellen Eigenschaften spezifisch gegen V. cholerae identifiziert. Anschließend erfolgten erste experimentelle Untersuchungen zur Targetidentifizierung der aktivsten Substanz vz0825, mit Hilfe einer Gesamtgenom-Sequenzierung des Genom-Pools aus 15 gegen diese Substanz resistenten Mutanten. Im Choleratoxin-Screening wurden sechs neue Substanzen identifiziert, die die Expression der Pathogenitätsfaktoren Choleratoxin und Toxin coregulated Pili inhibieren. Durch Anwendungen von Real Time-PCR-Analysen und Microarray-Analysen erfolgten die ersten experimentellen Untersuchungen zur Targetidentifizierung., Vibrio cholerae is a pathogenic Gram-negative bacterium that causes the diarrheal disease cholera. Cholera is an acute intestinal infection that leads to severe diarrhea and in short time to extended loss of water and electrolytes. The transmission of V. cholerae occurs mainly through contaminated water, and more rarely through intake of contaminated fish and seafood. Especially developing countries without adequate drinking water treatment and separation of sewage and fresh water suffer from cholera epidemics. Due to the increasing development of resistances against currently used antibiotics for treatment of V. cholera infections, there is an urgent need for the development of new specific therapeutics. To search for new drugs four different microtiter plate based high throughput screening assays were developed and established in this study. These assays were used to determine 1. growth rate of the bacteria, 2. cholera toxin expression, 3. activity of protoporphyrinogen IX oxidase and 4. bacterial adhesion to epithelial cells. Three new compounds with antibacterial activity specifically against V. cholerae have been identified in an extensive screening. The next steps were experimental studies of target identification for the most active compound vz0825 by developing a genome-sequencing of a genome pool out of 15 mutants resistant against this compound. In the cholera toxin screening six new compounds have been identified, which inhibit the expression of virulence factors cholera toxin and toxin coregulated pili. The first experiments for target identification in this case were real time PCR analysis and microarray analysis.