January 15, 2006 (Vol. 26, No. 2)

Novel Technological Approaches Take the Discovery of New Drugs to the Next Level

High-throughput screening has been around for a while, but it has only been in the last few years that doing so on a cellular level has been feasible. Screening using cell-based assays has been performed for many years, but recent technologies allow screening of cells that are alive and changing. Previously, working with cells would give you a picture of a cell that was frozen in a moment in time and of development, notes Judy Masucci, Ph.D., director of marketing at Cellomics (www.cellomics.com). Now it is possible to work with live cells in real time.

At Cambridge Healthtechs High Content Analysis conference, to be held in San Francisco later this month, companies will be presenting how their technology is making the bottom line a little thicker for major pharmaceutical companies.

Division-Arrested Cell Reagents

Cell-based assays are becoming increasingly popular for small molecule discovery in high-throughput screening. Advanced high-throughput screening technologies require standardized, consistent cell culture as reagents to generate reliable results.

Division-Arrest Technology, a scalable process developed and patented by Cell & Molecular Technologies (CMT; www.cmt-inc.net), makes it possible to use frozen assay-ready cells for screening as if they were ordinary assay reagents like enzymes or membrane preparations.

As cell-based assays are used more commonly in robotic high-throughput compound screening, cells themselves have become critical reagents. Thus, it has become essential to produce cell reagents with high consistency and quality.

We experimented with cells division-arrested with low level mitomycin C treatment and demonstrated that they perform with better consistency than non-division arrested counterparts in high-content screening imaging assays, says Zhong Zhong, vp of development at CMT. For cell-based screening, it is possible to uncouple the cell production process from the screening process.

The idea of a robust, cell-based assay is no longer an oxymoron. Cells are living entities. If you are not consistent in the way you handle them, the variability is going to be a big problem, Zhong notes. Cells can be produced en masse, treated to become irreversibly division-arrested, and cryopreserved. These ready-to-use reagents can be thawed, plated, and used in screening with improved consistency and convenience.

The advantage of our division-arrested cell lines is that it is a live cell, yet it is not dividing, Zhong explains. And all the other functions of the cell are preserved.

CMT also offers a cell-based assay technology based on Sentigen Biosciences (www.sentigen.com) Tango Assay System. CMT. Sentigen Biosciences developed Tango assays for GPCR profiling of targets of relevance to pharmaceutical discovery and development. This expanding panel of cell-based receptor assays, which currently includes 54 targets, is being offered by CMT for custom compound profiling to evaluate compound selectivity over a wide range of likely GPCR targets.

G Protein-Coupled Receptors

Redistribution assay technology relies on the imaging and quantification of intracellular protein translocation. For development of Redistribution assays, BioImage (www.bioimage.com) uses Aequorea victoria GFP and other fluorescent proteins fused to translocating targets of interest.

G protein-coupled receptors (GPCRs) account for some 50 percent of all current drug targets, says Arne Heydorn, scientist at BioImage, whereas marketed drugs only target about 30 percent of the GPCRs in the genome. We developed Redistribution cell-based assays for use in both primary screening and lead optimization of compounds targeting GPCRs.

Redistribution assays are quantitative assays that can be run in standard microtiter plates. The assay response is most commonly measured by capturing an image from an assay well, and the response is quantified by performing image analysis.

Redistribution assays are independent of the imaging platform and have been shown to give consistent results on a number of instruments on the market, including: IN Cell 3000 (GE Healthcare), Opera (Evotec Technologies), ArrayScan (Cellomics), Pathway HT (BD Biosciences), Discovery1 (Molecular Devices), and Icyte (CompuCyte).

Multiplexing refers to technology that enables the simultaneous measurement of multiple parameters in one test compound well. Redistribution assays can be multiplexed by either expressing two targets in the same cell line labeled with different colors, or by mixing different assay cell lines directly in the microtiter plate well.

These assays allow monitoring of GPCR desensitization, i.e., receptor internalization and recycling/degradation, by quantifying the intracellular translocation of GPCR-GFP fusion constructs.

A second class of assays is designed to report functional activity for Gq-, Gi-, and Gs-coupled GPCRs, by monitoring activation of downstream signaling pathways, such as nuclear factor of activated T cells (NFAT) or protein kinase A (PKA). For these assays, the GPCR of interest is transfected into a cell line expressing a fusion construct of GFP with the appropriate downstream target. Recent studies have shown that the effects of agonists and antagonists on receptor desensitization are variable, and that receptor desensitization plays a key role in drug behavior in vivo.

What we do is focus on the biology; we dont do the algorithms, Heydorn adds.

Live Cell Option

Capturing and quantifying biological changes in real time from nonfixable targets adds information content, increased flexibility, and further breadth to high-content assays. And having effective instrumentation to read the data is essential.

Cellomics aims to make automating cell-based assays possible. One way that it does this is through its Center of Excellence group. We are basically a services-based group, says Scott Keefer, senior application scientist. Our customers call and ask if it is possible to perform certain tasks on their instrumentation. And what we do is either adapt existing technology or troubleshoot. Ongoing development of instrumentation is heavily influenced by what our customers demand.

According to Keefer, Cellomics ArrayScan VTI was designed to address the need for better data and increased flexibility. It is available with modules such as optical sectioning, robotic plate delivery, and now a robust environmentally controlled unit that permits expansion and flexibility of both instrument and assay. The idea is that you dont have to make a huge up-front purchase, and you can grow as you need to.

Thanks to our customers, weve learned a lot about how to do live cell scans, Keefer said.

Genotoxicity Screening

GE Healthcare (www.gehealth care.com) launched a suite of new modules for its IN Cell analyzer 1000 and 3000. These systems allow screening for genotoxicity early in drug development, saving time and resources, according to GE.

Instead of testing compounds at the end of the drug discovery process, this technology enables companies to identify compounds with genotoxic activity early in development, saving both time and money, says Nick Thomas, principal scientist at GE Healthcare.

The micronuclei assay identifies DNA damage induced by genotoxic compounds. The manual method mandated by the FDA can take up to two weeks per compound to score and is consequently used only in the later stages of drug development. Manual analysis, which requires several different time points not to mention painstaking and subjective analysis, can take anywhere from two to three weeks.

When used with the IN Cell Analyzer 3000, the analysis software can image and analyze a 96-well plate of micronucleus assays in under 10 minutes, according to Thomas. In addition to data on micronuclei formation, the software provides cell proliferation and viability parameters which are invaluable in assessing cytotoxicity of test compounds. Combining this with our morphology image analysis software provides a multiparameter read-out for DNA damage and its phenotypic effects.

We are talking about getting this information earlier, not replacing FDA mandatory testing, notes Thomas. It is a steep curve, and developing drugs is expensive business. The sooner you have information on the genotoxicity of a compound, the less money you lose on less viable drug candidates.

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