Cell Assays – Present & Future (Part One)

Written by SelectScience Guest Editor, Dr Peter Simpson, AstraZeneca.

Assays form the backbone of the compound ’design-make-test-analyse’ cycle of data generation and decision making in drug discovery projects. To enable good decision making, they have to be designed, built and executed with skill and care. Assay scientists seek to build assays that reliably estimate the activity of compounds, while retaining relevance to the biological mechanism of the target under investigation.

Drug discovery screening assays
Historically, the majority of frontline drug discovery screening assays have relied on three optical signal measurements: absorbance, fluorescence and luminescence. These techniques continue to be used widely, as they are flexible, robust and well understood approaches. However, exciting developments in recent years have opened up new approaches with sufficient throughput, and excellent signal resolution, to provide a more extensive toolbox for assay design. For cell assays in particular, where scientists need not only good detection approaches, but to be able to query the biological mechanism downstream of the binding event, this improved assay toolbox is enabling better understanding of the consequences of different binding interactions and target types for biological consequences. Part one of this article looks at fluorescence based screening technologyand high content screening and its progression into high content imaging.

Fluorescence-based screening
After the invention of FLIPR (Fluorescent Imaging Plate Reader) in 1990s, fluorescence-based screening of ion flux became the predominant screening approach for ion channels. This is because conventional ion channel studies by electrophysiology is slow, requires skilled investigators, and can provide great detail but not true screening capacity. More recently, higher throughput electrophysiology platforms have proliferated, beginning with Ionworks from Molecular Devices and leading to a range of solutions from vendors such as, Sophion, Nanion, and many others. These platforms enable detection of the actual change in membrane electrical current, or voltage, that is triggered by the ion channel opening or closing; they do so without the requirement for expert manual ‘patching’ on to the individual cell membrane. They provide a more direct measure of the compound-channel events that enable more sophisticated understanding of the biophysics of the interaction and its consequences for ion mobility.

The Molecular Devices IonWorks Barracuda and Sophion Qube readers may provide the ability to screen voltage and ligand gated channels on a single platform. These platforms can be used by ion channel project scientists, and also by safety pharmacologists who are screening compound liability against panels of cardiac or brain ion channels, linked to cardiovascular adverse events, or seizure risks. Continual improvement in the scope throughput and accuracy of these machines should see previously laborious electrophysiology assays performed in high throughput as a matter of routine.

High content imaging
Fluorescence intensity was, as mentioned above, one of the earliest approaches to assays. There are now diverse ways of using fluorescence to enable more complex biology to be studied. This can include using fluorescence-based techniques such as fluorescence polarization, time resolved fluorescence etc on increasingly sensitive plate readers. Also there is the opportunity of studying subcellular events by high resolution cellular imaging and quantitation. The technology of high content imaging instruments has matured in terms of robustness and ease of use, offering a range of image resolution, speed, and informatics capabilities. Complex imaging assays are attractive to projects, as for example measuring changes in distribution of proteins that do not result in changes in protein level are easy to visualize.

Imaging assays unfortunately remain relatively burdensome, as expert staff can be required to provide guidance to maximize impact from imaging assays, and data storage and manipulation can be a challenge. Novel, multiplexed cell imaging assays can take relatively long to build, particularly if novel analysis algorithms are required. Simpler, two-fluorophore cell imaging assays provide lower information content, but can prove to be more robust in routine screening settings than the ambitious multi-endpoint imaging assay approaches project teams may prefer; either can be beneficial to the right project problem.

Chemotaxis and cell migration monitor the movement of cells in 2D and have been adapted from low throughput experiments to higher throughput modalities. The advent of high content imaging has led to these assays becoming easier to measure in reasonable throughput. In part two, the use of 2D and 3D cell cultures, bright-field imaging systemsand mass spectrometryin cell assays are discussed.

[A longer article on current and future assays, by Dr Peter Simpson & Dr Tim Hammonds, will be published later this year in A Handbook of Medicinal Chemistry, RSC Books.]