Drug discovery has undergone transformational changes in recent years. In the past purified cell membrane preparations were commonly used in receptor binding assays to identify compounds which bind to specific target receptors. This provided a homogeneous assay with end point readout which was easy to adopt in high throughput screen (HTS). As physiological importance of using the whole cell rather than the membrane fraction was realized, whole cell assays were adopted in drug discovery. This was done using immortalized, engineered cell lines representing specific disease models. These immortalized cell lines were easy to culture and propagate in large numbers needed for HTS. Results from these screens gave good signal to noise ratio, reproducibility, and good CV for the data because of the homogeneous nature of the cells. For several years this was the system of choice for cell based assay in drug discovery research.
During the last few years, pharmaceutical industry has seen an increasing number of failures of their lead compounds at late stage trials with various reasons including, lack of efficacy in target population, increased toxicity, and unpredictable side effects. This high rate of failures at late stage of development cost tremendously for the industry. This has led to re-evaluation of their HTS for target validation and later development efforts. They also questioned the physiological relevance of the immortalized cell lines for the cell based assay screen for the specific disease models and targets. This change in attitude has led to the use of disease and target relevant primary cells derived from corresponding tissue types in high throughput screen. In primary cells the target is expressed in physiologically relevant environment and closely resembles that found in human disease and at levels physiologically relevant. Consequently the results of the screen are more relevant to the disease they are targeting. The availability of numerous varieties of primary cell types from leading suppliers ( Life Technologies, Lonza) and optimized culture medium for the growth of these cells has made the transition at an accelerated pace. Primary cell based assays are now commonly employed in identification of compounds targeting GPCRs, kinases, nuclear receptors, and ion channels. In GPCR assays, most of them measure second messenger accumulation, or more complex, protein translocation, or activation of kinase signaling pathways.
Another convergence of technology is the highly sophisticated instrumentation available for HTS. This ranges from precision liquid dispensers to high content imaging platforms to complex software programs for the cell based assay image data acquisition, storage, and analysis. Some of the leading providers of the liquid dispensers include, Beckman Coulter, Caliper, and Tecan. Instruments offered by the leading manufactures can dispense into 96, 384, or 1536 well plates with precision and usually results in very good CV. This was instrumental in miniaturization of the assay saving costly reagents and the number of primary cells needed to screen a compound library.
Several leading instrument companies in life science space offer very complex high content image acquisition and analysis instrumentation. This includes Cellomics, now part of Thermo Fisher, Perkin Elmer, GE Health care, Evotec Technologies, Beckman Coulter and BD. The BD Pathway from BD Biosciences is an automated CCD imager using spinning disc confocal device. This system is designed to do end point assay or kinetic assay. It can also acquire several images per second even during the addition of drug compounds to the cells made possible with available liquid handling system. Evotec’s Opera, another high content screening instrument also can screen kinetic live cell assays. Screening can be done in 96, 384 or 1536 well format. Available environmental chamber controls CO2, humidity, and temperature within the cell chamber. This system also offers liquid handling unit to do kinetic assays.
Most of the high content imaging systems comes with very sophisticated software to acquire and analyze the data. Some of the leading software for image acquisition and analysis packages are provided by Cellomics, MetaXpress (Molecular Devices), Acapella ( Evotec Technologies ), and Harmony software from PerkinElmer.
In summary the convergence of technology in the form of efficient instrumentation for liquid handling, sophisticated high content imaging systems for end point as well as kinetic assays, readymade reagent kits for fluorescently labeling cells, software packages to analyze the data, and the use of primary cells in the HTS screen has all came together and made significant progress in the drug discovery in recent years and will continue to impact the field for the foreseeable future.
