Examples of Applications

In cases where the activation of one specific signaling cascade is under investigation, a synthetic promoter containing the respective response element may be the best approach. Multiple copies of the response element are usually coupled to enhance the regulatory effect. A whole variety of promoter elements that are responsive to the activation of different second messenger pathways are available. One such response element is TRE (tissue plasminogen activator (TPA) responsive element), which is activated by the signal transduction pathways mediated by PKC.45 GPCRs coupling to Gai and GaS can be investigated by CREs (using cAMP responsive elements),46,47 which bind CRE-binding (CREB) protein phosphorylated by PKA.35 Those coupling to Gaq signal through calcium, which activates the NFAT response element.48 Genetic engineering of a luciferase reporter cell line under the control of multiple copies of the hypoxia-responsive element (HRE) allow one to investigate the totally different mechanism of regulation of the response to hypoxia and to search for appropriate inhibitors.49,50 This mechanism is involved in the regulation of angiogenesis and anaerobic metabolism. Alternatively, natural promoter sequences can be used. The c-fos51 promoter contains several different responsive elements, which are regulated by various signaling pathways and hence can reflect the activity of different classes of receptors like GPCRs (histamine receptor, gastrin-releasing peptide receptor, unpublished results) and growth factor receptors (EGF, HGF, unpublished results). We have also successfully used the ICAM-1 receptor, which confers response to several signaling pathways,52,53 for the construction of reporter gene assays for different GPCRs, e.g. NK2 and 5-HT2.54 Reporter gene assays can also be a tool in the elucidation of virus host interactions and the identification of novel drugs for intervention. Reporter gene constructs carrying viral IRES (internal ribosome-entry site) sequences, such as that derived from the hepatitis C virus (HCV), allow the regulation of virus translation to be examined in host cells and provide the base for an assay to screen for virus-specific translation inhibitors.40 Engineering reporter genes into virus replicons of MNV41 or HCV55 generate powerful screening assays to identify specific inhibitors of virus replication. The technology is also applied to identify modulators of orphan GPCRs.56 An artificial multiple regulatory element containing the major response elements, through which different G-proteins can signal, allows a reporter assay to be set up without knowledge of the coupling mechanism of the orphan GPCR.57 The examples given above demonstrate the scope of applications of reporter gene assays in drug discovery, covering the whole process from gene to function, target identification and validation to lead identification, which includes primary HTS. Given the large number of compounds screened per target, which typically lies in the range of 600,000 to a million, the prerequisites for HTS

suitability are easy automation in a high-throughput mode and miniaturization to reduce reagent consumption and cell-culturing efforts. Since reporter gene assays, like those based on luciferase, require only one reagent addition step prior to measurement, they are readily amenable to automation. Miniaturization to 384-well plates and even 1536-well plates and beyond have been possible,57,58 but assay noise and variability due to edge effects, uneven distribution of cells and sensitivity of expression levels toward cell density and culture conditions are specific hurdles to be surmounted with cellular assays. Highly standardized cell-culturing procedures and suitable equipment on the automated screening system are required to cope with this challenge. Since the P-lacta-mase reporter gene delivers a ratiometric measurement that normalizes the result to the number of viable cells, this reporter gene seems to be specifically suited for HTS. One recent approach to obtain a unique stock of cells of uniform quality for HTS is to combine the BacMam technology with frozen stocks of cells for plating. This approach makes it possible to prepare cells in bulk and store them for up to 6 months before the start of HTS. Cell supply and HTS are thus uncoupled and assay quality is improved as a result (oral presentations at SBS conference 2004, Orlando by D. Finnigan, GlaxoSmithKline, and C. Cowan, Norak Biosciences). However, the examples referred mostly to cell lines measured by the fluorescent imaging plate reader (FLIPR) platform (Molecular Devices). The strategy is unlikely to be applicable to all kinds of cell lines, but may be an attractive approach in a number of cases.

One important issue with respect to the development of cell-based assays is the fact that cells display a multiplicity of targets in addition to the target of interest. This potential for additional interactions has to be considered specifically when using cellular assays in HTS to identify new hits. As the majority of compounds in a compound collection cannot be expected to be selective for the target of interest, many of them will hit additional targets within the monitored signaling cascade. These off-target interactions will affect the test results to different degrees and will usually lead to an increased number of false positive hits. It is therefore necessary to profile identified hits in another independent assay format to verify whether the compound has the desired mode of action. Reporter gene assays can be designed to monitor a gain or a loss of signal. Compounds interacting with molecules within the cell, which induce apoptosis, necrosis, or lead to reduced growth will reduce the measured signal as well. They will therefore show up as hits in assays with a loss of signal readout. As a result, significantly higher hit rates are found for assays with a readout involving reduction in the signal, as compared to assays that monitor a gain of signal (see Table 1). The effect becomes stronger with increasing incubation time and may already be obvious as early as 1-2 h of incubation. Generally speaking, assays with fast readouts, in the range of seconds or minutes, e.g. Ca2+ release, suffer less from this problem. In the P-lactamase reporter assay, interference from cytotoxic substances should also be reduced, since the results are normalized to the number of viable cells. There are several other strategies to reduce the number of false positive hits.

One approach is to test the compound in a control cell line in parallel. Ideally, this involves the same cell line in which the reporter gene is induced differently. Another option is to use a different cell line of the same origin as that of the target. It should express the same reporter gene driven by the same signaling cascade but involve another receptor, in case of GPCRs, or be driven by a promoter not regulated by the signaling cascade of interest. If two different cell lines are used, it is important that

Table 1 Primary hit rate of different luciferase reporter assays (los: loss of signal; gos: gain of signal) at the given hit criterion

Assay Type Incubation time Compounds tested Hit criterion Hit rate (%)

Table 1 Primary hit rate of different luciferase reporter assays (los: loss of signal; gos: gain of signal) at the given hit criterion

Assay Type Incubation time Compounds tested Hit criterion Hit rate (%)

(hours)

Translational

los

24

422100

<50%

17

regulation

Signaling

los

10

480959

<50%

11.6

Growth factor

los

5

254767

<50%

11

receptor

GPCR

los

5

460047

<50%

7.4

GPCR

gos

12

565259

>200%

0.04

both show similar sensitivity. Another strategy is to run a cytotoxicity assay in parallel on the target cell line and simply eliminate cytotoxic hits.

Was this article helpful?

0 0

Post a comment