LANCE cAMP Cell-based Assay is Ideal for Automating HTS of GPCRs
Use of ESC’s and Primary Cells Increasing in GPCR and Kinase HTS
Use of ESC’s and Primary Cells Increasing in GPCR and Kinase HTS
PerkinElmer continues to build its portfolio of cell-based assays in both GPCRs and kinases. Some of these involved genetically altering the cells to express biosensors; such as the AequoScreen® or PhotoScreen™ photoprotein technologies, whilst others can work with unaltered cells; such as LANCE® cAMP, AlphaLISA®, AlphaScreen® SureFire® assays and 3H- and 125I-radioligand binding assays. Historically, the use of genetically unaltered cells has been restricted to those cells that can be easily adapted to HTS protocols – such as PerkinElmer’s CHO and HEK cell offerings.
Recently, however, there is increasing interest in using primary cells for GPCR and kinase HTS. The cellular environment or, more correctly, the cell phenotype plays a predominant role in the pharmacology of compounds identified in HTS, and in turn defining the biology of drugs identified against those validated targets. Therefore, many drug discovery researchers have intense interests in identifying assays that can be employed for drug discovery in a HTS format using primary cells, as well as embryonic stem cells (ESC) that endogenously express a particular molecular target. The implicit assumption is that such cells more accurately reflect the cellular environment of the target in vivo. Consequently, compounds discovered against these cells will act in a more predictable pharmacological manner in human pathophysiology and disease.
CONVENTIONAL METHODS
Today’s principle method for primary screening uses cell-based assays with engineered immortalized cells. There are several advantages; first, cells transfected with a desired GPCR can be grown in almost unlimited quantities. Second, they provide a naïve background for target expression and subsequent measurement. Third, if care is taken with regard to passage number, such cells can provide a consistent, homogenous vehicle for HTS campaigns. Generally, using standard cloning and expression technologies, not only can the GPCR be stably expressed at physiologically relevant levels, but additional proteins can be engineered into these cells, thereby providing reporter protein readouts of compound/target interactions.
While most GPCR and kinase drug discovery HTS campaigns employ immortalized cells, there are clearly a number of issues with this approach which hinder identification of “hits” likely to become successful lead candidates for human testing and eventual therapeutic use. A prime problem arises from the artificial environment of the immortalized cell in comparison to the native cells expressing GPCRs. This artificiality includes abnormal expression of the receptor, the G proteins which mediate its cellular effects as well as the cellular signaling systems critical for mediating the physiological actions of the receptor. The artificial environment of the cells can result in drug profiles that work in a very predictable manner on the cell lines but whose activity in vivo may differ considerably.
PRIMARY AND EMBRYONIC STEM CELLS IN DRUG DISCOVERY
Currently in HTS, most primary cells and Embryonic Stem Cells (ESCs) are derived from rodent tissues and to date relatively few have been used in drug discovery, mostly in secondary screening campaigns and lead optimization studies. Primary cells are rarely used in initial HTS programs for the following reasons (see Table I for comparison of primary cells and immortalized cell lines for drug screening). Firstly, primary cells can generally only be grown in limited quantities, and the amount is usually insufficient for classical HTS approaches. Moreover, they cannot be readily frozen and subsequently thawed for later use in assay screening (in contrast to immortalized cells such as CHO or HEK 293 cells) and require much greater care in handling so as not to hinder or disrupt their physiological characteristics. Since primary cells are derived directly from animals, there is significant cost involved in their preparation in comparison to immortalized tumor cell lines.
Table I: Cells Used for Drug Screening and Discovery
| Properties | Immortalized Cells | Primary Cells |
| Use in HTS | Ubiquitous | Limited to 2' screening |
| Quantity | Unlimited | Limited except for ESCs |
| Cell Type | Homogenous | Heterogeneous |
| Cell Phenotype | Limited | Tissue selective |
| Ability to be engineered | Easy | Difficult except for ESCs |
| Handling Ability | Easy | Sensitive to injury |
| Cell Environment | Different from in vivo | Closer to natural cells |
| Assay Background | Generally low | May be high |
| Relation to target tissue in humans | Limited or none | High-Human ESCs |
Furthermore, while some primary cells are cultured from adult animals, including hepatocytes and pituitary cells, most primary cells need to be derived from embryonic tissues, particularly neuronal cultures. This raises the question whether the cells from embryonic tissues have similar phenotypes to that of the adult - in which most preclinical activities screening for drug efficacy are conducted and for which most drugs are developed in clinic for human use.
