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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5993_Библиотеки_им_академика_М_И_Перельмана
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100 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
requirement for effectively separate receptor signaling-mediated responses from
the background, and partly due to the fact that current biosensors all measure an
averaged response from a population of cells [25,98]. The background signal is
sensitive to the adhesion and states of cells. Higher confluency generally leads to
higher and more reproducible signal [100]. When the cells form a monolayer, the
sensor configuration can be viewed as a three-layer waveguide composite,
consisting of a substrate, waveguide thin film and a cell layer. Following a 3-layer
waveguide biosensor theory [99] in combination with cellular biophysics [104],
we found that for whole-cell sensing, a ligand-induced change in effective
refractive index, the detected signal
N, is governed by [99]:
N = S(C)n
C
= S(C)
d C
i
e
z
i
Z
C
e
z
i+1
Z
C
i
(1)
where S(C) is the system sensitivity to the cell layer, and
n
c
is the ligand-induced
change in local refractive index of the cell layer sensed by th e biosensor. Z
c
is
the penetration depth into the cell layer, is the specific refractive index
increment (about 0.18/mL/g for proteins), z
i
is the distance where the mass
redistribution occurs, and d is an imaginary thickness of a slice within the cell
layer. Here the cell layer is divided into an equal-spaced slice in the vertical
direction. We assumed that the detected signal is, to first order, directly
proportional to the change in refractive index of the bottom portion of cell layer
n
c
. The
n
c
is directly proportional to changes in local concentration of cellular
targets or molecular assemblies within the sensing volume, given that the
refractive index of a given volume within cells is largely determined by the
concentrations of bio-molecules, mainly proteins [104]. A weighted factor exp(-
z
i
/Z
c
) is taken into account for a change in local protein concentration occurring,
considering the exponentially decaying nature of the evanescent wave. Thus, the
detected signal is a sum of mass redistribution occurring at distinct distances away
from the sensor surface, each with unequal contribution to the over all response.
Eq.1 suggests that the detected signal with an RWG biosensor is sensitive
primarily to the vertical mass redistribution, as a result of any change in local
protein concentration and where and when it occurs. The detected signal is often
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GPCR Drug Discovery Advances in Combinatorial Chem istry & High Throughput Screening, Vol. 1 101
referred to as a dynamic mass redistribution (DMR) signal [105]. Thus, RWG-
enabled cellular assay is also termed DMR assays.
GPCR activation leads to a series of spatial and te mporal events, including ligand
binding, receptor activation, protein recruitment, receptor internalization and
recycling, second messenger alternation, cytoskeletal remodeling, gene
expression, and cell adhesion changes, to name bu t a few. Each cellular event has
its own characteristics regarding its kinetics, duration, amplitude, and mass
movement. Thus it is reasonably assumed that these cellular events may
contribute differently to the overall DMR signal, depending on the location where
they occur. Using a panel of agonists targeting a variety of GPCRs, we identified
three classes of DMR signals in human epidermo id carcinoma A431 cell, which
reflect the signaling pathways mediated [106] (Fig. 3). Since each is correlated
with the activation of a class of GPCR s depending on the G protein with which
the receptor is coupled, the DMR signals obtaine d were named G
q
-, G
s
- and G
i
-
DMR signals, respectively. Each class of DMR signals exhibits distinct kinetic
and dynamic characteristics, reflecting the unique signaling integration mediated
through different classes of GPCRs. Interestingly, G
q
-type DMR signal appears to
be rapid, whereas G
s
-type DMR is comparatively slow. Our recent chemical
biology and orthogonal fluorescence imaging studies suggest that G
q
-DMR signal
is downstream of Ca
2+
mobilization [107], whereas G
s
-DMR signal is downstream
of cAMP accumulation [108].
