ImpedanceRWG

Two very recently introduced technologies, based on impedance and RWG, have received considerable attention from cell biologists and, in particular, from researchers interested in GPCR signaling. Both platforms carry transla-tional potential and open the stage for new insights into GPCR- induced signaling pathways using a combination of label-free detection and real-time kinetics with high accuracy and throughput. In addition, these technologies offer enhanced information content over other assay technologies. Both platforms will be described here in more detail with respect to the underlying biological processes, assay principles, and their potential in GPCR research.

Eukaryotic cells rely on subcellular structures, such as the nucleus, mitochondria, endoplasmic reticulum (ER), and transport vesicles. They continuously adapt to changing environmental conditions or extracellular ligand stimulation through coordinated molecular and mechanical responses. These require a cytoskeleton- guided transport of molecules through intracellular regions with differing viscosity or elasticity. This leads to minute changes in cell morphology or induces adaptations of cell-cell and cell-substrate interactions. In the case of GPCR activation, dynamic molecular interactions at the plasma membrane initiate intense trafficking of subcellular structures, which includes the internalization of receptors in vesicles as part of the desen-sitization process. Microrheology experiments, for example, demonstrated that lysophosphatidic acid receptor (LPA) activation leads to transient changes in cytoplasmic stiffness and viscosity in serum-starved Swiss 3T3 cells [26, 27].

Figure 11.1 GPCR-induced signaling pathways measured with label-free detection technologies. Stimulation of GPCRs leads to activation of one or several downstream effectors, which induce alterations in subcellular compartmentalization and cytoskel-etal architecture. Label-free detection methods measure the integrated cellular response of a ligand-stimulated/agonist-stimulated GPCR.

Figure 11.1 GPCR-induced signaling pathways measured with label-free detection technologies. Stimulation of GPCRs leads to activation of one or several downstream effectors, which induce alterations in subcellular compartmentalization and cytoskel-etal architecture. Label-free detection methods measure the integrated cellular response of a ligand-stimulated/agonist-stimulated GPCR.

Cytoplasmic subcellular structures are highly organized by a dynamic cyto-skeletal scaffold that is controlled by effector molecules, which transmit extracellular signals received at the plasma membranes to induce changes in cellular structure (Fig. 11.1). It has been reported that heterologously expressed endo-thelin A receptor (ETA) or endogenously expressed LPA receptor stimulation induces stress fiber formation in Swiss 3T3 fibroblasts [28] , and it was shown that sphingosine - 1-phosphate (S1P) stimulation of S1P 2 receptor- expressing 3T3 fibroblasts leads to inhibition of insulin - -ike growth factor- 1- mediated chemotaxis and membrane ruffling, whereas stress fiber formation is stimulated [29] - The angiotensin II (Ang II)--nduced morphological effects were investigated in recombinant Ang II receptor subtype 1 (AT1 R) expressing HEK293 cells. Intense remodeling of the actin cytoskeleton and formation of membrane protrusions and ruffles were observed 10-15 min after Ang II treatment - 30] - These examples clearly demonstrate that GPCR stimulation or inhibition alters cell morphology and behavior of cells on a microscopic and also submicroscopic level.

On a molecular level, it is known that GPCR activation translates into changes in the actin cytoskeleton involving different Ga protein - induced pathways and downstream GTPases, such as Rho, Rac, or CDC42 [31-33] . Furthermore, GPCR-induced changes in second messenger levels (cAMP and Ca2+) modulate the cytoskeleton via protein kinase A (PKA) activation or by changing calmodulin activity. It was also shown that stimulation of some GPCRs leads to activation of focal adhesion kinase (FAK) and other well-known mediators of cytoskeletal rearrangement [34, 35]. More recently, it was demonstrated that dissociating GPy subunits can also influence cytoskeletal architecture via direct molecular interactions involving pleckstrin homology (PH) domains. The influence of GPCR-fnduced cytoskeletal modulation in pathophysiological conditions such as cardiac hypertrophy, hypertension, asthma, inflammatory disease, cancer, and neurological disease is well documented and highlights the physiological significance of such measurements in the context of GPCR drug discovery [36].

