Functional Assays

While the past success of the receptor binding assays cannot be understated, advances in assay methodologies that measure GPCR function have begun to circumvent limitations of the ligand binding assay, such as the inability to specifically screen for agonists versus antagonists, the inability to identify allosteric modulators and potentiators, or the requirement of a high-affinity labeled ligand. The use of functional assays for primary HTS in place of traditional receptor binding assays has been a paradigm shift in the pharmaceutical industry. In this scenario, functional information on the activity of newly identified structural classes of compounds is made available early on in the lead identification and lead optimization process, while the more traditional receptor binding assays with lower throughput are used as secondary assays for follow-up, with smaller subsets of compounds to confirm specific interaction with the target GPCR of interest.

With the current trend in the pharmaceutical industry focusing on assays that measure downstream effects of receptor activation, a wide variety of second messenger and reporter gene assays have been developed and made commercially available for HTS. These functional biochemical and/or cell-based assays are exploited as appropriate for HTS and uHTS as described in the following sections.

The categories of GPCR functional assays are as follows:

1. assays to measure G protein activation,

2. indirect downstream measure of GPCR activation by reporter gene assays,

3. second messenger assays indicative of effector activation,

4. receptor regulation/trafficking assays, and

5. other downstream signaling assays.

GTPyS Functional Biochemical Assay Activation of a GPCR by an agonist stabilizes a conformational change in the receptor, resulting in enhanced interaction between the GPCR and the Ga subunit of the hetero-trimeric G protein. Activation of the relevant G protein by the GPCR results in guanine nucleotide exchange on the a subunit (exchange of guanosine diphosphate [GDP] for guanosine triphosphate [GTP]) [7], which can be detected by measuring the binding of a radiolabeled nonhydrolyzable GTP analog (guanosine 5- -O-[gamma-thio]triphosphate) (GTPy35S) to the Ga subunit. The colocalization of the GTPy^S-bound a subunit with membrane-bound receptors upon GPCR activation can be measured by the classical filter binding assay (separation of bound from unbound) and also by SPA (similar to radioligand binding assays) (Fig. 13.3 ).

While the GTPyS assay can potentially be applicable to all classes of GPCRs irrespective of the signaling pathway activated by the ligand-receptor interaction, the higher affinity of the Gai subclass of heterotrimeric G alpha proteins to GPCRs, coupled with higher expression of Gai proteins in mammalian cells, makes the GTPy35S assay most suitable for Gai-coupled GPCRs [17]. Recently, the SPA GTPyS assay has been shown to be miniaturized into 1536-well plate format for uHTS of Ga- -coupled GPCRs [18] (Fig. 13.3). It is important to note that the ability to dispense SPA beads reliably into 1536-well plate format for uHTS is critical to the successful use of this assay type for HTS. Furthermore, while the GTPyS is a functional assay proximal to the GPCR of interest, it requires a priori knowledge of the receptor ligand (i.e., is not suitable for orphan receptors where the ligand is unknown) and is a radioactive assay with limited application suitable primarily to Gai-coupled GPCRs.

Reporter Gene Assays With the increasing popularity of functional assays to overcome some of the limitations of traditional ligand binding assays, reporter gene assays have made a significant impact on biological assays since the 1990s, especially in HTS laboratories. Reporter genes with readily measurable phenotypes have been used to measure the effects of signal transduction cascades originating from cell surface GPCRs on gene expression [19]. These assays offer high sensitivity, reliability, convenience, and adaptability to large-

Agonist

Agonist

3000

40 30

384 well

1536 well

3000

2000-

1000-

384 well

2000-

1000-

40 30

1536 well

-I-1-1-1-1-1-1-7 -6 -5 -4 -3 -2 -1 0

1536-well screening plate for agonist assay

600-

ro 300

1536-well screening plate for agonist assay

600-

ro 300

+

s

—if

m

-1 "x'

ililäj

m

Rflltj

Column no.

37 43

Figure 13.3 Schematic representation of OTPyS SPA assay for uHTS in 1536-well plate format.

13 19 25 31

Column no.

37 43

Figure 13.3 Schematic representation of OTPyS SPA assay for uHTS in 1536-well plate format.

scale measurements [20], as required for HTS. Commonly used reporter gene assays include chloramphenicol acetyltransferase (CAT) [21], P-galactosidase [22, 23], secreted alkaline phosphatase [24, 25] , luciferase [26, 27], green fluorescent protein (GFP) [28] , and P-lactamase (BLA) [29]. Table 13.2 summarizes the key features of some popular reporter gene assays and their suitability for use in HTS and miniaturized uHTS settings.