Following classical receptor biology, a numerical analysis suggests that protein
translocation and receptor internalization are two pr imary resources for the DMR
signatures observed for G
q
-coupled receptor signaling [99]. Unique to G
q
-
mediated signaling is the dramatic translocation of its signaling components,
including several protein kinase C (PKC) isoforms, GRKs, -arrestin, PIP-binding
proteins, and DAG-binding proteins, to name but a few [75,109]. The protein
trafficking mediated by the activation of G
q
-coupled receptors is relatively rapid
and become obvious shortly after agonist stimulation [75], leading to a rapid G
q
-
type DMR signal. On the other hand, we have found that an increase in cell
adhesion is the major contributor to the positive-DMR (P-DMR) event in the
2
-
AR signal induced by epinephrine [unpublished data]. The occurrence of the
initial negative-DMR (N-DMR) event reflects the fact that the majority of
102 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
downstream signaling components directly involved in the
2
AR signaling
complexes, with the exception (thus far) of AKAPs (A-kinase anchoring protein)
and -arrestins, are already compar tmentalized at or near the cell membrane [110-
112]. Thus, for G
s
-coupled receptor the initial recruitment of intracellular tar gets
to the activated receptors is much less pronounced than G
q
-coupled receptor
signaling, and is overwhelmed by other cellular events leading to the decrease in
local mass density in cells. Our recent confocal fluorescence imaging studies
suggest that the increase in cell adhesion appears to be only evident at least 10min
after the activation of G
s
-coupled receptors, consistent with the slow P-DMR
event of the G
s
-DMR signals [unpublished data]. Both G
q
and G
s
-DMR signals
are quite universal across multiple cell lines tested, although their fine features
differ, reflecting the important roles of cellular context in GPCR signaling
[106,108,113].
Fig. (3). The GPCR signaling and its DMR signal. (a) G
q
-signaling and its DMR signal (CHO
cells responding to 40unit/ml thrombin). (b) G
s
-signaling and its DMR signal (A431 cells
responding to 25 nM epinephrine). (c) G
i
-signaling and its DMR signal (A431 responding to
200nM LPA). The solid arrows in the kinetic graphs
indicate the time when the agonist solution is
introduce d (Reproduc ed with permission from ref. 106, Copyright Elsevier, 2007).
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GPCR Drug Discovery Advances in Combinatorial Chem istry & High Throughput Screening, Vol. 1 103
Bioimpedance Signals of GPCR Activation
The use of impedance measurements for whole cell sensing was first reported in
1984 [114]. These biosensors were later considere d to a cell mor phological s ensor
[115,116]. In a typical impedance-based cell assay, the total impedance of the
sensor system is determined primarily by the ion environment surrounding the
biosensor [95,117]. Under application of an electrical field, the ions undergo
field-directed movement and concentration gradient-driven diffusion. For whole
cell sensing, the total electrical impedance has four components: the resistance of
the electrolyte solution, the impedance of the cell, the impedance at the
electrode/solution interface, and the impedance at the electrode/cell interface. In
addition, the impedance of a cell comprises two c omponents - the resistance and
the reactance. The conductive characteristics of ce llular ionic strength provide the
resistive component, whereas the cell membranes, acting as imperfect capacitors,
contribute a frequency-dependent reactive component [118,119]. Thus, the total
impedance is a function of many factors, including cell viability, cell confluency,
cell numbers, cell morphology, degree of cell adhesion, ionic environment, the
water content within the cells, and the detection frequency. Electric biosensor
enabled cell assays can be viewed as bioimpedance assays.
In the RT-CES system, a percentage of the small voltage applied is coupled into
the cell interior. Such signals applied to cells are believed to be much smaller than
the resting membrane potential of a typical mammalian cell and thus present
minimal invasiveness to cell function cell function. The RT-CES system measures
these changes in impedance and displays it as a parameter called the cell index.
The cell index is calculated according to the formula [120].
CI = max
i=1,...,N
R
cell
( f
i
)
R
0
( f
i
)
1
(2)
where N is the number of frequency points at which the impedance is measured
(e.g., N=3 for 10 kHz, 25 kHz, and 50 kHz), and R
0
(f) and R
cell
(f) are the
frequency electrode resistance without cells or with cells present in the wells,
respectively.
