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Jun 1, 2010 (Vol. 30, No. 11)

Multiplexing Advances Redefine HTS

Novel Instruments, Comprehensive Services, and Streamlined Assays Boost Scientists' Efforts

  • Focusing on Targets

    BioFocus offers a  range of discovery services, principally aimed at target validation and developing molecules against targets, according to Doris Hafenbradl, Ph.D., senior director of biology and natural products. The company just reported the availability of four biologically focused libraries containing drug-like compounds that are aimed at kinases and protein-protein interactions. These compounds are modeled to bind to the kinase hinge, with allosteric sites, or to enter into other unspecified binding modes on the molecule.

    “We use surface plasmon resonance extensively in our screening platforms,” Dr. Hafenbradl explained. “This is especially relevant for screening of our diverse fragment library in which label-free technologies are especially convenient. 

    “We are also well-positioned for designing drugs using our chemo-informatics library of 900,000 compounds. Using high-throughput screening we have identified a large range of novel targets.” The use of radioactive tags is no longer a popular strategy in molecular analysis, but it has to be the starting point. “If you have an enzyme that is suspect as a candidate for a therapeutic role, you can find compounds that will bind to and inhibit it.”

    BioFocus also maintains an active screening program for epigenetic targets. Epigenetic modifications of the DNA form the basis of much phenotypic expression, and a wide range of disease states are now known to be associated with a group of modifications to DNA and the histone chromosomal proteins that include histone phosphorylation, methylation, acetylation, ribosyl transfer, ubiquitinylation, and proteolytic-driven cleavage.

    In this case, BioFocus uses an “intelligent selection” of library subsets for primary screening, rather than employ a large compound library, which would be more appropriate for an unknown target. The narrowing process uses in silico modeling of the enzymatic site in accordance with ADME. These parameters allow the 3-D structural information concerning the target to be maximized.

    “We are gradually accumulating a body of experience that will allow the elimination of futile compounds,” noted Dr. Hafenbradl. 

    A case study on histone kinase detailed the use of a targeted library of 60,000 members, which resulted in 82 validated hits after several rounds of verification. According to Dr. Hafenbradl, this project established the validity of the firm’s fast-track approach to novel chemical entities capable of inhibiting epigenetic targets.

    In addition to their ability to uncover drug targets, BioFocus screening platforms have the potential to uncover disease biomarkers that could be the subject of multiplex analysis, using label-free detection such as SPR, Dr. Hafenbradl concluded.

  • Cellular Responses

    Genetix’ CellReporter™ system was developed for assessing cellular responses, according to Jerry Williamson, president of U.S. operations. This technology can be used to study a variety of cell functions such as cell-cycle analysis, effects of cytotoxic compounds on cell function, and the process of protein translocation.

    Through the use of flexible image-analysis software, each object can be characterized, and individual cell responses can be identified. A number of assays can be run simultaneously for multiplexing functions.

    Tobin Dickerson, Ph.D., of Scripps Research Institute, is using a reconfigured CellReporter instrument to isolate compounds that block the toxic effects of botulinum neurotoxin. “The cell-based assay is an excellent format to analyze inhibitors of botulinum neurotoxin.”

    This extremely potent toxin is fatal to humans at a dose of 1 ng/kg. Its mechanism of action is through inhibition of exocytosis in neurons. Rather than outright killing, the toxin acts by internalizing and brings transmission within the nervous system to a grinding halt. The search for an effective small molecule inhibitor has been unsuccessful since most drug screens search out compounds that prevent cell death.

    As part of his approach, Dr. Dickerson screened a library of small molecules for allosteric modulators, as well as direct inhibitors. The screening system he uses reportedly works in a precise fashion, and his team has isolated a number of small inhibitory molecules. Two of these compounds are now being evaluated in animal models.

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