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Merck Team Uses Internally Developed Cell-Based Screening Tech on Intracellular Enzymatic Targets

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Researchers from Merck have developed a homogenous, 3,456-well plate, fluorescence-based cellular assay for identifying inhibitors of D-amino acid oxidase (DAO), an enzyme whose activity is implicated in the development of schizophrenia.

Besides successfully identifying several new potent, specific, cell-permeable DAO inhibitors, the work also suggests that high-throughput cell-based assays in general are a more efficient way than high-throughput biochemical screening assays to screen large compound libraries against intracellular enzymatic drug targets.

The research, which is published in the June 7 online edition of the Journal of Biomolecular Screening, was a two-year collaboration between scientists from Merck's departments of neurobiology and automated biotechnology in West Point and North Wales, Penn., respectively; and the department of molecular and cellular neuroscience in the Terlings Park, UK-based facility, which has since closed down, Wei Zheng, corresponding author and former Merck scientist, wrote in an e-mail to CBA News this week.

According to Zheng, who currently works in the National Institutes of Health's Chemical Genomics Center, the research also builds upon similar work conducted by the Merck scientists and published in the March 2004 issue of JBS.

In that paper, the abstract of which can be seen here, Zheng and colleagues used a homogenous cell-based assay to identify several inhibitors against another intracellular enzyme, inositol monophosphatase, which is a key target for developing alternatives to lithium as a treatment of bipolar disorder.


"The homogenous cell-based assay format is a better method [than biochemical assays] for screening intracellular enzyme targets."

Although that screening method used a radioactive scintillation proximity assay in only 384-well plates, its success inspired the researchers to develop the fluorescence-based cellular assay to identify DAO inhibitors, Zheng said.

"In this case, the previous intensive 10 years [of] work using cell-free enzyme assays for compound screening found a number of inisitol phosphatase inhibitors," Zheng said. "But none of these compounds were active inside cells or animal models, which resulted in the termination of that project in drug discovery.

However, using the homogenous cell-based approach, Zheng said that the group identified "a number of novel leads … for inositol monophosphatase which were not identified in the previous biochemical screening."

As the Merck scientists wrote in the most recent JBS paper, "enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein coupled receptors, ion channels, or other hormone receptors."

However, many important intracellular enzymes do play a role in a wide variety of diseases. The main reason that enzymes have been considered less druggable, the researchers argued, is due to a paucity of efficient methods for screening compounds against them.

Zheng said that for an intracellular enzyme target such as DAO or inositol monophosphatase, researchers have traditionally used biochemical assays. One of the major drawbacks to this approach, he said, is that an artificial cell-free environment is not physiologically relevant — in other words, it lacks specific co-factors and subunits, or doesn't quite have the proper pH or buffer components — which in turn can lead to the identification of an incorrect lead or structure-activity relationship.

Furthermore, "active compounds identified by cell-free screening are often inactive due to poor cell membrane permeability, metabolism inside cells, and active sites that are often highly polar," Zheng said.

As detailed in the paper, Zheng and colleagues developed an assay using CHO cells transfected with cDNA expressing the DAO enzyme. They added D-serine, which is oxidized inside the cells by DAO, producing hydrogen peroxide. The hydrogen peroxide then readily passes across the cell membrane and can be detected and quantified using a horseradish peroxidase/Amplex Red reporter system.

The researchers initially used a Tecan Safire plate reader to detect and quantify the resulting fluorescence from 96-well, and then 384-well plates after addition of various small molecule compounds. After these assay development stages, they moved on to a 3,456-well plate format enabled by an Aurora Discovery Island screening platform. This allowed the assay to be completely automated and miniaturized, resulting in screening volumes of 5 μM, with a throughput of 60 plates, or approximately 170,000 compounds per day.

After tweaking the assay to eliminate false positives, they identified 1,966 active compounds, which they then analyzed using computational structural clustering to reveal several representative compounds from clusters of similarly structured compounds. The development of these compounds is still ongoing at Merck, the researchers wrote. Zheng said he is no longer pursuing the research at NCGC.

"This approach underscores the tremendous value and simplicity of using whole-cell assays essentially as in situ enzyme assays," the researchers wrote in the paper. "We suggest that this general approach of a cell-based assay for screenings of enzyme and other intracellular targets simplifies and accelerates the lead discovery process and inherently leads to the selection of higher quality compounds.

More bluntly, Zheng wrote in his e-mail that "the homogenous cell-based assay format is a better method [than biochemical assays] for screening intracellular enzyme targets."

— Ben Butkus ([email protected])

 

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