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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5715_Библиотеки_им_академика_М_И_Перельмана
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90 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
Assays to Measure Changes in Second Messenger
GPCRs transmit signals mainly through their coupled G proteins, leading to
changes in intracellular levels of second messengers such as cAMP, inositol
triphosphate, and Ca
2+
. Direct measurement of intracellular levels of these
messengers upon stimulation is a popular functional assay format for GPCR
screening in mammalian cells. The activation of G
q
or G
i
coupled-receptors
promotes phospholipase C to hydrolyze phosphatidylinositol bisphosphate to form
two second messengers, inositol 1,4,5-triphosphate (IP
3
) and diacyl glycerol
(DAG). DAG then activates protein kinase C, while I P
3
activates the IP
3
receptor
on the endoplasmic reticulum, resulting in Ca
2+
mobilization from the
endoplasmic reticulum to the cytoplasm and consequently a transit elevation of
intracellular Ca
2+
. IP
3
is quickly hydrolyzed to IP
2
, then to IP
1
and finally to
inositol by a series of enzymatic reactions. For Ca
2+
mobilization, there are highly
sensitive calcium flux assays including FLIPR (fluorometric imaging plate reader)
and Aequorin assays for receptors naturally coupled to G
q
proteins or artificially
coupled to a chimeric G
qo5
or G
qi5
, or a promiscuous G
15/16
proteins [77]. For IP
3
generation and hydrolysis, there are traditionally radiometric scintillation
proximity assay (SPA) (GE Healthcare), and newer non-radiometric assays
including AlphaScreen™ (PerkinElmer), HitHunter™ fluorescent polarization
(DiscoveRx), and IP-One HTRF™ (Cisbio).
Today, most cAMP assays measure global changes in the concentration of
intracellular cAMP in the presence of a broad spectrum phosphodiesterase
inhibitor without and with the adenylyl cyclase activator forskolin for G
s
- and G
i
-
coupled receptors, respectively. In these assays, changes in intracellular cAMP are
generally detected by the competition between cellular cAMP and a labeled form
of cAMP for binding to an anti-cAMP antibody [3 0] . A wide var iety of as says ar e
commercially available; these include SPA (GE Healthcare), FlashPlate™ and
AlphaScreen™ amplified luminescent proximity homogeneous assay
(PerkinElmer), HitHunter™ enzyme fragment complementation (DiscoveRx), and
HTRF™ homogeneous time resolved fluorescence (Cisbio) cAMP assays. A gen e
reporter cAMP assay based on D-luciferin, Glosensor™ available from Promega,
provides a non-lytic live-cell-based and facile kinetic measurement of intracellular
cAMP dynamics [34]. Alternatively, a modified rat olfactory cyclic nucleotide
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GPCR Drug Discovery Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 91
gated (CNG) channel, which is engineered such that it enhances the cAMP
binding affinity but reduces the cGMP binding affinity, is used as a cAMP
biosensor for G
s
and G
i
GPCR screening [78] (BD ACTOne, BD Biosciences,
Rockville, MD). Amassing evidence now suggests that instead of uniform
alteration in cytosolic concentration, cAMP undergoes changes with unique
spatial and temporal gradients upon the receptor activation [76]. The spatial and
temporal compartmentalization of cAMP (and its target proteins) is central to the
ability of this second messenger to govern cellular activity over timescales
ranging from milliseconds to several hours. In the recent years, a variety of cAMP
biosensors have been developed to directly monitor rapid subcellular cAMP
dynamics. However, these single cell assays have not been fully developed for
HTS.
Assays to Measure Protein-Protein Interaction
The recent realization that GPCRs potentially function as homo-oligomeric and
hetero-oligomeric complexes [47-62] has given rise to rationale for developing
assays based on protein-protein interactions. These assays are useful not only for
elucidating the nature of receptor interaction within the oligomeric complexes, but
also for monitoring ligand-induced reorientation of an existing oligomer,
particularly relative to its bound G protein subunits. These assays have also been
used to study distinct protein interactions during GPCR signaling cycle, including
the interactions of ligand-activated receptor with its coupled G proteins, or other
regulatory proteins such as G protein-coupled receptor kinases (GRKs) and -
arrestins.
