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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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70 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Hanson and Hanson
(labeled with either the lanthanide complex or the fluorescent molecule) and a
labeled cAMP which contains the other TR-FRET partner. Free cAMP within the
cell competes for binding to the labeled cAMP antibody disrupting TR-FRET and
leading to a loss in signal. An FP based version of this assay is also available from
Perkin-Elmer in which the fluorescence polarization of a fluorescently labeled
cAMP is studied. When this labeled-cAMP is bound to an anti-cAMP antibody,
the fluorescence will be more polarized than when cAMP within the cell is bound
to the antibody leaving the labeled-cAMP free in solution. However, FP based
assays are more susceptible to compound interfere nce than the T R-FRET ver sions
of the assays. The HitHunter
®
assay from DiscoveRx also uses a cAMP antibody,
but in this case the labeled-cAMP contains an enzyme fragment of the beta-
galactosidase enzyme. The enzyme-complementing partner of the beta-
galactosidase enzyme is also present in the assay. Free cAMP now competes with
the labeled cAMP for binding to the antibody. When free cAMP is bound, the
labeled-cAMP is available for enzyme complementation and creation of a
functional beta-galactosidase enzyme which can then be detected with a
fluorescent substrate. All of these assays work wi th cell lysates and can be used
for HTS [25]. cAMP assays are reviewed in much more detail in several recent
reviews [26,27].
Although IP(1 or 3) accumulation assays were originally developed as radioactive
displacement assays, there are now also s everal fluorescent based kits to look at
IP accumulation which are amenable with HTS. Cis-Bio offers an IP-One assay
which utilizes a europium-cryptate labeled IP
1
antibody donor and a d2-labeled
IP
1
acceptor fluorophore to produce a TR-FRET signal. As intracellular levels of
IP
1
increase there is a reduction of signal in this assay format. Alternatively,
DiscoveRx offers a HitHunter
®
assay which is an FP assay dependent upon
competitive binding between intracellular IP
3
and a fluorescently labeled tracer.
The reporter gene assay group for GPCRs include assays which again link a
response element with a reporter gene of interest. In general, a cAMP response
element is utilized to look at Gs coupled receptors and some Gi/o coupled
receptors if first stimulated with forskolin, and an NFAT response element is u sed
to look at Gq coupled receptors and Gi/o/s coupled receptors when used in
conjunction with chimeric or promiscuous G proteins. The reporter genes utilized
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Fluorescent Probes Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 71
for fluorescent assays are usually beta-lactamase (which utilizes a ratiometric
FRET substrate) or beta-galactosidase (which utilizes various fluorogenic
substrates). These assays are highly sensitive as amplification of signal occurs
through the signaling pathways monitored and can be used with live cells instead
of cell lysates allowing further study of the cells after the cAMP assay. Several
HTS campaigns for GPCRs have been enabled by these technologies [28,29].
Beta-arrestin/translocation based assays constitute a third group of assays for
GPCRs. Upon GPCR activation, GPCRs will almost universally recruit beta-
arrestin and in most cases cause internalization of the GPPCR, so various assay
formats have been developed to capitalize on this phenomenon. These assays all
have the advantage in that the natural signaling pathway of the GPCR of interest
does not need to be known for the assay to be utilized, and a single assay format
can be utilized for different classes of GPCRs (Gs, Gq, Gi/o). The first assay
developed within this category was the Transfluor
®
assay now commercialized by
Molecular Devices. The Transfluor
®
assay uses a GFP labeled bet a-arrestin and
was developed as an assay for high content imagers. With this assay, the GFP-
labeled beta-arrestin starts out cytoplasmically localized, is recruited to the cell
membrane upon GPCR activation and then becomes localized to cytoplasmic pits
or vesicles upon GPCR desensitization. The location of the GFP is then utilized to
determine the activation state of the GPCR. A similar assay called Redistribution
®
(available from Thermo-Fisher) instead labels the GPCR with the fluorescent
protein and internalization of the receptor is studied. Again this assay works best
with an imaging device. A bioluminescence resonance energy transfer (BRET)
assay was developed as a modification of the Transfluor
®
assay. With the
BRET
2
/beta-arrestin assay commercialized by Perkin-Elmer, a GFP-tagged beta-
arrestin is again utilized, but with this assay format the GPCR of interest is also
tagged with a luciferase construct. In the presence of the luciferase substrate and
when the luciferase and the GFP are in close proximity, BRET occurs from the
luciferase to the GFP, resulting in a fluorescence signal. This fluorescence signal
is then able to be monitored using a fluorescence plate reader, removing the
necessity of having a high content imager to read out the assay. Two new assays
have also recently hit the market within the beta-arrestin arena. These are the
PathHunter
®
assay from DiscoverX and the Tango™ assay from Life
72 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Hanson and Hanson
Technologies. The PathHunter
®
assay relies upon enzyme complementation of
beta-galactosidase. For this assay a portion of the beta-galactosidase enzyme is
linked to the GPCR of interest and the complementary portion is linked to the
beta-arrestin. Upon GPCR activation, the beta-arrestin is recruited to the GPC R,
enzyme complementation occurs and the now functional beta-galactosidase is
available to act on its substrate. With the Tango assay, a non-native transcription
factor is linked to the GPCR N terminus with a protease cleaveage site between
the transcription factor and the GPCR. A protease is then attached to the beta-
arrestin. When the GPCR is activated and beta-arrestin is recruited, this protease
will now cleave the transcription factor and the transcription factor will activate a
beta-lactamase reporter gene [30].