Among the reporter gene assays, the BLA reporter gene assay is ideally suited for miniaturized uHTS and was the first of its kind to be introduced in the late 1990s when other reporter gene assays were amenable only to lower

TABLE 13.2 Reporter Gene Assays for Measuring GPCR Function in HTS and uHTS

Reporter

Amplification

HTS Plate

Ratiometric

FACS

Stable

Gene

Density (Wells/

Compatible

Cell

Technology

Assay Plate)

Lines

BLA

+

96, 384,1536, 3456

Yes

Yes

Yes

Luciferase

+

96, 384,1536, 3456

No

No

Yes

CAT

+

96

No

No

No

Secreted

+

96, 384

No

No

Yes

alkaline

phosphatase

Green

-

96, 384,1536

No

Yes

Yes

fluorescence

protein

p-galactosidase

+

96, 384, 1536, 3456

No

Yes

Yes

FACS, fluorescence-activated cell sorting.

FACS, fluorescence-activated cell sorting.

throughput assay formats (6-96-well formats) and required multiple washes, transfer steps, cell lysis, as well as longer assay time (>24 h). Development of a cell-permeable fluoregenic BLA substrate, CCF2/AM [29], was a key step in the use of BLA as a reporter gene in mammalian cells [30] . The CCF2 substrate/dye (or CCF4/AM) is cell permeable and consists of coumarin and fluo-rescein moieties connected by a P-lactam-containing cephalosporin core. Within cells, excitation of CCF2 (or CCF4) at 405 nm leads to fluorescence resonance energy transfer (FRET) from the coumarin moiety to the fluorescein derivative, resulting in green light emission at 530 nm [29]. In the presence of BLA reporter protein in the cell, the substrate is cleaved at the P-lactam ring, spatially separating the coumarin and fluorescein moieties, thereby disrupting the FRET. Hence, excitation of coumarin at 405 nm would result in blue fluorescence emission in the absence of FRET, detected at 460nm [13] . Thus, basal unstimulated cells appear green by fluorescence microscopy due to FRET, while cells producing BLA appear blue (Fig. 13.4). The BLA activity, reported as a ratiometric readout of 460nm/530nm emission, offers a significant advantage for HTS, primarily, to normalize for viable cell numbers per well [31] .

The BLA reporter gene assay is well suited for the study of cell surface GPCRs [31]. Receptor signaling through intracellular second messengers can be linked to corresponding modulation of the BLA gene via appropriate promoters. In the case of GPCRs coupled to the Gaq pathway, the BLA gene is engineered under the control of the nuclear factor of activated T cells (NFAT) [32] promoter, which is responsive to changes in intracellular Ca2+. Receptor

Green 530 nm fluorescence emission Blue 460 nm fluorescence emission

Ratiometric readout = 460 nm/530 mn

Green 530 nm fluorescence emission Blue 460 nm fluorescence emission

Ratiometric readout = 460 nm/530 mn

3456-well screening plate for antagonist assay

1.80

3456-well screening plate for antagonist assay

1.80

EC70 agonist IC50 antagonist Basal

0 5 1015202530354045505560657075 Column no.

EC70 agonist IC50 antagonist Basal

0 5 1015202530354045505560657075 Column no.

Figure 13.4 Principles and utilization of the BLA reporter gene assay for the detection of GPCR antagonists in 3456-well plate format.

activation and subsequent increase in i[Ca2+] results in the activation of the Ca2+-dependent phosphatase, calcineurin (Fig. 13.4). Activated calcineurin, in turn, dephosphorylates and activates the cytoplasmically located inactive NFAT. Dephosphorylated and activated NFAT translocates into the nucleus and initiates transcription of NFAT2regulated genes (i.e., the BLA gene in engineered cells). For GPCRs coupled to modulation of intracellular cAMP by stimulation or inhibition of adenylyl cyclase via the G proteins Gas or Gai, respectively, the BLA gene may be engineered under the control of the cAMP response element, CRE. Thus, Gas-coupled GPCRs increase intracellular cAMP, resulting in activation of the CRE binding protein (CREB), increased CRE activity, and BLA gene activation [33, 34] , while Gai-coupled GPCRs would result in a decrease in intracellular cAMP, CRE activity, and BLA gene transcription.

Additional details on the use of the BLA reporter gene technology to select and isolate functional GPCR clones and optimize the assay for HTS or uHTS, assay limitations, as well as example assay protocols for uHTS are discussed in Kunapuli [31].

Recent improvements in luciferase reporter gene technology, including improvements in substrates, have now enabled this reporter gene technology to be utilized more broadly for HTS and uHTS. Moreover, the availability of dual Glo luciferase systems using firefly and Renilla luciferase enzymes with appropriate substrate kinetics now provides the opportunity to normalize the luminescence readout in luciferase reporter gene assays to the number of cells per well, similar to the BLA technology. In addition, the luminescence mode of detection is sometimes favored over the fluorescence mode for HTS due to the potential for interference of colored compounds with fluorescent readout.

Although the reporter gene assays were beneficial for providing a functional response to GPCR activation, these assays are relatively long (several hours) with readouts that are quite distal from the actual target GPCR, allowing for potential compound interference at various stages of the signaling pathway when used in HTS [13, 31] . Hence, hit funneling strategies for post-primary screening should be planned appropriately, with the use of multiple upstream assays, such as a second messenger assay, in addition to a receptor binding assay to confirm direct compound interaction with the target GPCR of interest.

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