February 1, 2006 (Vol. 26, No. 3)

Suggestions for Overcoming Obstacles

The high-throughput screening (HTS) arena has been a hotbed of technological advances over the past few years. The ability to synthesize huge chemical libraries and the availability of automated robots to assist in the screening process has enabled drug development companies to routinely screen hundreds of thousands of compounds per day.

While these HTS advancements have made the screening aspect easier, researchers still need to grapple with a slew of challenges. Key among these are assay development for difficult or unknown receptors, use of innovative technologies to enhance productive discovery, smarter strategies to perform lead identification, and how to intelligently transform the deluge of data into good quality hits.

These issues will also be highlighted and addressed in detail at the ScreenTech conference to be held in March in San Diego.

Some general bottlenecks we come across are in developing good functionally coupled assays, distinguishing true hits from false ones, determining what appropriate secondary assay is needed to validate the hit, and ensuring target selectivity, says Bonnie Hanson, Ph.D., senior scientist and head of GPCR cellular assays group at Invitrogen (www.invitrogen.com).

When working with GPCRs, we sometimes also have to deal with receptor over-expression-related toxicity, receptor constitutive activity, or receptor inactivation.

Various Solutions

Invitrogen offers several solutions to address these problems and perform high-throughput receptor screening assays. The GeneBLAzer cell lines for GPCR assay development have a built-in beta lactamase reporter gene that allows functional sorting of cells (for example by flow cytometry), helps identify true hits from artifacts, and consistently produces assays with Z factors greater than 0.6 due to its unique ratiometric readout, according to Dr. Hanson.

Multiple choices exist for detection substrates, each with unique benefits, such as the ToxBLAzer DualScreen Kit that allows detection of beta-lactamase reporter gene-activity and cytotoxicity in the same assay. The Fluo-4 NW (No Wash) Calcium Assay is a calcium mobilization assay that measures cell-signaling events in a biological context and enables multiplexing of a secondary assay with the GeneBLAzer technology, she continues.

The company says target selectivity is made easier by Invitrogens multiple cell line offerings that allow one to run a panel of counter screen assays for a desired target. The GeneBLAzer technology platform can also be integrated with the Flp-In T-REx system that reportedly enables stable, site-specific integration and regulated expression of receptors, thus eliminating variability in readouts. It also helps in studies of toxic receptors, as it is an inducible and titratable system, allowing researchers to precisely manipulate the expression level of the receptor of interest.

Enhancing Discovery

Brute-force aptly describes how traditional HTS used to be performed, notes Auro Nair, Ph.D., vp of worldwide marketing at Caliper Life Sciences (www.caliperls.com).

But the drawback of this approach was lack of relevant and useful information on leads upfront, says Dr. Nair.

In collaboration with NovaScreen (www.novascreen.com), Caliper Life Sciences provides a suite of more than 600 assays in a variety of therapeutic areas to help define the mode of action, side effects, and other important activities of potential drug candidates. Caliper also offers the microfluidics-based LabChip Technology that can provide a direct measure of substrate and product.

Biochemical screening assays typically require speedy and bulk measurements in an experimental mixture. LabChip allows researchers to miniaturize the size of the experiment and rapidly separate out and measure individual molecular components from a sea of colored compounds. This enhances accuracy of the overall result.

With higher quality data, chemists can look at structure activity information early on and companies can make better decisions during lead identification. This technology also detects weak inhibitors, so we can cast a wider net in our quest to find lead compounds that are not already patented, explains Seth Cohen, Ph.D., director of application sciences at Caliper Life Sciences.

LabChip is being successfully applied in the cell-based assay arena, points out Dr. Cohen. Advantages of such cells on a chip include the need for only a few cells, ratiometric data measurements with cell marker and cell response dyes, and the abilities to test for receptor expression and to perform real-time cell monitoring.

