BioImage (www.bioimage.com) also creates cell-based assays that use fluorescence to monitor protein translocation. BioImage can make assays for "any protein in the cell that translocates within the cell in response to a pharmacological stimulus in a reproducible fashion," said Len Pagliaro, Ph.D., vp, business development.
The protein of interest is fused to GFP, the plasmid construct is transfected into cells, and a stable cell line is generated. Although "the idea is very simple," Dr. Pagliaro stressed that it is difficult to create a fully validated drug screening assay. "We make a big distinction between a cell line and an assay."
To become validated assays, cell lines must demonstrate consistent protein expression levels and respond appropriately to stimuli over time. "For every assay you develop, you need to have a reference compound," usually a small molecule that is used to confirm the biology of the cells, explained Dr. Pagliaro.
BioImage also uses RNAi technology to characterize its cell lines. RNAi "can provide excellent controls that users of high-content assays increasingly want to see," Dr. Pagliaro noted.
With a validated assay in hand, drug screening and profiling begins. Translocation-based screens can produce traditional drugs like kinase inhibitors. "Much more interesting," noted Dr. Pagliaro, is that these screens can also yield new compound classes that have "potentially better selectivity and novel modes of action."
BioImage identified one compound series as a moderate potency hit in a Forkhead oncology screen. Although its exact molecular mode of action is not yet known, BioImage has performed lead optimization and SAR and have some "impressive xenograft data," according to Dr. Pagliaro. The company is currently seeking partners to carry the project to an IND.
Daniel R. Rines, institute fellow, lead discovery, Genomics Institute of the Novartis Research Foundation (web.gnf.org/index. shtml), used automated fluorescence to conduct high-throughput screens for cell cycle and cancer targets. Rines' goal was to "identify new genes that would cause cells to arrest in metaphase."
A library of double-stranded siRNA was transfected into HeLa cells and U20S cells. The cells were fixed and stained with a fluorescently-labeled antibody against phospho-Histone H3 (p-His), an excellent marker for mitosis.
Since "histones are phosphorylated when chromosomes are being condensed, any cells stained positive for phospho-Histone H3 suggests they are in metaphase or G2/M," explained Rines. The percentage of cells staining positive for p-His is an indicator of the mitotic index.
In a normal cell population, about 5% of the cells are in metaphase. But if a specific siRNA inhibits a gene that allows progression through metaphase, more cells stain positive for p-His.
A key advantage of automated fluorescence microscopy for HTS with microtiter plates is that you "can do single-cell statistics because the microscope can distinguish individual cells in the well," said Rines.
The instrument calculated the total number of stained cells and the percent p-His positive, as well as data on cell morphology, such as the size of each nucleus, its brightness, and the cell cycle stage. On average, 2,000 cells were analyzed in each well.
Rines performed the genome-wide screen and found "six different phenotypes from eight novel genes," all involving defects in the mitotic spindle. Since the screen collected so much data, including morphological properties, such as the number of nuclei per cell, Rines was also able to identify cytokinesis genes in addition to those affecting the cell cycle and progression through metaphase.
"Because it's all based on cell morphology and sub-cellular protein localization, you can ask not only multiple questions in a single screen but the kind of questions that can't be addressed by any other technology. Standard high-throughput screens typically only ask one question, such as whether a protein is expressed or not," said Rines. That's what "makes this approach very powerful."
Frank Wunder, Institute of Cardiovascular Research, Bayer HealthCare (www.bayer.com), outlined a novel approach for identifying modulators of the cGMP pathway. Cyclic GMP "is a central mediator of vasorelaxation" so modulators may be beneficial for the control of blood pressure and hypertension.
Traditional assays for cGMP are labor-intensive and expensive. "We have a library of over 1million compounds, so we cannot perform the HTS by radioimmunoassay," Wunder said. To overcome this challenge, a bioluminescence-based cell line was created to enable high-throughput screening for cGMP modulators. The cells constitutively expressed soluble guanylate cyclase (a1/b1-heterodimer). When activated by a soluble guanylate cyclase activator or NO, soluble guanylate cyclase converts GTP to cGMP.
The cGMP then activates the cation channel CNGA2, which is also constitutively expressed in the cell and is selectively activated by cGMP. When activated, this channel conducts calcium ions from the extracellular medium into the cell.