Although these effects are well described in the literature, it is technically very difficult to obtain a highly accurate quantitative measurement using morphological changes and conventional microscopy. Both the impedance and RWG technologies make use of subtle ligand-induced cellular rearrangements and now provide a technical solution to measure changes in cytoskeletal architecture in a highly accurate and reproducible way. As the input of multiple signals or the activation of parallel signaling pathways induced by a single receptor converges at the cytoskeletal level to induce changes in morphology, an accurate readout provides information on an integrated response and thereby offers the potential to investigate the pharmacological effects of ligands or synthetic GPCR modulators in a physiological context (Fig. 11.1). The following describes findings in the GPCR field using either impedance or RWG, with particular emphasis on the correlation of data with results using established GPCR assay tools, including IC50 or EC50 values, and on partial or biased agonism and inverse agonism. We also examine the information content that is obtained by both technologies in comparison to other assay platforms.

Electric cell-substrate impedance sensing technology (ECIS™, Applied BioPhysics Inc., New York, NY) was originally invented by Giaever and Keese, who presented their first label-free biosensor to monitor cell morphology in 1984 [37, 38]. These morphological changes include cell locomotion and other behaviors directed by the cell'f cytoskeleton. In 1991, Giaever and Keese founded Applied BioPhysics Inc., a company that still commercializes and markets ECIS instruments. The ECIS system makes use of small circular gold electrodes (250 |im diameter) to which the cells adhere and a larger counter electrode. Both electrodes are deposited on the cell culture vessel bottom by a lithographic process. When a small AC (1 | A) is applied, the media electrolytes create an ionic environment that is influenced by the cells adhering to the smaller electrode. The ECIS instruments measure the impedance of the cell-covered electrode, which is determined by the capacitance of the cell membrane, the resistance between adjacent cells caused by junction

Figure 11.2 Principles of impedance and resonance waveguide grating (RWG). (a) Impedance is determined by the ratio of voltage to current as defined by Ohm's law (Z = V/I). Cells are seeded onto a cell culture vessel that contains electrodes at the bottom of the plate, and low voltage at various frequencies is applied. This causes a current to flow either through or around cells. Impedance assay technology is based on the complex impedance (Z) changes (Zs - Z) resulting from stimulation-induced changes in cell shape, cell-cell contact, and cell-substrate interactions, which influence the extracellular and transcellular currents. (b) RWG is the underlying principle that is used to measure changes of dynamic mass redistribution (DMR). Cells are seeded onto a cell culture vessel that contains an optical biosensor (waveguide) at the bottom of the plate. Stimulation-induced changes in subcellular structure localization, referred to as DMR, influence the refractive index of the measured reflected resonant wavelength in an area up to 150 nm distance from the biosensor.

Figure 11.2 Principles of impedance and resonance waveguide grating (RWG). (a) Impedance is determined by the ratio of voltage to current as defined by Ohm's law (Z = V/I). Cells are seeded onto a cell culture vessel that contains electrodes at the bottom of the plate, and low voltage at various frequencies is applied. This causes a current to flow either through or around cells. Impedance assay technology is based on the complex impedance (Z) changes (Zs - Z) resulting from stimulation-induced changes in cell shape, cell-cell contact, and cell-substrate interactions, which influence the extracellular and transcellular currents. (b) RWG is the underlying principle that is used to measure changes of dynamic mass redistribution (DMR). Cells are seeded onto a cell culture vessel that contains an optical biosensor (waveguide) at the bottom of the plate. Stimulation-induced changes in subcellular structure localization, referred to as DMR, influence the refractive index of the measured reflected resonant wavelength in an area up to 150 nm distance from the biosensor.

formation, and the distance between the cell surface and the electrode. Ligand-induced alterations in cell morphology, cell-cell contacts, or cell-substrate interaction affect the flow of extracellular and transcellular current, and this is quantitatively measured as changes in impedance (Fig. 11.2a).

Increased sophistication can be added by using ECIS instruments that are capable of monitoring both the voltage and the phase of the voltage relative to the current. Combining these parameters, the impedance can be broken down into two parts—one due to pure resistance and the other to the reactance of the system resistance and capacitance. These additional features are most useful for cell layer integrity assays. ECIS instruments can be used with a cell wounding module, which allows the creation of a well- defined, circular cell layer wound by a strong electrical pulse and the monitoring of healing through changes in impedance as cells migrate over the gold electrode to occupy the empty space [39]. More recently, other companies, such as ACEA Biosciences/ Roche, Molecular Devices, and Bionas, have introduced their own impedance-based technology products. The various instruments work on the same principle and differ mainly in the geometry and surface size of the electrodes, the magnitude and frequency of the applied AC signal, and additional features such as on-board pipetting capability. Table 11.1 provides more detailed information on this technology platform.