ION CHANNELS
Ion channels are a diverse group of membrane spanning, pore f orming proteins. In
general, ion channels control the flow of ions across biological membranes.
Ligand-gated channels are controlled by small molecules whereas voltage-gated
channels are dependant on membrane potential. Ion channels are not only
validated targets for drug discovery for epilepsy and other neurological disorders,
but are also safety targets. The human ether-a-go-go related gene (hERG), a
cardiac potassium channel, if blocked can lead to lethal ventricular arrhythmias
[31]. The ‘gold standard’ for evaluating ion channel activity is via
electrophysiology measurements or “patch clamping.” However traditional patch
clamping is time consuming and laborious, which has lead to the development of
fluorescent cell-based assays for ion channels including: direct ion detection,
surrogate ion detection, and voltage sensitive dye assays.
Direct ion detection of ion channel activity depends on the ability of a fluorescent
molecule to directly monitor the flow of ions through the channel and into the
intracellular space. Fluo dyes (as described in the GPCR section) are the most
commonly employed fluorescent dyes for direct ion detection of calcium flux
through channels. Other calcium sensitive indicators such as the fluorescent
protein based cameleon should also function to m onitor calcium flux through ion
channels. Since cameleon is a genetically encoded calcium sensor it can be
targeted to subcellular locations such as the intracellular side of the plasma
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Fluorescent Probes Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 73
membrane leading to more proximal detection of calcium flux through ion
channels.
Surrogate ion detection uses similar principles of the direct ion method with the
exception that non-native or surrogate ions are measured. Weaver and coworkers
noted the shortcomings of throughput, temporal resolution, sensitivity or
selectivity of traditional electrophysiology and radioactive rubidium flux assays
for potassium channels [32]. By exploiting the permeability of thallium through
potassium channels, Weaver and coworkers demonstrated ion channel activity can
be monitored by the fluorescent dye BTC-AM binding to thallium (Fig. 5). Cells
are loaded with thallium sensitive dye prior to compound addition. Compounds
are added prior to or at the same time as a thallium containing solution. Thallium
flux is monitored in kinetic mode analogous to a fluo-4 calcium assay on a
fluorescence plate reader with liquid handling capabilities. Functional ion channel
activity results in thallium influx. Thallium binding to BTC-AM results in an
Fig. (5). Schematic diagram of the FluxOR™ surrogate ion assay for potassium channels. Cells
are load ed with a f luoro gen ic thal lium sen sitiv e dye. Th alliu m is ad ded to th e extr acel lular m edia
surrounding the cells. In the “resting” state or closed conformation of the channel, then the
thallium remains segregated from the dye and little or no fluorescence is observed. Similarly if a
channel is blocked by a specific compound, little fluorescence increase is observed. However if a
potassium channel is open or “stimulated,” thallium flows into the cell, binds to the dye and
creates a highly green fluorescent complex.
74 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Hanson and Hanson
increased fluorescence signal compared to the dye alone. The thallium surrogate
ion detection assay is now commercially available as FluxOR™ from Life
Technologies.
Voltage sensitive dyes indicate ion channel activity by monitoring changes in
membrane potential. Voltage sensitive assay formats come in two flavors. The
single dye approach, which is commercialized by Molecular Devices, uses an
oxonol dye. This dye has the specialized property that it migrates within the lipid
bilayer of the plasma membrane depending on membr ane potential. In a resting
state, the cell interior has a relatively negative potential and the oxonol dye is
closely associated with the outer side of the plasma membrane. When the cell is
depolarized, the oxonol dye migrates to the inner side of the plasma membrane
where an increase in the fluorescence intensity of the dye takes place.
Alternatively, Life Technologies offers a voltage sensing assay technology that is
based on a pair of different wavelength dyes capable of FRET. The donor
fluorophore is a blue coumarin-phospholipid dye that is anchored on the
extracellular side of the plasma membrane. The acceptor dye is a red oxonol
(DiSBAC) dye that, like the single dye approach, migrates within the lipid bilayer
of the plasma membrane. In a resting state the cell interior has a relatively
negative potential and the oxonol dye is in close pr oximity to the coumarin donor ,
resulting in more efficient FRET. When the cell is depolarized, the oxonol dye
will migrate to the inner side of the plasma membrane, thus disrupting FRET.
Voltage sensitive dyes have the advantage of being applicable to any ion channel
target that changes the membrane potential and the assay can be performed in a
high throughput setting.
CELLULAR PATHWAY ANALYSIS
Cell signaling pathways are complex networks by which intracellular or
extracellular signals are detected and transduced into protein or gene expression
changes. Oftentimes, the signals are detected by receptors, mediated by kinases
through phosphorylation/dephosphorylation events, and terminated by
transcription factors altering gene expression via specific promoter elements.