These assays typically utilize either resonance energy transfer (RET) [62] or
protein fragment complementation [79,80]. The RET is a non-radiative transfer of
energy between a donor and an acceptor when they are in proximity, with an
efficiency that varies inversely with the sixth po wer of the distance between the
two molecules. The donor moiety can either be a fluorophore (FRET) or a
bioluminescent enzyme that emits light upon oxid ation of a substrate (BRET). In
both cases, the acceptor is a fluorophore. The changes in RET in response to
stimuli offers an effective means for real-time monitoring of dynamic protein-
protein interactions that are involved in GPCR activation and regulation in cells.
92 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
Alternatively, protein fragment complementation assays utilize a pair of
recombinantly engineered and inactive fragments of either an enzyme (e.g, -
galactosidase [79]) or a fluorescent protein (e.g., green fluorescent protein (GFP)
[80]), each being recom binantly attached to a pr otein. When the receptor a ctivation
leads to the interaction between the two proteins having the complementary
fragments, this interaction drives the functional complementation of the protein
mutant fragments. GPC R activation is meas ured directly b y quantifying the restored
activity of the enzyme or fluorescence of the fluorescent protein. Since protein
fragment complementation is an amplified signal detection system, exquisite
detection sensitivity is achieved, permitting analysis of protein interactions at
concentrations approximating their normal levels of expression. Although protein
interaction-driven functional complementation of 2 split fragments is reversible, the
rate of association of the two yellow fluorescent protein (YFP) fragments fused to Fos
and Jun has been reported to be much slower (t
1/2
of ~60sec) than the subunit
exchange rate of Fos-Jun heterodimer s (t
1/2
of ~10s ec) [80]. Such a slo w kinetics is
mainly due to relatively weak affinity between the 2 split YFP fragments, and is
typically slower than the reorientation of emitter/acceptor fluorescent proteins. Thus,
cautious is warranted to interpret the kinetics of protein-protein interactions measured
using protein fragment complement ation ass ays. Recently, Javitch and his colleagues
had combined protein complementation with resonance energy transfer to study
conformational changes in response to activation of the putative dopamine D
1
-D
2
receptor heteromer [81]. They found that the potency of the D
2
receptor agon ist R-(-)-
10,11-dihydroxy-N-n-propy lnoraporphine to change the G
i
conformation via th e D
2
protomer in the heteromer was enhanced ten-fold relative to its potency in the D
2
homomer, suggesting that GPCR dimerization can influence drug pharmacology.
Assays to Measure Protein Trafficking
GPCRs modulate diverse physiological signaling pathways by virtue of changes
in receptor activation and inactivation states. Functional changes in receptor state
lead to dynamic trafficking of the receptor [82] and many of its downstream
signaling molecules (e.g., arrestins [83], protein kinase C [75]) at various stages
of GPCR signaling cycle. Direct visualization of protein trafficking provides a
basis for high-content screening (HCS), which often provides a measure of
receptor-ligand interactions in multiple dimensions with spatial and temporal
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GPCR Drug Discovery Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 93
dynamics [27]. Receptor internalization and -arrestin recruitment assays are two
of the most common protein trafficking assays. Receptor internalization is an
essential component of receptor desensitization process, wherein GRKs
phosphorylate agonist activated receptors, which, in turn, recruit cytosolic -
arrestins to the cell membrane. The -arrestins then uncouple the activated
receptors from their cognate G proteins and target the receptor s to clathrin-coated
pits for endocytosis. A wide array of techniques has been adopted for these
protein trafficking assays [79,80,83].
These protein trafficking assays require no prior knowledge of the interacting G
protein(s), enabling screening orphan GPCRs. Furthermore, these assays are often
generic to all classes of GPCRs, thus overcoming the limitations associated with
typical G protein-dependent functional assays. However, these traff icking assays
suffer drawbacks associated with the fact that some agonists, particularly partial
agonists, could stimulate receptor signaling without causing the trafficking
event(s) seen when the receptor is stimulated by its native ligand [63,64]. For
example, morphine, a partial agonist, exhibits relatively poor activity to cause
internalization of the μ-opioid receptor [84].