The impedance readout is influenced by changes of cell number, changes in cell-substrate interactions, changes of cell-cell contacts, and subtle changes in individual cell morphology. Therefore, a significant number of different assay designs can be applied to this platform. The sensitivity of impedance measurements can reach the single-cell level, although most experiments are performed with 1000-50,000 cells. The original ECIS instruments have been most widely used to measure extracellular ligand- i nduced changes in endothelial or epithelial layer integrity in immortalized and primary cells t40-42] . Despite the very attractive assay platform that was offered by the ECIS instrument and the ensuing appearance of numerous applications in many areas of cell biology, impedance has not been considered a breakthrough in GPCR research and has not drawn the attention of drug discovery programs in the pharmaceutical industry. One reason for this lack of attention was the unavailability of 96-well assays, which were only recently introduced, and the lack of G protein-specific pathway information. Furthermore, the surface area offered by the ECIS electrodes was apparently not sufficient to generate the required accuracy to perform quantitative pharmacology in the context of drug discovery. A new generation of impedance technology-based instruments, such as CellKey™ (Molecular Devices) and xCELLigence™ (Roche Applied Sciences, Indianapolis, IN), formerly known as RT-CES® (Acea Biosciences, San Diego, CA), provide improved electrode designs, onboard pipetting capabilities (CellKey), standard 96-well or 384-well formats, and improved sensitivity, reproducibility, and throughput of the assay system. However, these features alone have not been sufficient to significantly raise the attention for impedance technology, especially among GPCR-related drug discovery programs.

This has changed with the remarkable finding that impedance measurements can be used to discover G protein-specific patterns of ligand-stimulated GPCRs [43] . The distinct signatures were observed during the first period of data acquisition, that is, during the first few minutes and within a time frame that is known to involve changes in Cat+ release or cAMP levels. In a given cellular background, such as CHO-K1 cells, characteristic patterns of known Gaq-, Gas-, and Gai-coupled receptors were clearly shown, although it must be pointed out that the observed pattern for a given receptor is not necessarily conserved across cell lines. Figure 11.3 illustrates the three prototypic response patterns in CHO-K1 cells that can be observed for recombinant GPCRs, which

Gai (S1P1)

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Gai (S1P1)

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Figure 11.3 Prototypic Ga subunit signatures are detected in impedance measurements (RT-CES/xCELLigence). Stable CHO-K1 cells expressing either recombinant ETA, S1P1; or prostanoid EP4 receptor (EP4) were used to characterize pathway-specific signatures. Cells were stimulated with their cognate ligands (solid lines) or vehicle (dotted line), and impedance changes were monitored during 60 min. Characteristic signatures were identified for Gaq (ETA), Ga- (S1P), and Gas (EP4) protein - mediated signaling.

Figure 11.3 Prototypic Ga subunit signatures are detected in impedance measurements (RT-CES/xCELLigence). Stable CHO-K1 cells expressing either recombinant ETA, S1P1; or prostanoid EP4 receptor (EP4) were used to characterize pathway-specific signatures. Cells were stimulated with their cognate ligands (solid lines) or vehicle (dotted line), and impedance changes were monitored during 60 min. Characteristic signatures were identified for Gaq (ETA), Ga- (S1P), and Gas (EP4) protein - mediated signaling.

are known to signal exclusively through Gaq-, Gas-, or Gai-mediated pathways. It is immediately obvious from this figure that Gaq-coupled responses are characterized by an immediate and transient reduction in impedance, which is followed by a larger increase in impedance and a gradual decline during a period of recovery. In contrast, a prototypic Gai response is characterized by an immediate rise in impedance, which decreases over time, suggesting that it lacks the initial decrease in impedance that is seen in a prototypic response in Gaq-mediated pathways. Gas-induced responses are characterized by a gradual decrease of impedance upon ligand stimulation, which is long lasting and requires several hours for full recovery to baseline. It is noteworthy that such patterns can be observed using CellKey or xCELLigence (RT-CES) equipment, provided that measurements are started immediately after ligand addition and that the interval of data collection in each well is maximally reduced. These findings also imply that the assay temperature, usually room temperature or 37°C, does not critically influence the response pattern and that the number of frequencies that is used to apply the AC current can be reduced without significant loss of information.

