High-Throughput Screening HTS

To be effective, a given compound must dissolve completely in the assay medium. It is common to add a small amount (1%) of dimethyl sulfoxide (DMSO) to the a.ssay to as i.st solvation. The best concentration of compound to use is somewhat debatable. High concentrations (10 /uM and above) often lead to more fal.se po.sitivcs than. screening at 

Just as there arc. several ways to detect and identify members of a combinatorial library, there are many ways to measure activity in NTS assay.s. Any such method must be accurate, reproducible, and have a high signal-to-noi.se ratio (S/ N). Typically, the result of HTS is a qualitative (ye.s/no) or semiquanlitative one (high-medium-low), rather than a precise value (e.g.. KIjn or LDso). The methods for detection in HTS fall into the categories of lumradiometric and radio-metric. 

Nonradiometric methods iticlude absorbance, fluorescence. and luminescence spectroscopy. Enzyme assays are a common example. The as.suy is usually run at or below the value of thq substrate, with only about S% of the substrate consumed during the assay, and multiple enzyme turnovers occur during the a.ssay. Sometimes enzyme reactions arc coupled, especially if the target reaction does not produce a product that can be detected directly in the assay. An example is carboxypeptidase. which is coupled to the teduction of NADP to NADPH. giving ri.se to absorbance at 340 nm. 

SPA is a newer, simpler method (Fig. 3-13b). Wc start with the same radioactive substrate, which may not necessarily need a capture group. The enzyme and potential drug are added, causing the cleavage of the substrate to some degree. Now. instead of filtering, a special resin bead coated with a 

Other HTS assay advances include the us-e of microorganisms such as bacteria and yeast, the cloning and expression of mammalian receptors in microorganisms, probing protein-protein inlcraclions. and veiy importantly. DNA and protein arrays. These are loo involved to discuss here, but excellent reviews exi.sl.- - The increasing use of HTS to screen fora molecule s absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties has been covered as well.  

Another class of readout measures RNA expression levels, with the three most common methods being chip-based hybridization/fluorescence techniques, realtime polymerase chain reaction (RT-PCR) and quantitative nuclease protection assays (QNPA) [48, 49]. Chip-based methods are widely used for whole-genome scans (discussed in more detail below), but have a disadvantage that they are relatively expensive and so are not really high throughput. The quantitative reproducibility and dynamic range of these chip-based methods are also lower than for the other RNA readout techniques. RT-PCR is a more quantitative technique for measuring transcript levels, and is typically run for up to 40 transcripts at a time. QNPA is another 

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Along with all the data generated by combi-chem and high-throughput screening (HTS) came the need to manage and analyze the data. Hence, computers and the science of informatics became increasingly vital. 

The potential sources of the compounds that are evaluated in a hit triage process are described in other chapters in this volume. These sources may include a high throughput screen (HTS) of a diverse compound collection, a targeted screen of... 

High-throughput screening (HTS) is today the most commonplace method for identifying lead compounds that can be subsequently optimized to generate drug candidates. 

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