Fluorescent inhibition screen

The DDI fluorescent inhibition screen is a high-throughput screen, primarily as a result of reduced analysis time, and can range from a few hundred compounds per week to a thousand per week. The assay uses recombinant microsomes prepared from insect cells infected with recombinant baculovirus containing a human CYP enzyme and individual fluorogenic substrates. In this instance the substrates are not specific and hence cannot be used in a mixed enzyme system such as HLM. In addition, this assay is more prone than the conventional inhibition screen (Section 8.3.2) to NCE interferences within the assay (i.e., inherently fluorescent NCEs, fluorescent quenching by the NCE). 

In early discovery determination of percent inhibition at a single concentration is sufficient to compare compounds vfithin a chemical series. Typically concentrations in the 1-5 j,M range are used [27, 28]. Test compounds and vehicle control are incubated with the probe substrate and the amount of metabolite formation from the probe substrate determined. The metabolic rate can then be expressed as a percentage of the 

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Figure 14.8 (A) Screening of a glycopeptide library using a fluorescent-labeled lectin and ligands bound to PEGA beads. The acbve compounds are analysed by mass spectrometry. (B) FITC-labeled lectin binding to resin bound mannose could be inhibited by soluble glycopeptides obtained from library screen. Percent inhibition was quantified by recording of lectin fluorescence. Only every second well of the microtiter plate was used and nonfluorescent beads indicated good inhibitors.44...

One-third of these appeared in the actives for the assay, which was defined using a 30% inhibition threshold. The overall hit rate for the assay was 2.0% in other words, 20% of the actives were false positives due to extreme optical interference with the assay readout. This number rises depending on the level of initial fluorescence used as a cutoff in the selection scheme used by the project team at the time, almost half of the actives were rejected as false positives on the basis of the initial fluorescence readings. The corporate screening collection has been reformatted since then, and many of these problem compounds have been removed. 

Next to the detection of enzyme inhibition, ESI-MS can also be used to monitor protein-ligand interaction, employing an assay format similar to fluorescence-based receptor assays. Using a similar continuous-flow analytical screening system as shown in Fig. 5.2, a competitive assay can be set up using ESI-MS to measure the interaction of the analyte(s) with an affinity protein such as an antibody, receptor or enzyme [28]. Figure 5.10 shows the equilibrium reactions that form the basis of the assay concept. In a first step, the sample was injected into a con-... 

Dorn, A., Hermann, F., Ebneth, A., Bothmann, H., Trube, G., Christensen, K. and Apfel, C. (2005) Evaluation of a high-throughput fluorescence assay method for hERG potassium channel inhibition. Journal of Biomolecular Screening, 10, 339-347. 

The assay was used to screen over 215 randomly selected extracts of plants from the National Center for the Development of Natural Products repository of plant extracts (half from South America and the other half from the U.S.). Plant extracts that inhibited the increase in fluorescence by 50% were considered positive. Seven extracts (3% total)... 

Residues of TCs were quantified via the MCAC-HPLC method (26) in pork and chicken muscle tissue that had been previously screened with both a microbiological inhibition test using B. subtilis and an ELISA method. The correlation between the mean area of the inhibition zones and the DXC levels found in 28 samples by HPLC was 0.82 the correlation between the ELISA results and the DXC levels in the same samples was 0.73. The results indicated that an inhibition test was well suited to screen the mentioned samples for TCs residues. The authors found the more expensive ELISA screening test unnecessary, because only a minority of analyzed samples did not contain TCs. Confirmation with HPLC method was necessary because of the presence of some false-positive results. Moreover, the positive results from LC-fluorescence assay were confirmed using LC-MS-MS assay with electrospray ionization working in positive-ion mode (31). 

Some plant pathogenic bacteria and their phytotoxins have been screened in bioassays that monitor the effects of their toxins (antibiotic and phytotoxic) on other sensitive bacteria. For example, several fluorescent Pseudomonas syringae pvs. produce extracellular phytotoxins.76,106,116 Tabtoxin is produced by P. syringae pv. tabaci and pv. coronafacines, and this natural product inhibits glutamine synthetase.34,46,116 Phaseolotoxin, produced by P. syringae pv. phaseolicola... 

The presence of the DOM samples was found to inhibit the carbofuran photolysis. The inhibition ranged from 19 to 70% for a concentration of DOM of 5-30 ppm. This decrease cannot be explained by a screen effect alone indeed, at the highest DOM level (30 ppm), the decrease in light absorption by carbofuran was only of 27%, while the rate was decreased by 70%. Using the fact that carbofuran is fluorescent, the authors determined its binding to... 

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