Most published protocols describe a change from cell growth medium to buffer followed by an equilibration period of 15min just prior to ligand or compound addition. However, recent experiments show that this may not be required in all cases, and comparable dose response curves can be established without any medium change before the onset of the experiment (J. Gatfield, unpublished observation). This observation suggests that these systems can be used in conditions that closely resemble standard cell growth conditions. This can be considered another significant advantage of this technology compared to other assay platforms, especially when using primary cells.

Subsequent reports further substantiated these initial findings and demonstrated pathway-specific signatures in various cellular backgrounds using recombinant systems [44]. However, it should be again emphasized that signatures for a given receptor coupling do not necessarily translate from one cell type to another, although they likely remain constant in a given cell background. Based on the initial data using GPCR signatures, impedance technology was applied to identify endogenously expressed receptors in several cell lines using receptor-panning experiments, and pathway analysis was performed to successfully identify particular receptor subtypes that were known to have different G protein coupling.

In the U2OS cell line, receptor-panning experiments showed a variety of functionally expressed GPCRs, including muscarinic, prostanoid, thrombin, and histamine receptors. A careful analysis of the receptor signature of the histamine-stimulated response then identified a Gaq-coupled process, and this correctly pointed to the histamine receptor subtype 1, as it is the only known Gaq- coupled histamine receptor subtype [ 45]. This study not only demonstrated the successful use of impedance technology to perform GPCR receptor subtype identification based on pathway signatures, but also demonstrated the use of this technology to perform extensive GPCR receptor panning in a multitude of cell lines in parallel. Previously, such data were laboriously acquired using quantitative polymerase chain reaction (Q-PCR) analysis combined with functional analysis. It is therefore conceivable that impedance technology will not only help to profile cell lines of interest for the functional expression of GPCRs, but could also be integrated in approaches to deorpha-nize GPCRs, which often rely on the use of promiscuous or chimeric G proteins in addition to the recombinant GPCR of interest to make the cells amenable for conventional high-throughput assay formats. The significant advantage of measuring pathway-specific signatures of GPCRs in recombinant cells or in cells expressing the endogenous receptor opens the field to perform pharmacological studies in a noninvasive fashion and with limited assay development time.

As many GPCRs are known to mediate signals through activation of multiple G proteins simultaneously, impedance technology is also predisposed as a tool to dissect the individual coupling pathways. As an example, Fig. 11.4 shows the activation profile of the endogenous bradykinin B2 (BKB2) receptor in the human epithelial carcinoma cell line A431 treated with 10nM BK

No inhibition

No inhibition

0 10 20 30 40 50 60 Time (min)

Adenylate cyclase inhibition 1 mM SQ22536

Ca2+ sequestration 50 |M BAPTA

Ca2+ sequestration 50 |M BAPTA

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Figure 11.4 Impedance technology can be used to dissect coupling to multiple pathways (RT-CES/xCELLigence). A431 cells endogenously expressing the BKB2 receptor were triggered at the indicated time point (arrow) with 10 nM BK (solid line) or vehicle (dotted line), giving rise to a specific biphasic response (top left panel). Pretreatment with 1 mM SQ22536 (adenylate cycle inhibitor, top right panel) or 50 |M BAPTA-AM (Ca2+ chelator, bottom panel) specifically blocked either the second phase or the first phase of the response, allowing a clear assignment of Gaq and Gas coupling to the two response phases.

Time (min)

Figure 11.4 Impedance technology can be used to dissect coupling to multiple pathways (RT-CES/xCELLigence). A431 cells endogenously expressing the BKB2 receptor were triggered at the indicated time point (arrow) with 10 nM BK (solid line) or vehicle (dotted line), giving rise to a specific biphasic response (top left panel). Pretreatment with 1 mM SQ22536 (adenylate cycle inhibitor, top right panel) or 50 |M BAPTA-AM (Ca2+ chelator, bottom panel) specifically blocked either the second phase or the first phase of the response, allowing a clear assignment of Gaq and Gas coupling to the two response phases.

