Problem: Pharma and biotech companies are striving to reduce drug attrition rates within the drug discovery process. G-protein coupled receptors, a major target class within drug discovery, have traditionally been assayed in recent years using fluorescence calcium flux assays. However, inherent to these assays is the generation of false positives due to fluorescence compounds within compound libraries. Potential drug candidates can therefore be missed. There is a need for highly sensitive counter-screening across different targets within hit follow-up to ensure potential lead compounds are fully characterized prior to decisions regarding their progress.

Solution: Orthogonal screening approaches are now being deployed earlier in the drug discovery process. The incorporation of label-free Epic® technology has improved the process both in assay development and post- HTS screening for structure-activity relationship (SAR) and hit profiling studies.

Optical label-free technology measures changes in light refraction resulting from dynamic mass redistribution within the cell. This occurs in response to receptor activation or deactivation in a zone within the cell’s monolayer. The response is indicated by a wavelength change in the emitted light. Labeled technologies such as fluorescence, however, measure a particular biomarker within an individual signaling pathway. The integrated cellular response obtained from a label-free assay enables characterization of the signaling pathways involved that can be affected by biased agonism, dimerization and allosterism.

The PerkinElmer EnSpire® Multimode Plate Reader now incorporates Epic® optical label-free technology, offering an orthogonal approach to the drug discovery process.

An example of the benefit of incorporating label-free in the drug discovery process is that of a fluorescence Ca2+ flux assay using a FLIPR^TETRA® High Throughput Cellular Screening System and confirmation studies using the Corning® Epic® label-free system.

Novel compound activities that are missed by a traditional labeled method can be revealed using Epic® technology. In the top figure, a compound was tested in a FLIPR® Ca2+ flux assay and shown to behave as a neutral antagonist. The Epic® DMR response shown in the middle figure similarly demonstrates the antagonist activity of the compound and identifies inverse agonist activity after the first addition. No such response was observed in the FLIPR® Ca2+ flux assay. The dose response data for antagonist activity (yellow curve) and inverse agonist activity (blue curve) on Epic® is shown in the bottom figure. For assay development, the process benefited from being both more rapid and generic.

Label-free technology is ideal for use in drug discovery, enabling the study of GPCRs and cell signaling. It has already been adopted within the hit-tolead process for target confirmation and lead optimization within secondary and orthogonal screening, SAR studies, biophysical testing and ADME/toxicity.

Pathway-unbiased, label-free technology offers more fully characterized information about cellular and biochemical systems, pathway-independent analysis, non-invasive, more physiologically relevant data and the ability to study difficult targets or weak biological interactions. Rich information can be obtained for difficult targets, endogenously expressed receptors and recombinant cell lines in 384- or 96- well plate formats.

For more information, visit www.perkinelmer.com