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Columns : Nov 15, 2008 ( )
Aiming to Accelerate Discovery Process
BellBrook Labs Designed Generic Transcreener Platform for Efficient Drug Target Detection!--h2>
Aberrant proteins cause most diseases, and pharmaceutical companies use high-throughput screening (HTS) to identify drugs that interfere with these targets. The creation of new assays to detect individual drug targets can take several months, however, creating bottlenecks in drug discovery pipelines.
BellBrook Labs says it has developed a universal method that gets around this obstruction. “We’re focused on introducing products that solve significant problems: products to accelerate drug discovery,” says Robert Lowery, Ph.D., president and CEO. Dr. Lowery and John Majer, COO, started BellBrook Labs in 2002.
The company’s technology allows an entire family of enzymes to be screened with the same reagents. In January 2005, BellBrook Labs introduced the first product in its Transcreener HTS Assay Platform.
The current types of Transcreener Assays measure nucleotides released by glycotransferases, kinases, ATPases, GTPases, ligases, and phosphodiesterases, which are linked to cancer, cardiovascular disease, inflammation, and metabolic disorders. Assays for acetyltransferases, and methyltransferases are in development.
Since its release, researchers at pharmaceutical companies have validated the Transcreener Kinase Assay in primary screens, selectivity profiling, and lead optimization.
This assay was recently renamed the Transcreener ADP Assay, to emphasize that it detects ADP (adenosine 5'-diphosphate). Numerous targets, including protein kinases, lipid kinases, and ATPases, can be screened using this single assay.
Truly Generic Assays
Group transfer reactions, such as phosphorylation, glycosylation, and methylation, are important on/off switches for signaling proteins in a variety of disease pathways. The activity of drugs and hormones is also controlled by group transfer reactions. The enzymes that catalyze these reactions are generally assayed by detecting the amount of product formed or substrate depleted. This requires individual assays and reagents, however, since most enzymes within a group transfer family have different substrates and products.
BellBrook Labs’ Transcreener platform takes a different approach. The generic assays detect nucleotide products of a donor molecule cleavage, such as ADP for kinases and ATPases; UDP (uridine diphosphate) for glycosyltransferases; coenzyme A for acetyltransferases; and AMP (adenosine monophosphate) or GMP (guanine monophosphate) for phosphodiesterases.
Each type of group transfer reaction generates just one donor product, so a single set of Transcreener reagents can be used for all family members, regardless of the starting substrate or end product. “The assays are truly generic,” says Dr. Lowery.
The Transcreener HTS assays are based on competitive fluorescence polarization immunoassays to monitor the binding of a donor product to a specific antibody. This detection method has less interference and a lower false positive rate than assays that measure substrate depletion.
Moreover, the same assays can help pharmaceutical companies to predict adverse side effects, which are responsible for nearly half of all clinical failures. Glycotransferase enzymes, for instance, play an important role in the metabolism of drugs and the production of toxic metabolites. The development of new assays for targeted classes of proteins will enable the screening of many more enzymes in emerging target families, such as ubiquitin ligases, which produce AMP.
“We want to give people access to targets that they otherwise would not be able to screen, to make a real difference,” comments Dr. Lowery.
The main advantage of the Transcreener assays over existing HTS methods is the wide diversity of targets that can be addressed with just a few assays, says Dr. Lowery.
For instance, there are about 2,000 human proteins that utilize ATP. Since most of them generate AMP or ADP as a product, just two Transcreener assays are needed to detect them. The assays come in a single-addition, mix-and-read format, making them ideal for automated HTS. The reagents and consumables work with existing HTS equipment, and the results are read on standard multiwell plate readers. The nucleotide product of a group transfer reaction is usually generated in stoichiometric amounts, giving a direct measure of enzyme turnover.
3-D Cell-Based Assays
Whereas the Transcreener assays measure biochemical reactions, a second technology platform focuses on cellular assays. The core technology was invented in the laboratory of David Beebe, Ph.D., at the University of Wisconsin, Madison.
Iuvo™ (Latin for aid), the microchannel plate consists of MicroConduit Arrays that support the 3-D growth of cultured cells. The MicroConduit Arrays replace wells in a microtiter plate, where cells typically are grown as monolayers on the bottom of wells covered with culture media. This condition does not mimic the three-dimensional architecture of cells in the body. Consequently, such nonphysiological cellular assays do not give accurate predictions about how drugs will behave in humans.
Instead, MicroConduit Arrays use a microfluidics technology that allows researchers to reconstruct microenvironments that underlie many diseases. For example, scientists at BellBrook Labs built an angiogenesis model using primary endothelial cells and fibroblasts. Cell migration within a chemo-attractant gradient also can be modeled.
“Iuvo lets people incorporate physiologically relevant biology into screening assays,” explains Dr. Lowery, who knows of no other microfluidics platform that incorporates 3-D cells.
The MicroConduit Array system blends with standard HTS equipment like automated liquid handling and detection instruments. Using a passive pumping technology, all of the steps for operating Iuvo are accomplished by placing and removing droplets from the surface of the plate. This task can be accomplished with automated liquid handling dispensers. The process does not disturb cells in the channels, making Iuvo suitable for immunoassays that require many wash steps. BellBrook Labs expects to launch Iuvo next month.
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