104 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
In the CellKey system, a change in sen sor system’s impedance is attributed to a
change in complex impedance (delta Z or dZ) of a cell layer that occurs in
response to receptor stimulation [93]. At low frequencies, the small voltage
applied induces extracellular currents (iec) that pass around individual cells in the
layer. However, the conduction currents through cell membrane due to ion
channels may also be important at low measurement f requencies [119]. At high
frequencies, the small voltage applied induces transcelular currents (itc) that
penetrate the cellular membrane (Fig. 1b). The ratio of the applied voltage to the
measured current for each well is its impedance (Z) as described by Ohm’s law.
When cells are exposed to a receptor ligand, signal transduction events are
activated, leading to complex cellular events that cause changes in cell adherence,
cell shape and volume, and cell-to-cell interaction. These cellular changes
individually or collectively affect the flow of extracellular and transcelular
current, thus resulting in the alteration in the magnitude and characteristics of the
impedance. Fig. (4) shows three types of impedan ce signal s mediated through the
activation of three classes of GPCR s, depending on the G protein with which the
receptor is coupled [93,121]. The profiles are obtained using CellKey system.
Similar profiles were also recorded using the RT-CES system [122]. It is believed
that these impedance signals are due to the different effects on the actin
cytoskeleton that affect the cellular parameters measured by impedance, in
Fig. (4). The impedance signals of G
q
, G
i
and G
s
GPCRs. The profiles are obtained in Chinese
ovary hamster (CHO) cells stably expressing rat muscarinic receptor subtype 1 (CHOm1) using
the CellKey system. The agonists are carbachol (M
1
), 5-hydroxytryptamine (5HT
1B
) and
prostaglandin E2 (prostanoid EP
4
). Both 5HT
1B
and EP
4
receptors are endogenously expressed
(Reproduced with permission from ref. 95, Copyright Elsevier, 2005).
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GPCR Drug Discovery Advances in Combinatorial Chem istry & High Throughput Screening, Vol. 1 105
response to the activation of different classes of GPCRs. It has been shown that
activation of G
q
[123,124] and G
i
GPCRs [125,126] leads to increased actin
polymerization, while stimula tion of G
s
GPCRs leads to actin depolymer ization [127].
LABEL-FREE CELL-BASED ASS AYS F O R GPCR SCREENING
Receptor Pannin g
Label-free cell-based assays are capable of monitoring the activity of different
classes of GPCRs with high sensitivity, offering a universal assay platform for
assaying endogenously expressed and functionally active receptors in a cell
system. Receptor panning with a GPCR agonist library, known to activate many
families of GPCRs, would allow one to reliably map endogenous receptors in a
cell system [106].
It is known that more than one family member of many GPCR families are
endogenously expressed in a single cell system, such as purinergic P2Y receptors
in A431 cells [106] as well as human embryonic kidney (HEK) cells [128]. In
addition, a ligand including naturally occurring agonists could have cross-activity,
but different efficacies, to activate distinct members of a receptor family [129].
Since the biosensor measures an integrated cellular response [24,25,99], a ligand-
induced biosensor signal may represent the signaling mediated through the
activation of multiple receptors. Determining the potency and efficacy of panels
of non-selective and selective agonists against the r eceptor family should enable
the assessment of which family member is dominated in the cell system, wherein
multiple family members are co-endogenously expressed [128]. The cell system-
or tissue-specific functional activity of a GPCR might indicate its physiological
roles, or hold potential for the side-effect evaluation of a drug candidate. The
ability of receptor panning at both the cell system and receptor family levels
allows one to easily screen disease-relevant cell types, to determine the
differentiation states of stem cells, and to permit more physiologically relevant
selectivity and specificity screens. DMR profiling of endogenous receptors in
A431 led to the identification of three types of DMR signatures (Fig. 3),
consistent with the primary signaling pathways of their cognate receptors [106].
Interestingly, the maximal amplitudes of the DMR signals arising from the
activation of endogenous protease activated receptors by distinct agonists are
106 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
found to be correlated well with their respective maximal increase in intracellular
Ca
2+
level [130].
