In the liver, glucokinase (GK) regulatory protein (GKRP) negatively modulates the metabolic enzyme GK by locking it in an inactive state in the nucleus. Here, the authors established a high content screening assay in the 384-well microplate format to measure the nucleus-to-cytoplasm translocation of GK by reagents that destabilize the interaction between GK and GKRP. As a cellular model system, primary rat hepatocytes endogenously expressing both GK and GKRP at physiological levels were used. The GK translocation assay was robust, displayed limited day-to-day variability, and delivered good Z' statistics. The increase of the glucose concentration in the extracellular medium from a low glucose situation (2.8 mM) to beyond its physiological set point value of 5 mM was found to drive GK from the nucleus into the cytoplasm. Likewise, both fructose (converted intracellularly into fructose-1-phosphate) and a known allosteric GK activator were found to induce the export of GK from the nucleus and to synergistically enhance the effects of medium or high glucose concentrations with respect to GK translocation. Transfer of the high content screening format to a semiautomated medium throughput screening platform enabled the profiling of large compound numbers with respect to allosteric activation of GK. (Journal of Biomolecular Screening 2008:837-846)

Historically, only relatively low-throughput or expensive methods have been available to measure cell migration. Hepatocyte growth factor (HGF) is a ligand for the tyrosine kinase receptor Met that, in addition to mediating proliferation and survival, increases cell motility and metastasis. The authors have developed a high-throughput imaging assay for measuring inhibition of HGF-induced scattering in human HPAF-II pancreatic adenocarcinoma cells. Following treatment with test compounds and HGF for 24 h, cells are labeled with a nuclear stain and imaged at 10x magnification. The proximity of neighboring nuclei is measured, and the distribution of internuclear distances across each field of view is used to calculate the fraction of scattered cells. This method of analysis can be extended to other cell types and signaling pathways and, compared with other membrane-based migration assays currently available, the assay is significantly lower in cost, is less labor intensive, and provides higher throughput. (Journal of Biomolecular Screening 2008:847-854)

As natural peroxisome proliferator-activated receptor- (PPAR) ligands, high levels of fatty acids and glucose could lead to hyperactivation of PPAR, like that seen in diabetes. Important diabetes research goals are to uncover new metabolic or signaling pathways involved in hyperglycemic cellular injury and to develop therapeutics for preventing or reversing this injury. Consequently, 1040 putative antidiabetic agents were screened for their ability to 1) affect PPAR lipid binding, 2) directly bind PPAR, and 3) alter PPAR transactivation in the presence of high glucose. A high-throughput fluorescent binding assay was developed to examine each compound's ability to restore fatty acyl-CoA binding to PPAR in the presence of high glucose concentrations. Approximately 1% of the compounds restored acyl-CoA binding by 60% or more. These compounds directly interacted with PPAR with high affinity (nM K_ds), validating the primary screen. Furthermore, these compounds altered PPAR transactivation, and 1 strongly reversed the hyperactivation of PPAR found in the presence of clofibrate and high glucose levels. (Journal of Biomolecular Screening 2008:855-861)

In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. (Journal of Biomolecular Screening 2008:862-869)

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of β-amyloid (Aβ) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Aβ. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Aβ with IC₅₀ values of <10 µM and showed little or no toxicity at concentrations up to 30 µM, none inhibit the calpain enzyme directly. (Journal of Biomolecular Screening 2008:870-878)

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The "hit" rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI₅₀ = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI₅₀ > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (Journal of Biomolecular Screening 2008:879-887)

The bioluminescence resonance energy transfer (BRET) technique has become extremely popular for studying protein-protein interactions in living cells and real time. Of particular interest is the ability to monitor interactions between G protein–coupled receptors, such as the thyrotropin-releasing hormone receptor (TRHR), and proteins critical for regulating their function, such as β-arrestin. Using TRHR/β-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET¹, BRET², and eBRET) by using the novel forms of luciferase, Rluc2 and Rluc8, developed by the Gambhir laboratory. Furthermore, for the 1st time it was possible to use the BRET2 system to detect ligand-induced G protein–coupled receptor/β-arrestin interactions over prolonged periods (on the scale of hours rather than seconds) with a very stable signal. As demonstrated by our Z'-factor data, these luciferases increase the sensitivity of BRET to such an extent that they substantially increase the potential applicability of this technology for effective drug discovery high-throughput screening. (Journal of Biomolecular Screening 2008:888-898)

The nonstandard molecular beacon described in this article consists of 2 fragments, each built of a short single-stranded oligonucleotide sequence and a double-stranded sequence. One of these hybridization probes, labeled with a fluorescence donor (fluorescein), is solid phase immobilized. The second nonimmobilized probe is labeled with a fluorescence quencher (dabcyl). Annealing of both probes via single-stranded sequences was possible only in the presence of a specific protein molecule that recognized the response element sequence initially separated between the immobilized and nonimmobilized fragments. The system was applied successfully to detect the sequence-specific interaction of a natural hsp27 response element from the promoter of the hsp27 gene with the DNA binding domains of 2 nuclear receptor proteins: ultraspiracle Usp (UspDBD) and the ecdysone receptor EcR (EcRDBD). Measured in the absence of EcRDBD, the dissociation constant, K_d of the UspDBD-hsp27 complex, was determined to be 3.26 nM, whereas for UspDBD devoid of the A-box (UspDBDAhsp27 ), the dissociation constant was 4.81 nM. The respective K_d values in the presence of EcRDBD were 2.43 nM and 10.80 nM. The results obtained with the immobilized molecular beacon technology were in agreement with those obtained by conventional fluorescence titrations and by fluorescence resonance energy transfer measurements with nonimmobilized beacons. ( Journal of Biomolecular Screening 2008:899-905)