Dissecting the biased agonism of GPCR ligands has led to a hypothesis that G
protein and -arrestin pathways are distinct and could be pharmacologically
modulated independently; and both receptor internalization and -arrestin
recruitment assays are considered to be G protein-independent functional assays
[34,63,66]. However, in a recent elegant study, Sauliere et al. used a set of new,
highly sensitive and direct BRET-based G protein activation probes specific for
all G protein isoforms to evaluate the G protein-coupling activity of [
1
Sar
4
Ile
8
Ile]-
angiotensin II (SII) [85]. SII has been described as a G protein-independent -
arrestin biased ligand for angiotensin AT
1A
receptor. Results showed that SII
promotes G
q
and G
i
protein-dependent signaling in both heterologous and primary
cells, but stabilizes a specific AT
1A
receptor conformation distinct from that of
Ang II and triggers a different intracellular signaling pattern. The most notable
finding is that the -arrestin biased agonism of SII is not truly G protein
independent, as evidenced by the partial G
q
protein dependence on the -arrestin 2
recruitment to AT
1A
receptor upon SII stimulation in HEK293T cells, as well as a
G
i
- and G
q
-dependent -arrestin-mediated ERK1/2 activation in MEF cells. The
94 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
observed G protein-dependent -arrestin activation as a specific signature of SII
may put G protein pathways back in the spotlight.
Assays to Measure Changes in Gene Reporter Activity
GPCR activation is well known to alter gene transcription. Several genes contain
elements responsive to second messengers upon the activation of GPCRs; these
elements includ e the cAMP response element ( CRE), the nuclear factor of activate d
T-cells response element (NFAT-RE), the serum response element (SRE) and the
serum response factor response element (SRF-RE), thus providing the basis for
developing reporter gene assays [86]. Reporter gene constructs usually contain
second messenger responsive elements upstream of a minimal promoter, which in
turn regulate the expression of the reporter protein. Modulation of intracellular cAMP
by G
s
or G
i
GPCRs is detect ed wit h a re porter ge ne, whose transcripti on i s regulated
by the transcripti on factor cAMP respon se-element binding protein (CREB) bin ding
to upstream CREs. Calcium flux, mobilized by G
q
-coupled receptors, is detected
using a reporter gene, whose transcription is regulated by the calciu m-sensitive AP1
(activator protein 1) or NFAT (nuclear factor of activated T cells) elements.
Commonly used reporter g enes include alkaline phosphata se, - galactosidas e, GFP,
luciferase, and -lactamase, all of which can lead to detectable colorimetric,
fluorescent or luminescent signals. Different gene reporters exhibit distinct sensitivity
and dynamic range. Owing to the high sensitivity, broad dynamic range and
amenability to HTS, luciferase has become the most popular gene reporter. However,
beside the requirement for long incubation periods and the gene expression signal
quite distal from the receptor activation, these reporter assays also have disadvantages
associated wit h the measurem ents far distin ct from nat ive systems due t o the use of
highly artificial systems. The substrates used may also introduce interference.
Recently, D-luciferin, the m ost widely used lucifer ase substrate, ha s been found to
pose partial agonist activity at the GPR35 [87].
Assays to Measure Changes in Phenotype
Receptor activation ultimately leads to changes in cell phenotype. One example is
the melanophore technology (Arena Pharmaceuticals, San Diego, CA), which
uses GPCR targets expressed in frog skin cells containing a pigment that is highly
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GPCR Drug Discovery Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 95
sensitive to changing levels of cAMP [3, 26]. The pigment disperses throughout
the cell and causes the cell to appear black when the intracellular cAMP level
increases; conversely, when the cAMP level decreases, the pigment aggregates at
the center of the cell, causing the c ell to appear clear. In a recent study, Marsh all
and his colleagues have used flagella of the green alga Chlamydomonas
reinhardtii as a model to screen chemicals that may regulate signaling pathways
critical for ciliogenesis and length regulation [88]. Based on the flagellar length,
motility, and cell viability, they found that the most frequent tar get found to be
involved in flagellar length regulation was the family of dopamine binding
GPCRs, and cilium length was altered with expression of the dopamine D
1
receptor in mammalian cells.