Nonetheless, cultured primary cells provide a more physiologically relevant environment for the molecular target under examination than that same target recombinantly expressed in an "artificial" immortalized cell environment. This is notably the case with primary neuronal cells, for which the complex interplay of endogenously expressed ion channels, second messengers and other cell signaling proteins, can be better recapitulated than in transformed immortalized cell lines. Therefore, HTS campaigns using primary neurons, notably neuronal networks, may be an essential step in the identification of novel compounds to treat neurological disorders that could not be as easily identified using more standard screening approaches of the past.
The limitations of cell abundance that hinder the use of primary cells in HTS could be overcome with the use of ESC. Pluripotent ESCs can be grown in unlimited supplies and even frozen in stock solutions much like immortalized cell lines and thawed at some later time in a manner that retains their appropriate functions to allow them to be used for drug screening. Several studies have now shown that these cells can be induced to differentiate into selective cell types such as neurons, hepatocytes or myocytes. By recombinantly engineering into ESCs, GFP or other protein tags, one can use cell sorting techniques to isolate and purify homogenous cultures of specific cell types from the general ESC population. Furthermore, the insertion of antibiotic resistance genes under cell type specific promoters, such as inserting the neomycin resistance gene under the control of the Sox-I promoter to induce neural phenotypes, or the ?-cardiac myosin heavy-chain promoter for directing to cardiomyocytes, allow scientists to select lineages. Specifically, these transfected ESCs can be selected, expanded, and then, using specific growth factors, induced to differentiate into populations enriched for a selective image such as neurons, myocytes and hepatocytes.
ESCs are generally obtained from either mice or humans, and although human ESCs provide obvious advantages in drug screening, there are some disadvantages as they do not grow as well and are more difficult to maintain and expand than murine cells and the procedures for directing them to differentiate are less defined than those for murine ESCs. Consequently, much more work is necessary to provide human ESCs that call be employed in a practical manner for compound screening. One alternative to the use of human ESCs are human adult multipotent stem cells. These can be obtained from cord blood, bone marrow and other tissues. However, a limitation of these cells is that their expansion capabilities are not as well understood as ESCs even though they are quite capable of differentiating.
Collectively, stem cells are very useful for in vitro drug discovery because they allow a greater range of tissue types to be studied than hitherto possible with standard techniques. Furthermore, human stem cells allow for optimal pre-clinical evaluation of compounds directly on "relevant" human cells prior to clinical testing. This is important because relatively little information is usually obtained during pre-clinical development procedures or the manner in which novel drugs act on human tissues.
How do you increase HTS efficiency?
The question arises as to how to increase efficiency of HTS screening campaigns to bias discovery more toward identifying compounds likely to be effective in vivo? In some respects this question is already being answered. In some GPCR drug discovery programs, “hits” identified in HTS campaigns are retested on secondary screening programs using primary cells for effects on more physiological responses such as changes in electrophysiological readouts or changes in other cellular responses such as hormone or transmitter secretion. This secondary screening can start to better identify those compounds that selectively bind to a given GPCR and elicit a response predictable of in vivo activity. By bringing natural tissues back into the drug discovery process, the likelihood of increasing the efficiency of identifying molecules that will be effective therapeutics should be greatly enhanced.
In summary, as HTS for GPCR and kinases moves into a strong cellular context, PerkinElmer is ideally positioned to offer a range of solutions for both immortalized and primary cell users. As shown in Table 2, this broad offering will continue to expand in our portfolio and going forward, not only by offering cells themselves in our product line, but also by generating extensive application data in primary cells. Consequently, the marriage of data from PerkinElmer assays with studies undertaken in immortalized and primary cells provides a compelling reason for customers to view PerkinElmer as the complete solution provider.
Table 2: The Right Solution for Your Cell-based Assay
| Target Application | PerkinElmer Technology | PerkinElmer Detection Instruments |
| Cellular GPCRs | LANCE cAMP | EnVision, VICTOR, ViewLux® |
| AlphaScreen cAMP AlphaScreen SureFire ERK |
EnVision | |
| britelite™ plus & steadylite™ plus | EnVision®, VICTOR™, MicroBeta® JET | |
| cAMP [125sup>I] RIA Kit FlashPlate [125sup>I] Microplates |
MicroBeta, TopCount® | |
| Cell Lines | AequoScreen (Aequorin) and PhotoScreen (Photina®) cAMPZen FroZen Cell Lines Validated GPCR Classical Cell Lines |
LumiLux®, EnVision®, VICTOR™, MicroBeta® JET |
| Cellular Pathway Analysis — Kinases | AlphaScreen SureFire | EnVision® |