Assay HTS

Sigma LOPAC Pharmacologically active. 1208 Major target classes GPCRs, kinases, ion channels, nuclear receptors, metabolic enzymes, cell signaling, apoptosis, cell cycle, etc. Used extensively for assay/HTS validation. ... 

A study at Novartis compared hits obtained by screening 30,000 compounds for tyrosine kinase activity in three different assays a scintillation proximity assay (SPA), a homogeneous time-resolved fluorescence energy transfer assay (HT-FRET), and a fluorescence polarization (FP) assay.Library compounds were screened as mixtures of five in a well, which meant that after active wells were identified, the five constituents would be deconvoluted by screening each compound individually or using other methods to establish which of the compounds was active. " When this was done SPA, HT-FRET, and FP turned up 30, 59, and 64 hits respectively. A low degree of overlap was observed between the sets. Only four or seven compounds, depending on where the activity bars were set, would have been identified... 

Virtual screening allows the scope of screening to be extended to external databases. When this is done, increasingly diverse hits can be identified. The application of virtual. screening techniques before or in parallel with HTS hclp.s to reduce the assay-to-lcad attrition rate observed from HTS. In addition, virtual screening is faster and less expensive than experimental synthesis and biological testing. 

Calcium Hypochlorite. High assay calcium hypochlorite [7778-54-3] was first commercialized in the United States in 1928 by Mathieson Alkali Works, Inc. (now Olin Corp.) under the trade name HTH. It is now produced by two additional manufacturers in North America (Table 5). Historically, it usually contained about 1% water and 70—74% av CI2, so-called anhydrous product, but in 1970, a hydrated product was introduced (234). It is similar in composition to anhydrous Ca(OCl)2 except for its higher water content of about 6—12% and a slightly lower available chlorine content. This product has improved resistance to accidental initiation of self-sustained decomposition by a Ht match, a Ht cigarette, or a small amount of organic contamination. U.S. production in the 1990s consists primarily of partially hydrated Ca(OCl)2, which is sold as a 65% av CI2 product mainly for swimming pool use. Calcium hypochlorite is also sold as a 50% av CI2 product as a sanitizer used by dairy and food industries and in the home, and as a 32% product for mildew control. 

HTS is usually carried out it multiwell plates and the industry has settled on certain standard formats. One key standard is that most screening assays are carried out in 96 (8 12) or 384 (16 24) well plates with a standard footprint. This standard is important because most automation is optimized for use with these plates and attendant assay volumes in the 100 pL range. Furthermore, source plates containing test compound are typically stored in a similar configuration, allowing for more efficient transfer of test compound from the source plate to the assay plate. Various types of plates are available to match different assay formats, including... 

An important breakthrough in HTS ee assays came from the group of Reetz in late 1990, with the introduction of mass spectroscopy (MS)-based procedures [90]. These methods use special asymmetrically isotope-labeled compounds. Enzymatic transformations of these compounds usually lead to two pseudoenantiomeric compounds whose relative concentration can be estimated using MS techniques. 

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

This approach of combining shape-matching and conformahonal analysis proved a useful complement to HTS. Some of the compounds identified by the computational screen were not detected in the original experimental screen. This was because their relative weak activity was difficult to separate from the noise of the assay. Nonetheless, these compounds had different scaffolds (i.e. were lead-hops ) compared to the previously known inhibitor. The key contribution from conformational analysis was that the newly discovered inhibitors were not found by the corresponding searches based on 2D methods. 

HTS false positives that are nonstochastic (nonrandom) are mostly chemistry related. There is something about the chemical structure that leads to an HTS assay readout... 

A second approach to handling false negatives relies on a computational analysis of actives in the primary HTS. Were there analogs or similar compounds to actives that appeared inactive in the original HTS These inactives are retested perhaps in a more careful screen and some of the original inactives will now be found to be active. Most commonly these were truly active compounds that appeared inactive in the original screen because they were not in solution under the initial HTS assay conditions. 

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