Importance Of Target Cells In Gpcr Hts Assays

Generally, cell-based assays involve the expression of recombinant GPCRs in modified tumor cell lines. By expressing the molecular target in such cells, large quantities of the target are generated for testing in HTS assays. A major advantage of these approaches is that the immortalized recombinant cells provide a relatively naive background for target expression and a relatively homogenous target expression system that facilitates consistency in screening. Furthermore, not only can the GPCR be stably expressed at physiologically relevant levels, but other proteins can be engineered into these cells that can provide reporter readouts of drug-target interactions. Additionally, unique molecular targets can also be expressed in tumor cells in order to identify specific types of drugs, including small molecules selectively interacting with GPCR oligomers as well as allosteric regulators [3, 11, 39] . Collectively, due to their versatility, wide applicability, natve background, and ease of use, a limited number of tumor cell lines expressing recombinant molecular targets provide the main systems for drug screening against GPCRs.

Despite their extensive use, there are limitations in screening compounds using tumor cell lines, particularly with regard to the applicability of the data generated on the action of the compound to the human physiological setting. A major limitation is related to the levels of expression of the transfected target protein. In the case of GPCRs, the expression levels in tumor cell lines employed in HTS are frequently much higher than occurs with endogenous levels found physiologically or even patho-physiologically. Such high expression levels significantly change the ratio of GPCR to G protein and, consequently, the inherent efficiency in receptor activation [40-42]. This may cause misleading interpretation of drug actions on GPCRs such that compounds can act as full agonists on the recombinant receptor while being only a partial agonist or antagonist at the receptor endogenously expressed in cells and in vivo.

Another important issue with regard to GPCR overexpression in tumor cells concerns the creation of constitutively active receptors, that is, receptors producing a functional response in the absence of activating ligand [3, 11, 20, 39, 40]. Since constitutively active receptors exhibit an intrinsically high basal activity, antagonists that reduce basal activity are designated as inverse agonists. Kenakin [41] suggests that many antagonists in clinical use today act as inverse agonists, and it has been suggested [43] that the use of constitutively active GPCRs can provide the means to identify an important new class of drugs.

These postulates are based on pharmacological studies using recombinant receptors, without concern for potential physiological relevance. Importantly, it is not clear whether there are GPCRs that are constitutively active in natural tissues and whether inverse agonists identified in HTS assays act as inverse agonists on endogenous receptors -n vivo . If in fact, the drugs identified on recombinant GPCRs overexpressed in tumor cell lines act differently on GPCRs in vivo, it raises questions as to whether most drugs identified using immortalized tumor cells are clinically relevant.

In addition to the concerns of GPCR overexpression, a number of studies have shown that the overall cellular environment of the receptor profoundly influences the properties of GPCRs and the drugs targeted to those receptors. Thus, the rank order of agonist potencies as well as efficacy at a given GPCR can differ depending on the cell line in which the receptor is expressed. This can be due in part to differences in the G protein expression patterns in the different cell lines used in GPCR drug discovery, and Kenakin [41] suggested that cell: cell variations in G protein association affects receptor reserve and thus the efficacy of agonists and other ligands to modulate signaling pathways. Such issues create a dilemma in determining whether the cell line choice employed for HTS does or does not express the "correct" portfolio of G proteins that couple endogenously to the GPCR under examination. Thus, the cell line selected can significantly affect the pharmacological profile leading to identification of leads that will possess different activities in vivo when compared to their effects in clonal cell lines.

These potential disparities between the physiological environment of screening systems using recombinant tumor cell lines compared to natural tissues has led to a growing interest in the use of primary mammalian cells for drug screening and discovery [44-46]. In primary cells, the endogenous molecular targets are tacitly assumed to be expressed in an environment that more closely resembles that found in the patient, and with levels that reproduce those found naturally and endogenously. Consequently, novel drugs characterized using these primary cell systems are presumed to act in a more predictable fashion in clinical evaluation than those characterized in tumor cells in which recombinant targets are overexpressed.

Primary cell lines may consist of cells derived from embryonic tissues, including neuronal cultures, as well as those from adult tissues such as pituitary cells or hepatocytes. Primary cells can be used to evaluate endogenous targets for drug discovery or be generated to express recombinant targets using viral vector systems as well as employing tissues from transgenic animals. As with recombinant cells, assays using primary cells may employ responses such as transient changes in intracellular calcium using fluorometric dyes, detection of second messenger accumulation or other proteins reflective of cell function, including reporter enzymes via response elements recombinantly engineered into cells derived from transgenic animals as well as the use of electrophysi-ological approaches that measure changes in membrane potential.

A major limitation of the use of standard primary mammalian cells in HTS is the limited availability. To some extent, this is compensated for by using highly sensitive assay techniques, and miniaturized detection systems, collectively allowing the use of very few cells per assay. Alternatively, the broader availability of embryonic stem cells (ESC) or adult pluripotent stem cells (APSC) provides for cells that can be grown in relatively high abundance in a similar manner to tumor cells, yet which retain several phenotypic characteristics of the "natural" cells [45, 47, 48]. Furthermore, ESCs and APSCs can be induced to differentiate into distinct cell types, each reflective of specific organs and tissues, such as hepatocytes, cardiomyocytes, kidney cells, and neurons [49]. In some cases, these differentiated cells develop the characteristics of the mature cell, such as cell. cell networks, and in many ways, return compound screening to classical pharmacological approaches used 30 or so years ago, in which organs and tissues were used to assess drug actions and interactions.

