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Apr 15, 2009 (Vol. 29, No. 8)

Focused Assays Lead to More Potent Drugs

Label-Free Remains No.1 Cell-Based Trend, but Electrophysiology Is Not Far Behind

  • Brighter TR-FRET Assays

    Through a recent partnership with Lumiphore, Cisbio Bioassays now incorporates Lumi4™-Tb cryptate for its HTRF® platform for GPCR screening. This molecule’s structure is a lanthanide tightly embedded in a surrounding macrocyle and provides enhanced brightness and greater stability. “This is important in kinase and GPCR research,” explains Francois Degorce, head of marketing, “where you have to use serum or a complex medium.” Other features include enhanced assay performance in terms of sensitivity, assay window, and robustness.

    Two new assays are using Lumi4-Tb: the IP-One Tb and cAMP Tb. Originally launched in 2005, the IP-One was the first cell-based HT system to detect inositol phosphate (IP1), which closely correlates with Gq-coupled activity. By replacing Europium cryptate with Lumi4-Tb, the new assay offers increased sensitivity and enhanced signal-to-noise ratio.

    Another advantage to this molecule is that it is compatible with a variety of acceptors, including near infrared and green acceptors like fluorecein. This enables multiplexing assays, while maintaining high throughput. “You can multiplex IP-1 with a cytokine assay to investigate whether a unique GPCR can signal through both pathways. You can also multiplex several cytokines at once.” This assay is called HTplex™ and allows simultaneous codetection of IP-1 and cAMP in one well.

    Researchers at St. Jude Children’s Research Hospital are combining cell-based assays, high-content screening, and primary cells to discover new compounds that increase understanding of the pathophysiology of pediatric disease. Taosheng Chen, Ph.D., director of chemical biology and therapeutics, says there are specific challenges with pediatric diseases. “We don’t usually have a validated target to start with, so this must be done first. This is different from big pharma, where every target is fully validated.”

    His group uses ACEA Biosciences’ label-free screening and a time-lapse microscope (IncuCyte™) that provides real-time growth of cells. Dr. Chen claims  his group started using primary cells to do small-scale, proof-of-principal cell-based assays. The primary cells provide an advantage in that they are physiologically relevant, but do pose some limitations. These include: difficulty in culturing, variability, low fluorescence, and minimal availability.

    The hospital has an on-site library of 525,000 commercial compounds, with ongoing efforts to develop its own library of unique compounds more specific to its  targets. Dr. Chen explains that when they need to validate a target they use a similar approach as big pharma. He adds that in oncology, however, the target and pathway are often unknown; only the phenotype to achieve is known.

    In addition, pediatric diseases have  unique targets, such as fusion transcription factors. These are associated with chromosome translocations and common in pediatric oncology. “When these transcription factors are knocked down, you don’t necessarily kill the cells. This indicates there is another mutation involved, which has to be identified,” adds Dr. Chen.

  • Cancer Drug Combinations

    In order to assess the effects of combining two anticancer drugs, researchers at Amgen (www.amgen.com) have enhanced a commercially available cell-based HT assay system. Sharon Zhao, Ph.D., senior scientist, lead discovery, says her group adjusted the automation to fit their assays and plate layout, made it high throughput, and developed their own data analysis calculations.

    “We test two compounds mixed together to see if they work synergistically or if there is some other effect. This helps provide information on what two drugs for which patient will work best.” Dr. Zhao reports that the assay monitors how well the drugs kill cancer cells.

    This assay can be applied during early- stage drug discovery to see if two drugs can work better together, and during later-stage drug development for clinical compounds. The platform can test 30–50 cancer cell lines (versus about 10—the standard for academia) and more combinations. Primary cells are difficult for this application because they are not easily expandable, so her group uses standard cancer lines like CHO.

    Some of the challenges of cell-based assays in oncology include relating in vitro data to in vivo predictions, cell specificity, and off-target effects. Dr. Zhao says they are focusing on expanding the system and trying more cell lines and combinations. “We’re trying to collect information more thoroughly about the cell lines, like expression and mutations. We’re trying to see whether we can correlate this data with other data like gene expression profiling or mutation characteristics. These connections will make our data more useful and more predictable.”

    There is no doubt that cell-based assays provide invaluable information for drug discovery. The trends now are disease-relevant and primary cells to create a physiological environment to obtain optimal leads earlier. In addition, there is a push to develop more ways to screen functional activity of endogenous receptors forward in the discovery process instead of downstream after HTS. Researchers are hoping that these assays may help bridge the gap between in vitro assays and in vivo predictions, which remains a big challenge.


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