(solid line) or vehicle (dotted line). Using conventional assays, it was shown that the activated BKB2 receptor couples through Gaq and Gas proteins in A431 cells [46]. In the impedance assay, a biphasic response to BK is observed, consisting of a rapid but transient decrease in cell index, followed by an increase of the cell index to a stable plateau. Pretreatment of the cells with either the adenylate cyclase inhibitor SQ22536, to block the Gas pathway, or the calcium chelator BAPTA-AM, to block the Gaq pathway, specifically abolishes either the plateau phase of the response or the initial transient decrease. Impedance technology, in combination with selective pathway inhibitors, is therefore a useful tool to separate and clearly assign the Gas- and Gaq-mediated coupling of the endogenous BKB2 receptor in A431 cells. It is also interesting to note that the Gas signature induced by the endogenous

BKB2 receptor in A431 cells differs from the prototypical Gas pattern observed in CHO-K1 cells (see above), which again highlights the cell background dependence for specific GPCR signaling pathway signatures. In extrapolation, the unique feature of an integrated readout of multiplexed coupling allows a characterization of GPCR agonists and antagonists on recombinant and nonrecombinant cells with respect to pathway bias, a beneficial feature that is presently of large interest to the pharmaceutical industry.

Since impedance technology provides information on an integrated cell response upon ligand stimulation, it is of critical importance to compare the pharmacological parameters of cognate ligands and synthetic GPCR modulators measured by impedance assays with measurements using existing in vitro assays. A recent report describes three examples of Gai-coupled GPCRs to evaluate impedance measurements for intra- experimental and inter- experimental precision, and the reproducibility of a known structure—activity relationships [ 47] . Agonists and antagonists spanning a range of potencies and efficacies were used, and compounds displaying positive allosteric modulator activity were included in this study. The investigators chose Gai-coupled receptors because they pose a particular problem in drug discovery, as the relevant functional cellular assay that measures cAMP levels involves the combined use of forskolin and test compound, which often leads to lack of assay precision. Using the dopamine D2 receptor expressed in CHO-K1 cells, they found a generally good agreement between impedance measurements and preexisting cAMP values of synthetic agonists, including efficacy and potency values. The M4 muscarinic receptor was used to investigate three known positive allosteric modulators, and they found a conserved rank order between these compounds in impedance and GTP.S assays with a greater dynamic range in impedance. Other studies on a wider range of GPCR coupling types, including Gaq- and Gas-coupled receptors, suggest that this technology delivers values for natural ligands and synthetic compounds that are highly comparable to existing technologies and highlight the possibility to measure inverse agonism in a real-time and in a quantitative manner [48] . The described z values for impedance measurements are usually above 0.5, which is indicative of a robust assay with the potential to be used in screening campaigns.

Peters et al. also investigated the pharmacological responses of an endogenous GPCR with low expression levels in CHO-K1 cells [47]. The expression level was insufficient to establish a robust assay using traditional assays. Even under these difficult conditions, impedance technology was successfully used to establish a robust assay and to measure compound potency. Thus, impedance technology was clearly superior in measuring endogenous receptor activity under a situation of low receptor abundance. It should be noted, however, that an accurate evaluation of experimental compounds requires well-characterized tools (agonist or antagonists) to fully attribute the response to the receptor of interest. However, provided that these tools are available, impedance technology has a significant potential to be used in compound characterization in physiologically relevant cells, even with low GPCR expression levels, not only in immortalized cell lines but also in primary cells from healthy or diseased conditions. There are also ongoing efforts to combine impedance electrodes with additional pH and oxygen sensors in a single well (Bionas, Rostock, Germany); however, this is still done in a low throughput format, and a change of buffer prior to the experiment is mandatory. Nevertheless, it will be interesting to see if these combined measurements allow an even more detailed pharmacological characterization of synthetic compounds, especially in the context of partial or biased agonists.

Summarizing, there is sufficient evidence to show that impedance measurements are a useful addition to the repertoire of existing GPCR assay tools, with the possibility to investigate receptor pharmacology and compound characteristics in physiologically relevant cell systems. Based on the current literature, impedance technology does not suffer from the known problems using microphysiometry, thus offering higher throughput and increased reproduc-ibility. Clearly, it is a significant advantage that impedance technology does not involve prelabeling or postlabeling of cells, that it can be performed without changes of the cell growth medium, and that it provides real-time data to allow acquisition of highly reproducible and accurate quantitative data. In this context, impedance technology can be applied in drug discovery settings that are aimed at compound characterization in secondary assays using physiologically relevant target cells.

