Two critical areas of drug discovery are the development of assays and their subsequent progression to the HTS environment where identification of leads among potential targets generated occurs. Kristian Enkvist reports.
There is a growing need for a smooth transfer of assays from the development stage to the fully automated HTS stage avoiding bottlenecks.
Similarly, in another very active area of drug discovery today, that is, in the later stages of a project such as secondary screening, greater efficiency in testing the ever-increasing numbers of compounds from HTS screens is sought.
To test the ease of moving between detector platforms, we studied a homogeneous Wallac Lance time-resolved fluorescence resonance transfer assay for tyrosine kinase activity using both instruments.
A biotinylated peptide substrate was phosphorylated by kinase activity with ATP as the phosphate group donor. APC-streptavidin and a europium-labelled anti-phosphotyrosine antibody were added to the wells.
The streptavidin APC-acceptor conjugate will bind to all peptides, whereas the Eu-conjugated anti-phosphotyrosine antibody will only bind to phosphorylated peptides, in which case it is close enough to excite the APC, whose emission-signal is then measured.
Data comparison
Comparison of data from the plate reader Victor, and the whole plate imager ViewLux, shows that both instruments gave similar results reading the same plate. This clearly demonstrates the simplicity with which an assay can be developed on the plate reader and subsequently run on the high capacity imager without any changes.
The principle for a homogeneous Lance time-resolved fluorescence resonance transfer assay for tyrosine kinase is shown in Fig. 1.
Functional cell-based assays to look at multiple parameters in cellular response end-points are now seen as an integral part of secondary screening. Real-time cell-based assays using multiple indicators and/or ratiometric readouts are often used at this stage in drug discovery. Utilising one of the new features of the latest Victor2 V plate reader, we have developed a dual wavelength ratiometric Ca2+ assay using Fura-2.
The advantage of using dual wavelength Ca2+ indicators, such as Fura-2 is the ability to calculate a signal ratio, which is proportional to the intracellular Ca2+ concentration, but independent of cell number and of the variability in loading of cells.
Additionally, the signal can be calibrated into actual Ca2+ activities, enabling real quantitation, and comparison of experimental data. This is in contrast to the qualitative output seen with the single wavelength plate readers in general use today. We used Chinese hamster ovary (CHO) cells, which express a purinergic P2Y receptor.
Cells were grown on Wallac white 96-well Isoplatesa to confluency and then loaded with 5 µM Fura-2 AM. The plates were read on Victor2 V giving one excitation ratio pair (340/380 nm) every second when monitoring emission at 510 nm.
The purinergic receptor ligand, ATP, was added using the instrument's integral injector in a volume of 50 µl to the 150 µl already in the plate.
The left-hand panel shows the traces of emission at 505 nm from the two individual excitation wavelengths, 340 and 380 nm. 100µM ATP was added to the cells using the Victor injector.
Signal increase
An increase in intracellular Ca2+ is represented by an increase of the signal when exciting at 340 nm and a decrease in signal when exciting at 380 nm. The right hand panel shows the calculated ratio with one data point per second.
The new technology enables real quantitative Ca2+ readouts and other multi-wavelength fluorescence determinations in microplate format using ordinary Victor2 V plate readers.
Today these types of experiments are mostly done in single-sample systems such as microimagers or cuvette fluorometers.
This new feature, together with the other built-in measuring technologies should make the Victor2 V an attractive instrument, both for assay development and HTS, and for secondary screening and mechanistic studies in drug discovery projects.
ENQUIRY No 67
M O Kristian Enkvist is product manager, Lead Optimisation and Cellular Assays, PerkinElmer Life Sciences, Turku, Finland. www.perkinelmer.com