DMR assay is a phenotypic whole cell assay with wide pathway coverage, yet
permit molecular mechanistic deconvolution [24]. Thus, phenotypic profiling
using DMR assays could be used to characterize the activity and specificity of a
library of known agonists targeting known GPCRs in a native cell line or cell
system including cancer cells and stem cells; and the resultant patterns of these
agonists could be used to characterize the origin and states of the cell line or cell
system [131]. Surveying functional receptors in t he human epidermal squamous
cancer cell line A431 with a library of agonists for most of all known GPCRs (~
220) showed that a number of GPCR agonists led to robust DMR in A431,
consistent with the expression pattern of their cognate receptor(s) [131]. For a
subset of agonists tested, hierarchical Euclidean cl ustering analysis bas ed on their
real time kinetic responses led to a heat map consisting of two major classes at the
low resolution, consistent with the primary pathways mediated through these
receptors (Fig. 5). The G
s
-coupled receptor agonists are clustered together, and
separated from the G
q
-coupled receptor agonists. These results suggest that DMR
assays faithfully report functional receptors in the cells.
The recent years have witnessed increasing interests in the regulated
differentiation of stem cells into tissue specific cells for tissue repair, and to
produce disease-relevant cells for drug discovery [132]. Essential to comprehend
the potentials of stem cells for regenerative medicine and drug discovery is to
determine the precise types and quality of differentiated cells derived from a
specific stem cell or progenitor cell. However, challenges remain to quantitatively
measure the functions of stem cells and their differentiated products. Recently, we
applied DMR assays to characterize the differentiation process of ReNcell VM
human neural progenitor stem cell [133]. The neuroprogenitor stem cell line can
differentiate into dopaminergic neurons, astrocytes and oligodendrocytes after
growth factor withdrawal, thus creating a neuronal cell system. Together with
quantitative real time PCR, DMR profiling using an agonist library targeting over
100 known GPCRs revealed that a subset of receptors including dopamine D
1
and
D
4
receptors underwent marked alterations in both receptor expression and
signaling pathway during the differentiation process. These findings suggest that
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GPCR Drug Discovery Advances in Combinatorial Chem istry & High Throughput Screening, Vol. 1 107
DMR assays can decode the differentiation process of stem cells at the cell system
level.
Fig. (5). The heat map classification of the DMR signals of quiescent A431 cells induced by
diverse GPCR agonists. The heat map was generated using the Euclidean hierarchical cluster
analysis. The real responses of all DMR signals at discrete time points, as indicated, were used as
the basis for similarity analysis. The amplitude and direction are indicated by color: red indicates a
positive value, green a negative value and black a value close to zero (Reproduced with
permission from ref. 131, Copyright Informa Healthcare, 2010).
Bioimpedance assays have also been used to identify functional endogenous
receptors in cells commonly used in dr ug discovery, such as HEK293, and U-2
OS cells [93,121]. Receptor panning has resulted in the identification of several
functionally active, differently coupled endogenous receptor s in these cell lines,
some of which have not been previously documented in the liter ature, suggesting
that these assays also have high sensitivity, enabling pharmacological profiling of
endogenous receptors in native cells including primary cells.
Systems Cell Biology Studies of GPCR Signaling
Cells rely on highly dynamic network interactions in their decision-making
processes. An exogenous signal typically affects the functionality of specific
108 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Ye Fang
target(s), thus resulting in the execution of various cellular machineries in order
for the cell to accommodate such a signal. GPCRs are competent to elicit a
diverse array of cell signaling pathways [134]. Much information is available for
the signaling of GPCRs via either G protein-dependent or independent
mechanisms [35,36]. However, owing to the signaling complexity the network
interactions of GPCR signaling cascades are still far from being completely
understood [44,46]. Label-free cell-based assays are well positioned for dissecting
the signaling pathways of GPCRs in distinct cellular backgrounds, because they
oftenmeasure an integrated whole cell response, such as DMR signals obtained
with RWG biosensors, or the bioimpedance signals obtained with electrical
biosensors [24,25,44,99,105,107]. Schroder et al. demonstrated that a small set of
well-established G protein modulators including the G
i
irreversible inhibitor
pertussis toxin (PTx), the G
s
irreversible activator cholera toxin (CTx), the
selective G
q/11
inhibitor GYM-254890, and the pan-G protein activator aluminum
fluoride (AlF
4
) can be used to deconvolute the signaling pathways of all four
major classes of GPCRs [113]. The free fatty acid receptor FFA1, previously
believed to signal only through the G
q/G11
pathway, was found to also activate the
G
i
pathway. In another study, Tran and Fang demonstrated that the adenylyl
cyclase activator forskolin can be used as a probe molecule to dissect GPCR
signaling, based on the experimental observations that the activation of adenylyl
cyclase can partially desensitize G
q/11
-mediated pathway, but cause complete
heterologous desensitization of G
s
-mediated pathway, and potentiate G
s
-
mediated signaling via heterologous sensitization [108].
