Fluorescence dose-response assay

Figure 15.7 shows an analysis of the selectivity of2094 recent hits in high-throughput screens at Roche. Of these, almost half were specific for the screen in which they were identified as a hit - there were no other screens where these compounds had been titrated in a dose-response assay. However, 8% of the compounds had been tested in dose-response assays for more than five projects. These compounds are frequent hitters, and the most commonly occurring substructures are shown in Figure 15.8. Several of these compounds either are electrophilic, or can give electrophilic species on oxidation. The nitrobenzoxadiazoles are known fluorescent compounds. The oxindoles...

The processes of both seed formation and fibril extension are dependent on temperature and on peptide concentration, with 37°C being required for establishing equilibrium within 24 h with 30 pM Pi 4o- A full description of the assay system may be found elsewhere [97,117], A 4 h reaction time is typically within the linear portion of the time course. This nucleus-dependent assay detects mainly inhibitors that are substoichiometric with the monomeric peptide, which is present at high concentration. It is relatively insensitive to inhibitors that target the monomeric peptide. Whether the inhibitors interact with the growing end of a seed or with a low abundance conformational form of the p peptide that is competent to add to the seed is difficult to determine at this time. Similar dose-response curves are obtained for Congo Red as an inhibitor with either thioflavin T (ThT) fluorescence or filtration of radioiodinated peptide readouts (Fig. 4) Caveats in the interpretation of both the ThT and radiometric filtration assays for the evaluation of putative inhibitors are discussed elsewhere [97]. 

FIGURE 2.3 Dose-response curves for protease inhibitor with autofluorescence characteristics, (a) Result of standard fluorescence intensity-based assay employing an AMC-labeled substrate at a concentration of 2 pM. Profiling data could not be obtained due to the interference of the compound s fluorescence with the readout, (b) Result obtained from assay employing a RhllO-based substrate at a concentration of 0.5 pM. The profiling data (IC50 value of 335 nM and Hill coefficient of 1.0) were obtained for the depicted data set. 

It should be noted that the relationship between the final signal output and concentration of the analyte (dose-response) may be one of direct or inverse proportionality, and is dependent on the specific assay format. In addition, a number of different reporter enzymes may be used (e.g., horseradish peroxidase, alkaline phosphatase, p-galactosidase), along with a number of different signaling systems (e.g., substrates that yield chromogenic or fluorescent or chemiluminescent products, activation of signaling enzymes, amplification by biotin-avidin system or polymerase chain reaction). 

---