Second Messenger Assays

cAMP The concept of transmembrane signaling via GPCRs, which transduces extracellular signals into intracellular messages, was established with the discovery of cAMP by Sutherland et al. [35]. Specifically, binding of an agonist to a GPCR could either increase (via coupling through Gas) or decrease (via coupling through Gai) the rate at which cAMP is generated in cells through activation or inhibition of adenylyl cyclase, respectively [7]. There are a variety of assays designed to directly measure levels of cAMP. The original cAMP assays almost 50 years ago consisted of radiolabeling of adenine nucleotides, incubating with hormones, followed by separation on Dowex columns (Sigma-Aldrich, St. Louis, MO). The current cAMP detection methodologies have come a long way in offering a wide selection of assay types, detection, and plate formats suitable for all modes including uHTS. The currently popular cell-based cAMP assays used as a measure of GPCR activation are all based on competition between endogenously produced cellular cAMP and exoge-nously added labeled cAMP for interaction with anti-cAMP antibodies using a variety of detection technologies [36, 37].

The Flashplate (PerkinElmer) assay is a modification of the original radio-immunoassay using cAMP antibodies and radiolabeled cAMP in a classical enzyme-.inked immunosorbant assay (ELISA), converted into a plate mode with scintillant coated wells of the assay plate for implementation in HTS laboratories. While successfully used in HTS, this assay type has limitations in further miniaturization beyond the 384-well plate format, in addition to the disadvantages of using large quantities of radioactivity for HTS.

The HTRF (Cisbio, France) and Lance (PerkinElmer) assays are based on time-resolved FRET (TR-FRET) between a labeled antibody to cAMP and a labeled cAMP molecule. The Lance cAMP technology utilizes energy trans fer from europium to an acceptor like Alexa Fluor 647. This principle has been utilized in a competitive binding assay to measure cellular cAMP. The signal in this assay is inversely proportional to the amount of cAMP. TR- FRET assays exhibit relatively low background and high signal/background, and are thereby suitable for HTS. The HTRF assay is similar to the TR-FRET and measures competition between biotinylated labeled cAMP and endogenous cAMP using ratiometric detection of fluorescence at 665nm and 620 nm.

The enzyme fragment complementation technology uses the principle of complementation between an inactive fragment (enzyme donor, ED) tagged with cAMP and a larger complimentary portion of the P - galactosidase enzyme. Competition between endogenous cAMP and ED-labeled cAMP for binding to an anti- cAMP antibody determines the amount of the reconstituted P -galactosidase active enzyme generated, which is measured by substrate conversion into a fluorescent or luminescent product. This technology has successfully been miniaturized into 1536-well and 3456-well plate formats for uHTS for both Gas and Gai-coupled GPCRs [38, 39] . In addition, the availability of the assay technology in luminescence mode offers the opportunity to overcome fluorescence interference often encountered during HTS of large sample collections.

Another technology to measure cellular cAMP is the cAMP-Glo™ assay from Promega (Madison, WI), which is a homogeneous, bioluminescent assay suitable for uHTS in 3456-well plate format. This assay is based on the activation of PKA by cAMP, thereby decreasing the cellular adenosine triphosphate (ATP), and consequent decrease in light production in a coupled luciferase reaction.

Other detection modalities for measuring cAMP generation as a means of monitoring GPCR activation include FP technology, electrochemilumines-cence technology (ECL) by Meso Scale Discovery (Gaithersburg, MD), and AlphaScreen technology by PerkinElmer using beads coated with anti- cAMP antibodies. The latter two technologies, albeit being HTS compatible, require a specific detector for the measurements.

Given these wide varieties of cAMP assays currently available to study GPCRs in HTS, the selection of an assay type for use in general laboratory or in HTS mode should be made with care based on assay sensitivity, dynamic range, the use of whole cell or membrane assays, and so on. Some other critical considerations are the use of the assay for Gas-coupled versus Gai-coupled receptors (Gai-coupled receptors are typically more challenging to analyze), detection of agonists or antagonists, and the need for pharmacological agents like forskolin for Gai-coupled receptors (to increase basal cAMP levels, allowing visualization of the actions of a Gi- coupled GPCR agonist, which will decrease basal cAMP). Among these different scenarios for measuring cellular cAMP downstream of GPCR activation, detection of antagonists of a Ga i -coupled GPCR is by far the most challenging for uHTS, with the need for titrating the optimal amount of both forskolin and agonist stimulation that would allow for a sufficient assay window (signal/basal) while maintaining

Treatment
Rank order of potency of antagonists of a Gi-coupled GPCR in HitHunter cAMP assay in 3456-well uHTS plate format