LABEL-FREE CELL-BASED AS SAYS
Label-free cell-ba sed a ssays gen erally employ a bio sensor to m onitor lig and-induced
responses in living cells. A biosensor typically utilizes a transducer such as an optical,
electrical, calorimetric, acoustic, or magnetic transducer, to convert a molecular
recognition event or a ligand-induced change in living cells into a quan tifiable signal.
These label-free biosensors are commonly used for molecular interaction analysis,
which involves charact erizing how molecular complexes form and disassociate ov er
time. Many comprehensive reviews are available for different aspects of these
biosensors for bio-molecular interaction analysis [89-91]. Thus, this section only
highlights the application s of these biosen sors for w hole cell sensing, particul arly for
functional GPCR screening. Fig. (1) highlight two types of biosensors that are
currently used as the basis for label-free cell-based assays - resonant waveguide
grating (RWG) biosensors [92] and electrical biosensors [93-95].
RWG Biosensors and Systems
An RWG biosensor consists of a substrate (e.g., glass), a waveguide thin film
with an embedded grating structur e, and a cell layer (Fig. 1a). The RWG biosensor
utilizes the resonant coupling of light into a waveguide by means of a diffraction
grating, leading to total internal reflection at the solution-surface interface, which
in turn creates an electromagnetic field at the interface. This electromagnetic field
is evanescent in nature [96], meaning that it decays exponentially from the sensor
96 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
Fig. (1). Principles of two types of biosensors for living cell sensing. (a) A RWG biosensor for
monitoring ligand-induced dynamic mass redistribution in living cells. Cells are directly cultured
onto the surface of a biosensor. The biosensor consists of a glass substrate, a waveguide thin film
within which a grating structure is embedded. Only the mass redistribution within the bottom
portion of cells is directly measured. (b) An electric biosensor for monitoring the ionic
environment surrounding the biosensor and the cells. Cells are cultured on the surface of a
biosensor having arrayed gold microelectrodes. Both flows of extracellular (iec) and transcelular
(itc) current are measured, and a low AC voltage at variable frequencies is a pplied to the cell layer.
surface; the distance at which it decays to 1/e (e is a numerical constant that is
equal to 2.71828) of its initial value is known as the penetration [97] and is a
function of the design of a particular RWG biosen sor, but is typically on the or der
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GPCR Drug Discovery Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 97
of 150 to 200nm [98]. Similar to biomolecular interaction analysis, RWG
biosensor also exploits such an evanescent wave to characterize ligand-induced
alterations of living cells at or near the sensor surface [99].
RWG instruments can be subdivided into systems based on angle- or wavelength-
interrogation measurements. In a wavelength interrogation instruments, polarized
light covering a range of incident wavelengths with a constant angle is used to
illuminate the waveguide; light at specific wavelengths is coupled into and
propagates along the waveguide [98]. Alternatively, in angle-interrogation
instruments, the sensor is illuminated with monochromatic light and the angle at
which light is resonantly coupled is measured [99]. In the recently developed
swept wavelength interrogation RWG imagers, a light beam from a swept tunable
light source is used to illuminate a partial or whole 384well biosensor microplate,
resulting in distinct spatial resolution down to ~10 μm so single cell can be
resolved [100,101]. A high speed digital camera is used to record the reflected
resonant lights. During a single cycle of wavelength sweep from 825 to 840 nm
total 150 spectral images are acquired every 3 sec, and the spectral image stack
are then processed to generate signals. Since the wavelength sweeping is stepwise
in 100 pm every 20 ms, this imaging system enables a time resolved reson ance,
leading to improved spatial resolution. However, for RWG biosensors the
resonance conditions are influenced by the physical properties of the cell layer
that contacts with the surface of the biosensor (e.g., cell confluency, adhesion and
status such as proliferating or quiescent states). When a ligand or an analyte
interacts with a cellular target (e.g., a GPCR, a kinase) in living c ells, any change
in local refractive index within the cell layer can be detected as a shift in resonant
angle (or wavelength) [98,99].