Dr. Cohen cites the example of a collaboration with Millennium (www.millenni um.com), where the team successfully developed platelets on a chip for a study with cardiovascular platelets. Caliper Life Sciences is further developing this technology with a variety of cell lines.

Multiple Screens

Screening for a family of targets rather than a single target is becoming more common in HTS, says Patrick Zarrinkar, Ph.D., director of molecular biology and technology development at Ambit Biosciences (www.ambitbio.com). The firm offers a new technology to screen compounds against a large panel of kinases as opposed to the traditional approach of screening compounds against a single kinase in a single assay at a given time.

The KinomeScan platform screens small molecule compounds against a panel of 235 kinases simultaneously, providing a fast, accurate, uniform, and quantitative kinase profile for each compound, claims Dr. Zarrinkar. KinomeScan turns the cross-reactivity issue of kinase inhibitors into a discovery advantage, revealing how well compounds bind not only to their intended targets, but also to mutations and unintended targets that may cause side effects or offer new uses for existing drugs, he adds.

Our proprietary technology enables kinase researchers to efficiently perform a high-content and high-throughput screening assay under standardized conditions. We are currently working on expanding our technology to include more kinases in our assay panel, says Dr. Zarrinkar.

HTS Trends

Traditional HTS approach is typically a sequential and iterative process starting with a full library screen, followed by dose-response and counter screen(s), and multiple rounds of hit-to-lead activity. It could take many months to make a lead determination.

At Boehringer, we are trying out an intensive parallel approach, where we apply a lot of resources upfront including assay development, screening (both of the general library and more focused libraries), virtual screening, and medicinal chemistry. Our screening, assay development, and chemistry groups work hand in hand during this process. The hope is to collapse the timeline and enable quicker lead decisions, says E. Michael August, Ph.D., senior principal scientist, biomolecular screening group at Boehringer Ingelheim Pharmaceuticals (www.boehringer-ingelheim.com).

As HTS throughput has increased, the volume, complexity of data, and the content level of information output, data processing has become harder and more time-intensive. It can take several days to sift through all this information in a conventional way, maintains Stephan Heyse, Ph.D., project head of screening informatics at Genedata (www.genedata.com).

The basic question a compound screen seeks to answer is: What are the true biological effects of a compound? The challenge for current data analysis tools is analyzing multiple readouts for signs of compound activity, specificity, and cross-reactivity to determine real effects from artifacts. An additional goal is to interpret compound effects from multiple assays that use diverse technologies to gain a comprehensive picture of a compounds mode-of-action.

Precision and accuracy in data interpretation are key factors in HTS. Genedata offers integrated solutions in a multistep workflow to enable customers to manage their throughput and the data analysis complexity of HTS, says Dr. Heyse.

The companys Genedata Screener enables the analysis of millions of data points and provides precise data that represents biological reality, he explains, noting that the product performs large-scale processing (>10,000 samples) of dose response curves and reduces the complexity in separating out true response from false positives.

Genedata Screener also performs high-level interpretation of high-content screens, such as cell-based assays, by aligning multiple test responses to provide a complete picture of compound activity, according to Dr. Heyse.

Balancing Speed and Cost

A good chunk of technology costs in HTS are due to HTS cell culture, assay-associated costs, and the use of versatile platforms that enable scale-up. Hence, companies continue to make investments in technologies that allow HTS to be performed faster and cheaper.

While speed is desirable, one must constantly evaluate how we can produce quality hits that have true clinical relevance. Use of innovative technologies, automation, informative assays performed upfront, and real-time monitoring of drug activity greatly increase the potential to find good hits, explains Dr. Nair, from Caliper Life Sciences.

All involved with HTS agree that the need to translate hits to clinically relevant compounds while maintaining speed and efficiency is needed now more than ever. In spite of technological advances and increased pharmaceutical research spending, the rate of bringing new drug entities to market has continued to decline.

We must continue to strive to make HTS more therapy centric, so that HTS can function as a good-quality operation that is fast and cost-effective in the long run, emphasizes Dr. Cohen.

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