300-

EC/IC50

S/N

250-

5.7 ^M 138 nM

17.1 2.6

200-

Agonist ~T

Folskolin **]!// SV Agonist

RLU

iL! j/»?/T Antag 1 ft he/ • Antag 2

7.8 nM 10.5 nM

2.8 2.6

100-

Antag 3

7.6 nM

2.9

50-

i * Antag 4

217 nM

2.6

-13-12-11-10 -9 -8 -7 -6 -5 -4 • Log (compound), M

-13-12-11-10 -9 -8 -7 -6 -5 -4 • Log (compound), M

3456-well screening plate for Gi antagonist assay

Basal

175

150

on

125

100

75

c

50

ss

25 0

-25

-50

3x stdev kolin + agonist

Forskolin

0 5 10152025303540455055606570 75 Column no.

3x stdev kolin + agonist

Forskolin

Basal

Figure 13.5 cAMP assays for Gai-coupled GPCRs in 3456-well plate format for uHTS.

suitable assay sensitivity (Fig. 13.5). Some cAMP technologies are being used routinely in this complex setting for uHTS in 3456-well plate format [39].

Calcium Upon receptor activation, GPCRs known to couple to the Gaq class of G proteins result in the activation of phospholipase CP , with inositol phosphates (InsP) turnover at the plasma membrane, followed by an increase in intracellular calcium (i[Ca2+]) [40-42]. The transient increase in intracellular calcium from internal stores, like the endoplasmic reticulum, can be detected by fluorescent dyes sensitive to Ca2+ ions, bioluminescent photoproteins like aequorin, or Photina (PerkinElmer), as well as reporter genes via the NFAT transcription factor (see previous section on BLA reporter gene).

Bioluminescent photoproteins, such as aequorin, can be used as a reporter for GPCR-mediated Ca2+ signaling, as an alternate to direct Ca,+ measurements ,43] , Although aequorin is an indirect, albeit sensitive, "reporter" of i[Ca2+] and is dependent on transcription and translation (often transient) of the apoaequorin gene, this technology differs from the classical reporter gene technology in that the reporter gene transcription/translation does not depend on cellular activation or the signaling pathway of interest.

Measurements of intracellular Ca2+ with fluorescent Ca2+ dyes upon GPCR activation has been a popular method for studying Gq-coupled GPCRs for HTS since the identification of FURA-2 [44]. More recently, newer generation

A = EC-ioo agonist B = EC50 agonist C = DMSO D = Basal

A = EC-ioo agonist B = EC50 agonist C = DMSO D = Basal

4.0-1

3.5-

ni

F

3.0-

*

E

2.5-

=3 2.0-

rr

1.5-

1.0-

EC50

S/B

■ Agonist 1

5.0 pM

3.2

♦ Agonist 2

1.9 pM

2.8

Column no.

Figure 13.6 Second messenger Ca2+ screen in 1536-well plate format for Gaq-coupled GPCR.

calcium indicators like Fluo4 offer greater sensitivity and larger changes in fluorescence intensity upon calcium binding and are widely used in HTS. In the past few years, this methodology has been miniaturized into a 1536-well screening format with the development of CCD-based plate imaging detectors like the fluorescent imaging plate reader (FLIPR, Molecular Devices, Sunnyvale, CA) (Fig. 13.6). In addition, fluorescent dye-based calcium assays have also evolved into homogenous formats with "no-wash" dye protocols (primarily aimed at reducing the background fluorescence in the wells) for further adaptability in uHTS [45].

In addition to being a common methodology for studying Gaq-coupled GPCRs, the measurement of intracellular Ca2+ has also been utilized for numerous orphan GPCRs with unknown second messenger signaling through the use of chimeric (Gqi5) and/or promiscuous G proteins like Ga15 and Ga16 signaling [31, 46].

Inositol Phosphates GPCR signaling through Gaq, Gao, or GPy activate phospholipase CP, which hydrolyzes phosphatidyl-inositol-4,5-bisphosphate (PIP2) to diacylglycerol and inositol 1,4,5 - triphosphate [ 47] . InsP3 binds to calcium channels on the endoplasmic reticulum, causing a release of the internal Ca-+ stores within the cell. Unlike assays to measure intracellular Ca-+, few assays exist to measure InsP3 that are suitable for automated HTS, in part due to its inherently short half-.ife. The HitHunter InsP3 assay (DiscoveRx, Fremont, CA) is a competitive binding assay wherein cellular InsP3 displaces a fluorescent derivative of InsP3 from a binding protein. This assay is based on the principles of FP. An alternative HTRF assay from CisBio is based on the measurement of IP1, a downstream metabolite of IP3, which accumulates in cells following Gaq receptor activation and is stable in the presence of LiCl, thus allowing a suitable format for a functional HTS assay. The HTRF assay for measuring IP1 is relatively new but potentially applicable even for uHTS in 1536-well plate formats.