While primary cells are rarely, if ever, used in HTS for drug discovery, they have been employed extensively for drug toxicity screening. Indeed, HTS approaches using microfluidic technologies to examine large numbers of compounds for toxic effects on liver cells can now be routinely assessed either as release of lactate dehydrogenase (LDH) from injured cells or scanned as changes in respiratory cellular activity. Such assays are employed as a secondary screen to test potential "hits" for potential toxicity and can also be employed as a primary screen to exclude compounds.

Toxicity screening is now employed using primary neuronal cultures to predict side effects of potential novel CNS drugs. Peripheral neurons, such as motor neurons or central neurons derived from embryonic tissues can be cultured using microtiter plate format. Here, the toxicity of novel compounds is assessed in terms of cell viability. For example, the effect of drugs on these neurons can be quantified by counting the living fluorescent cells in automated fashion using systems such as a FlashCytometer (Trophos, France) which image and quantify the number of luminescent cells per well. Many other dyes are now used to measure oxidative stress (ThermoFisher/Cellomics, Waltham, MA), changes in redox potential, and mitochondrial function as initial measures of neuronal toxicity.

Although this approach is employed to study neuronal drug toxicity, a similar approach is used for drug discovery, notably to identify novel neuro-protective agents [ 50] . Such drugs have the potential to treat degenerative central or peripheral nervous system diseases, such as Alzheimer- disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Essentially, the approach involves stressing neurons, either by removal of growth factors, or addition of toxic agents such as glutamate, to induce neuronal death. Consequently, the stressed neurons are used to screen small molecule libraries for compounds that can reverse neuronal loss. This technique has been most useful in screening for compounds that protect motor neurons from necrosis.

I mportantly, Rogers et al. [ 51] have recently generated transgenic mice expressing a green fluorescent protein (GFP)-aequorin fusion protein targeted to the mitochondria to measure intracellular Ca++. These authors employed this approach to measure Ca++ transients in vivo by detecting bioluminescent images using either a "photon imager" or the "video imager" consisting of intensified charge-coupled device (ICCD) cameras. Their studies suggested that the aequorin probe was expressed in most if not all cells and that Ca++ signaling in many different cells could be detected both under basal physiological conditions and in response to drugs. This is critical because primary cells, including ESC derived from such animals, could be employed for drug screening in much the same manner that immortalized tumor cells are used for GPCR drug discovery. This is particularly important since aequorin is such a highly sensitive detector of Ca++ transients that relatively few cells per well (500 or less) can be used for drug screening, possibly making the system adaptable for HTS format.

Furthermore, changes in intracellular signaling can be readily measured in primary cells using calcium- sensitive dyes such as Fluo3 or Fluo4 just as in immortalized recombinant cell lines. Viero et al. [52] has now developed an imaging system to measure calcium ion signaling in single adult ventricular myocytes—measuring not only Ca++ ion transients, but also myocyte contractility. These cells can be maintained in culture for extended periods with only minimal changes over time in calcium ion signaling or electrical "pacing" activity or contractility. These parameters are unaltered even when the cells are engineered to express calcium ion sensors that allow for rapid, continuous calcium ion measurements in HTS. Consequently, these systems, particularly when linked to HTS imaging systems, allows for high-throughput drug screening against individual, physiologically active, cardiomyocytes.

Classical microplate reader assay systems that detect calcium ion transients are also used in HTS campaigns with cultured brain neurons [53] . Moreover, imaging-based systems using FRET technologies to detect membrane potential changes in an HTS format have also been employed. Indeed, most, if not all, pharmaceutical companies have now developed automated microscopic HTS systems that measure discrete changes in protein translocation, and many of these assays have been adapted for primary cells. Therefore, despite the limited abundance of primary cells, the recent growth in available sensitive imaging technologies facilitates HTS against a range of molecular targets expressed in primary neuronal or cardiac myocyte cultures.

Thus, primary cells may provide major advantages over immortalized tumor cell lines for GPCR drug screening. In particular, the use of primary neuronal cells provides advantages because they can maintain an environment that replicates the complex interplay of endogenously expressed ion channels, second messengers, and other cell signaling proteins to which central nervous system GPCRs are normally exposed. The result is that the primary cells with the endogenous receptors better recapitulate the physiological milieu than when the GPCR is recombinantly expressed in a tumor cell line. Furthermore, using primary cells from transgenic animals expressing disease phenotypes may provide the basis for developing drugs targeting the disease condition rather than the normal conditions providing the means to develop more effective drugs with fewer side effects. Therefore, in the future, HTS campaigns using primary cells, and perhaps even cellular networks, could serve as the basis for identification of novel compounds and become an essential component of future GPCR drug discovery programs.

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