One of the problems that must be addressed to fully exploit this technology in high-throughput screens is the theoretical difficulty to detect false positives due to activation of nontarget-related receptors, which is a particular concern if agonists are sought. However, the technology will be very useful to identify cytotoxic compounds in a high-throughput screen without the need for additional assays. The coming years will show how this new technology will fully establish itself, although it is already evident today that impedance technology became a valuable tool in many research facilities, especially those with a particular focus on GPCR pharmacology.

Almost concomitant with the appearance of impedance technology in GPCR signaling research, RWG became known as a novel and interesting technology to examine cell signaling in a noninvasive, real-lime, label-free, and high-throughput format [49, 50]. Similar to impedance technology, RWG makes use of ligand-induced changes in cellular composition; however, instead of measuring changes in cell shape, cell-substrate, or cell-cell interaction, RWG makes use of the process of dynamic redistribution of cellular content, also referred to as dynamic mass redistribution (DMR). DMR is measured with an optical biosensor that is integrated into each well of the cell culture vessel, and it uses a microplate reader. Cells are grown on the biosensor, and the microplate is illuminated with broadband light at 830nm. Measurements are made of the reflected light, which is sensitive to changes in refractive index caused by the cells and their content at a distance of up to 150nm from the biosensor surface. DMR is determined as a result of GPCR stimulation by its ligand or synthetic compounds (Fig. 11.2b). Based on this principle, RWG measures integrated and converging signaling events, similar to impedance technology. It is therefore not surprising that RWG shares many characteristics with impedance technology. Currently, the best known system that is based on the RWG principle is the EPIC® System (Corning, Lowell, MA), which features a 384-well format, and because it is compatible with standard pipetting robotics equipment, it is suitable for large-scale HTS campaigns. Similar instruments, such as BIND® Reader (SRU Biosystems, Woburn, MA) or Owls (Microvacuum, Budapest, Hungary), are available from other providers.

Using the DMR readout, GPCR activation revealed a G protein-dependent response pattern in a given cell background [50], and analysis of the prototypical response curves showed a rapid response onset with a peak at 10 min after stimulation. The kinetics are therefore slower than those measured in impedance assays, and the shape of the characteristic response curves for Gaq-, Gai-, or Gas-mediated responses is quite different from impedance curves. Based on a most recent publication, it was, however, not possible to distinguish a Gaq- from a Gai-mediated response in CHO-K1 cells that expressed two known recombinant GPCRs (dopamine D3 or muscarinic M1 receptor). To resolve the issue, forskolin was used to correctly identify the Gai proteinmediated coupling [51] . In the same publication, a small collection of compounds was used to examine the pharmacological behavior of known GPCR modulators, and the measured IC50 and EC50 values were compared with data derived from conventional assay platforms. In most cases, especially those using antagonists, the measured DMR responses were of comparable magnitude and potency. However, there were some exceptions that highlight the potential problems and opportunities in using an integrated readout. The agonist potencies measured in DMR assays in the dopamine D3 receptor-expressing cells were slightly lower than in cAMP assays, with the exception of the selective synthetic agonist PD 128907, which showed a more than 1000-iold shift to lower potency in the DMR assays compared to the cAMP assay. It was speculated that the discrepancy could be caused by unknown activation pathways that desensitize or inhibit the D3 receptor-mediated response induced by PD 128907. This clearly shows that results from DMR measurements, or results from any other label-iree technology using integrated end point signals, need careful analysis to avoid misleading conclusions.

The potential of DMR assays to measure GPCR responses in cells expressing endogenous receptors was explored in A431 cells using a selection of well-known p2-adrenergic receptor modulators [52]. In this study, a multiparameter analysis revealed distinct receptor activation patterns that were induced by different agonists, demonstrating the sensitivity and utility of DMR assays to investigate pharmacological characteristics of compounds. In this context, DMR will be particularly useful in screens that are designed to identify pathway-biased compounds, provided that the required signature is well known and sufficiently correlates to a desired pharmacological output. The results of a full-scale high-throughput screen using the EPIC System recently became available [53], and they clearly demonstrate the applicability of this technology in this specialized area. The reported data indicate good z values >0.6 and an acceptable false positive rate.

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