Receptor Trafficking/Translocation Assays Recently, cell imaging assays monitoring GPCR trafficking have become increasingly popular. GPCR trafficking assays are independent of receptor signaling and are thus ideally suited for orphan receptors. In addition, these assays provide a valuable measure of receptor desensitization, an important feature for the use of GPCR agonists as potential therapeutic agents. The most popular GPCR imaging assays are based on the principles of receptor desensitization and internalization monitored directly or indirectly by GFP [28].

Elucidation of the mechanism of regulation of GPCR function by receptor desensitization in the 1990s laid the foundation for the receptor internaliza-tion/trafficking assay. The approach is unique, being independent of the second messenger signaling modulated by the receptor-ligand interaction. GPCR desensitization (waning of the receptor responsiveness with time) is mediated primarily by two protein families: the GRKs and the arrestins [48] . Agonist stimulation of GPCRs promotes the phosphorylation of serine/threonine residues located predominantly in the carboxyl-terminal tail and/or the third intracellular loop of the receptor by the family of GRKs. The activated, phos-phorylated GPCRs are a substrate for the arrestin family of proteins, which translocate from the cytoplasm to the receptors at the plasma membrane. Arrestin binding to the activated and GRK-phosphorylated receptors effectively uncouples the receptor-G protein interaction, thereby terminating receptor signaling [49] .

In addition to offering a novel mechanism to study GPCR activation, P -arrestin/GPCR interaction appears to be a distinct signaling cascade independent of classic G protein signaling. Recent publications have shown that a "ligand bias" may channel a compound to preferentially block the P-arrestin signaling cascade over the classic G protein-evoked second messenger signaling cascade [50].

The arrestin proteins bound to the activated and phosphorylated receptor subsequently target GPCRs for endocytosis via clathrin-coated pits [51] . The movement of activated GPCRs from the plasma membrane into either pits or vesicles can be monitored as an indication of receptor activation and forms the basis of a functional cell-based assay that employs a fluorescent microscope. Such methodology is also amenable for HTS/uHTS with the advent of automated microscopy and is now being used in high-content screening (HCS) in HTS laboratories. These HCS assays are typically conducted in 384-well plate format, although advances in cellular imaging detectors have enabled miniaturization into 1536-well plate format with readers like the MDC Ultra (Molecular Devices, Sunnyvale, CA), Evotec Opera (PerkinElmer), and so on. An advantage to microscopy-based technologies like Transfluor™ (Molecular Devices) is their ability to analyze potential toxicity associated with screening compounds, as the technology is essentially based on visual inspection of cellular morphology. The use of specific algorithms to monitor cell shape and size, nuclear shape and size, and so on can be used to infer compound toxicity [52] (Fig. 13.7).

Among the different GPCR functional cell-based assays currently available, the receptor trafficking assay offers value as a receptor "proximal"

■ % inhibition, BAM15 477 nM(IC5o) * % Inhibition, SP

Log (antagonist), M

Figure 13.7 High-content cell imaging GPCR trafficking assay measuring receptor specificity and toxicity.

Log (antagonist), M

Figure 13.7 High-content cell imaging GPCR trafficking assay measuring receptor specificity and toxicity.

readout (unlike the reporter gene assays) and is particularly advantageous for receptors that lack a suitable radioligand for use in receptor binding assays. In addition, trafficking assays can be used for most GPCRs without prior knowledge of the agonist-induced signaling cascade. Another advantage of this functional assay is the potential to identify different categories of ligands: classical competitive antagonists (also identified by the receptor binding assay) as well as allosteric modulators (i.e., compounds that do not inhibit the binding of a radioligand to the receptor) that presumably inhibit receptor function by binding to sites on the receptor distinct from the agonist binding pocket.

The most direct method to monitor GPCR trafficking in cells is to express the GPCR of interest recombinantly as a chimera with GFP in the carboxyl-terminal tail and monitor the fluorescence localization in the cell upon receptor activation. The disadvantage of this method is the use of a recombinant fusion receptor, albeit at the C-tail [52].