The Epic
®
system (Corning Inc., Corning, NY) is a wavelength interrogation
reader system tailored for RWG biosensors in microplate for both label-free
biochemical and cell-based assays. This system consists of a RWG plate reader
and Society for Biomolecular Screening (SBS) standard microtiter plates (Fig. 2a)
[102]. The detector system in the plate reader exploits integrated fibre optics to
measure the shift in wavelength of the incident light, as a result of ligand-induced
changes in cells. A series of illumination/detection heads are arranged in a linear
fashion, so that reflection spectra are collected simultaneously from each well
98 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
within a column of a 384-well microplate, leading to a temporal resolution of ~15
seconds. The whole plate is scanned so that each s ensor can be addr e ssed multiple
times, and each column is addressed in sequence. The wavelengths of the incident
light are collected and used for analysis. A temperature-controlling unit is built in
the instrument to minimize spurious shifts in the incident wavelength due to the
temperature fluctuations. The EnSpire system from PerkinElmer is a multimodal
plate reader containing a label-free RWG module from Corning, which is based
on a single light head to scan the whole plate, resu lting in relatively low temporal
resolution (~85 sec). The whole plate RWG imager from Corning can
simultaneously image the whole plate with a temporal r esolution of 3 s econds and
a spatial resolution of 80 to 100 μm [100]. Both Epic and EnSpire are mostly
operated at room temperature, whereas the imager can be placed inside standard
cell culture incubators so assays can be performed under physiological conditions
[101].
Fig. (2). Schematic drawings showing the surface configurations of three types of biosensor-
embedded microplates used for label-free cell-based assays. (a) A well of a 384-well Epic
®
RWG
biosensor microplate. The inset shows a scanning electronic microscopic graph of a portion of the
biosensor. (b) A well of a 96-well CellKey gold electrode-embedded microplate. (c) A well of a
96-well RT-CES gol d ele ctrode-embedded micr oplate.
Electrical Biosensors and Systems
Electrical biosensors consist of a substrate (e.g., plastic), an electrode, and a cell
layer (Fig. 1b). In this electrical detection method, cells are cultured on the
substrate having an array of small gold electrodes. A small constant voltage at a
fixed frequency or varied frequencies is applied to the electrode or electrode
array, and the electrical current through the circuit is monitored over time. The
ligand-induced change in electrical current provides a measure of cell response.
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GPCR Drug Discovery Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 99
The first generation impedance systems suffer from high assay variability due to
use of a small detection electrode and a large reference electrode [103]. To
minimize this variability, the latest generation of systems, such as CellKey system
(MDS Sciex, South San Francisco, CA) and RT-CES (ACEA Biosciences Inc.,
San Diego, CA), utilize an integrated circuit having a microelectrode array.
The CellKey system consists of an environmentally controlled impedance
measurement system, a 96-well electrode-embedded microtiter plate, an onboard
96-well fluidics, and custom acquisition and anal ysis software [93]. T he cells are
seeded in the culture wells; each well has an integrated electrode array (Fig. 2b).
The system operates using a small-amplitude alternating voltage at 24
frequencies, from 1 KHz to 10 MHz. The resultant current is measured at an
update rate of 2 sec. T he system is thermally regulated and experiments can be
conducted between 28°C and 37°C. A 96-well head fluid delivery device handles
fluid additions and exchanges onboard.
The RT-CES system is composed of four main co mponents: electronic microtiter
plates (E-Plate™), E-Plate station, electr onic analyzer, and a monitoring system
for data acquisition and display [94]. T he electronic analyser sends and receives
the electronic signals. The E-Plate station is placed inside a tissue culture
incubator. The E-Plate station comes in three throughput varieties: a 16x station
for running six 16-well E-Plates at a time, a single 96-well E-Plate station, and the
Mult-E-Plate™ station, which can accommodate up to six 96-well E-Plates at a
time. The cells are seeded in E-Plates, which are integrated with microelectronic
sensor arrays (Fig. 2c). The system operates at a low-voltage (less than 20 mV)
AC signal at multiple frequencies.
Optical Signals of GPCR Activation with RWG Biosensor
Cells are dynamic objects with relatively large dimensions - typically tens of
microns. RWG biosensors enable detection of ligand-induced changes within the
bottom portion of cells, determined by the penetration depth of the evanescent
wave [99]. Furthermore, the spatial resolution of RWG biosensors is dependent on
illumination scheme of the detection systems used. Generally, a confluent layer of
cells is used in order to achieve optimal assay results, partly due to the
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