Label-free biosensor cell-based assays can be used to delineate the molecular and
cellular mechanisms of receptor signaling at syste m cell biology level [131]. This
is possible for several reasons. F irst, the biosensor response of a receptor-ligand
interaction is a whole cell phenotypic response, and consists of contr ibutions of
multiple signaling pathways and proteins. Second, HTS-compatible biosensor
systems have made it feasible to probe the impact of a wide variety of probe
molecules on the receptor-ligand biosensor signal at a unprecedented scale. Third,
owing to its non-invasiveness in measurement, label- free can be easily integrated
with chemical biology approaches. Chemical biology uses probe molecules
including RNA interference to intervene cell-signaling components one at a time
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GPCR Drug Discovery Advances in Combinatorial Chem istry & High Throughput Screening, Vol. 1 109
[135]. Combined with chemical biology approaches, we have studied the systems
cell biology of endogenous bradykinin B
2
receptor in A431 cells using RWG
biosensors [107]. Stimulation with bradykinin of quiescent A431, obtained by
continuous culturing using 0.1% fetal bovine serum for 20 hrs after the cells reach
~90% confluency, resulted in a dose-dependent and saturated DMR response,
which can be inhibited by a B
2
specific antagonist HOE140 in a dose-dependent
manner, suggested that the DMR signal is a direct result of B
2
receptor activation.
The sensitivity of the DMR signal to cholesterol depletion by methyl--
cyclodextrin argued that B
2
receptor signaling is dependent on the integrity of
lipid rafts; disruption of these microdomains hinders the B
2
signaling.
Modulations of several important intracellular targets with specific inhibitors
suggested that B
2
receptor activation results in signaling via at least dual pathways
- G
s
- and G
q
-mediated signaling, and the two signaling pathways counter-regulate
each other (Fig. 6). Several critical downstream targets including protein kinase C
(PKC), protein kinase A (PKA), dynaminn, and epidermal growth factor receptor
had been identified to involve in B
2
receptor signaling. These findings suggest
that the RWG biosensor appears enable the analysis of GPCR signaling.
Similarly, impedance-based biosensors have been used for studying complex
signal transduction of GPCRs including human melanin-concentrating hormone
receptor 1 (MCHR1) expressed heterologously in CHO and U2-OS cells and
endogenously in MOLT4 cells. The biosensor detects both pertussis toxin-
sensitive and Ca
2+
-coupled events downstream the MCHR1 activation [93].
Label-free cell assays can be used to discover new pathways in GPCR signaling.
Cellular metabolism is essential for cell proliferation, with abnormal metabolism a
principal hallmark of human cancer. De novo nucleotide biosynthesis is often
upregulated in cancer cells to provide building blocks for the synthesis of RNA,
DNA and other effectors [136]. However, nothing is known about the role of
GPCR signaling in modulating de novo purine biosynthesis. The two kinase
inhibitors, TBB and DMAT, have been found to regulate in an opposite way a
reversible multienzyme complex (i.e., purinosome) participating in de novo purine
biosynthesis in HeLa cells [137]. In conjunction with RNA interference, DMR
profiling showed that the two kinase inhibitors triggered opposite types of DMR
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