An indirect method to measure GPCR activation using localization of an arrestin-GFP chimera was initially developed by Norak Biosciences (Morrisville, NC) and commercialized by MDC (Molecular Devices) as the Transfluor assay. The primary advantage of this assay over the direct GFP labeling of the receptor is the expression of native unaltered receptors in the recombinant cells. In this assay, the arrestin-GFP fluorescence is localized in the cytoplasm as a diffuse signal when receptors are inactive at the plasma membrane. Upon receptor activation, the arrestin- GFP first translocates to the activated receptors at the plasma membrane and is then subsequently internalized into either small pits near the plasma membrane (as occurs with the MRG-X1 receptor) [52] or larger vesicles (as occurs with the NK-1 receptor) [52] . Thus, the assay is based on the affinity of the arrestin for the activated and phosphorylated receptor [53] (Fig. 13.7). An advantage of the Transfluor assay is that it does not require manipulation of the receptor sequence. Several GPCR screening campaigns have been conducted with this indirect GFP tracking mode [52].

P-arrestin translocation to the activated receptor can also be measured by the reporter gene assay technology, TANGO (Fig. 13.2). The TANGO assay system from Invitrogen (Madison, WI) is a cell- based assay technology for monitoring protein-protein interaction and is applicable to study GPCRs based on the interaction of P-arrestin with the activated receptor. In this assay, the GPCR of interest is expressed as a recombinant fusion protein expressing the tetracyclin- controlled transactivator (tTA) in the C- t erminal tail of the receptor with a protease consensus sequence at the intersection of the C- t erminal tail of the GPCR and the tTA site. The P - arrestin molecule is expressed as a fusion with a protease. Upon GPCR activation, P - arrestin binding to the receptor brings the protease in close proximity to the protease cleavage site, thereby cleaving and releasing the tTA transcription factor, which translocates to the nucleus and transcribes the downstream reporter gene (luciferase or BLA). The TANGO reporter gene assay is suitable for HTS and uHTS.

P2-AR TANGO assay

5000 4000 5 3000 : 2000 1000

P2-AR TANGO assay

5000 4000 5 3000 : 2000 1000

EC50 S/B

■ Ritodrine No Response Fenoterol 22 nM 127 Metaproterenol 142 nM 18 Procaterol 17 nM 104

■ Terbutaline 270 nM 15

■ Isoproterenol 27 nM 165

EC50 S/B

■ Ritodrine No Response Fenoterol 22 nM 127 Metaproterenol 142 nM 18 Procaterol 17 nM 104

■ Terbutaline 270 nM 15

■ Isoproterenol 27 nM 165

ß2-AR PathHunter

ß2-AR PathHunter

-11 -10 -9 -8 -7 -6 -5 -4 Log (agonists), M

EC50

S/B

Ritodrine

No

Response

Fenoterol

38 nM

7.5

Metaproterenol

1.1

3.1

Procaterol

73 nM

7.3

Terbutaline

1.5

3.4

Isoproterenol

100 nM

14.2

ß2-AR (HitHunter) cAMP assay

ß2-AR (HitHunter) cAMP assay

-13 -12 -11 -10 -9-8-7-6-5-4 Log (agonists), M

EC50

S/B

No

Response

Ritodrine

1.5 nM

3.4

Fenoterol

30 pM

3.5

Metaproterenol

1.3 nM

3.1

Procaterol

26 pM

3.9

Terbutaline

40 pM

4.3

Isoproterenol

300 nM

4.2

Figure 13.8 Rank order of potency of p2-adrenergic receptor ligands in TANGO, PathHunter, and HitHunter cAMP assays in 3456-well plate format.

The PathHunter P-arrestin assay from DiscoveRx is based on the principles of enzyme fragment complementation (similar to the cAMP assay). The target GPCR is expressed as a recombinant fusion protein with a fragment of the P -galactosidase enzyme and is coexpressed with a P-arrestin fusion protein containing the complementary portion of the P-galactosidase enzyme. GPCR activation and consequent translocation of P-arrestin to the cell membrane results in complementation of the two parts of the P - galactosidase enzyme to regenerate active enzyme capable of cleaving a chemiluminescent substrate that can be detected as light emission. The amount of light produced in this system is directly correlated to the amount of the receptor that is activated.

Both the TANGO and the PathHunter assays offer limited off-target activity during HTS, since they both utilize recombinant tagged receptors; moreover, both approaches are applicable for miniaturized screening in 3456-well plate formats (Fig. 13.8). It is important to bear in mind that comparing different GPCR assay technologies may reveal differences in compound sensitivities (IC50 or EC50 values). This may be due, in part, to the inherent differences in assay sensitivities or the differences in assay conditions (preincubation of compounds vs. coincubation), buffer and serum concentrations, incubation time and temperature, presence or absence of washes, and so on (Table 13.3).

0 0

Post a comment