Defects in cell signaling pathways can lead to a variety of disorders including
autoimmune disease and cancer.
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Fluorescent Probes Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 75
An important cell signaling pathway that is often studied is the apoptosis pathway.
Apoptosis is a process of programmed cell death that is mediated by intracellular
signaling pathways. There are numerous assayable apoptosis markers including
mitochondrial function, plasma membrane integrity, and phosphotidylserine
exposure. However, caspase activation remains one of the hallmark features of
cells undergoing apoptosis. Caspases or cysteine-aspartic acid specific pr oteases
can be monitored in cell lysates via peptides tagged with coumarin based
fluorophores. For the detection of caspase-1 activity, the peptide Trp-Glu-His-
Asp-AFC is often used. This peptide emits blue light (
max
= 400 nm) until
cleaved by caspase-1 or related caspases at which point free 7-Amino-
Trifluoromethyl Coumarin (AFC) emits a yellow-green fluorescence (
max
= 505
nm), which can be quantified using a fluorescence spectrophotometer or a
fluorescence microtiter plate reader [33]. Comparison of the fluorescence of AFC
from an apoptotic sample with an uninduced control allows determination of the
fold increase in caspase activity. Millipore, R&D Systems, and Calbiochem
provide commercially available fluorogenic assays for caspase detection from cell
lysates.
Life Technologies uses a fluorescent inhibitor of caspases (FLICA™)
methodology in their Vybrant
®
assay platform for studying caspase activity in live
cells. The FLICA reagent is comprised of three parts: 1) a fluoromethyl ketone
(FMK) moiety, 2) a carboxyfluorescein group (FAM) and 3) a valine-alanine-
aspartic acid tripeptide linker. The FLICA reagent is cell permeant and
noncytotoxic. The tripeptide region of FLICA is recognized by the caspase as a
potential substrate. Upon close association of the reagent with the caspase, the
FMK moiety covalently reacts with cysteines in the active site. Unbound FLICA
molecules diffuse out of the cell and are washed away; the remaining green-
fluorescent signal from the FAM group is a direct measure of the amount of active
caspase present in the cell at the time the FLICA reagent was added. FLICA
reagents are used widely to study apoptosis with flow cytometry and microscopy
and have even been applied to high content screening [34].
Detection of caspase 3,7 activation can be perfor med in a cellular context via a
fluorgenic substrate. The substrate contains the canonical caspase 3,7 peptide
cleavage sequence Asp-Glu-Val-Asp (DEVD), connected to a non-fluorescent
76 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Hanson and Hanson
DNA binding dye. Upon caspase activation, the peptide sequence is separated
from the DNA binding dye. The dye is then free t o migrate into the nucleu s, bind
DNA, and fluoresce. This assay methodology results in the illumination of
apoptotic cell nuclei in a green fluorescent color. The assay is commercially
available from the Life Technologies as C ellEvent™ caspase 3,7 reagent or from
Biotium as NucView™ 488 caspase-3 substrate.
Translocation or image based assays are another means to interrogate cell
signaling pathways. GFP is often the work-horse for translocation assays. For
example, fusion of GFP to a protein of interest, such as extracellular signal-
regulated kinase (ERK), allows the mitogen-activated protein kinase
(MAPK)/ERK signal transduction pathway to be studied. The basic pathway
couples intracellular responses to the binding of growth factors to cell surface
receptors. In many cell types, activation of this pathway promotes cell division. A
key step in the pathway is the migr ation of E RK from cytoplasm to the nucleus of
the cell. Therefore a GFP-ERK fusion results in diffuse cytoplasmic green
fluorescence being concentrated in the nucleus, allowing the pathway to be
followed by manual or automated fluorescence microscopy. Thermo Fisher
commercializes the Redistribution
®
technology, which couples GFP to cellular
targets for oncology, inflammation and other pathway analysis. Alter natively, the
Tango™ assay commercialized by Life Technologies or enzyme fragment
complementation (EFC) using beta-galactosidase and a fluorescent substrate as
commercialized by DiscoveRx can be used to study cellular pathways [30,35].
Cellular pathway analysis is also enabled by the use of beta-lactamase as a
reporter gene. Beta-lactamase is placed downstream of a particular promoter
sequence or response element. Upon external stimuli a signaling cascade is
initiated which terminates in beta-lactamase production. The extent of signaling is
evaluated by using a fluorescent substrate specific for beta-lactamase.
Alternatively, beta-galactosidase with an appropriate fluorescent sub strate can be
used, but without the ratiometric advantages of beta-lactamase.
The beta-lactamase approach commercialized by Life Technologies under the
name CellSensors
®
has been used to study the combined affects of RNAi and
small molecule inhibitors of epidermal growth factor receptor (EGFR) pathway
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Fluorescent Probes Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 77
components. O’Grady et al. showed 2.5-12 fold increased potency of small
molecule inhibitors of EGFR and MEK-1 in combination with RNAi directed
towards EGFR when stimulated with EGF [36]. The experiments were performed
using beta-lactamase downstream of an AP-1 response element. They postulated
that since the combination of these two targeted agents was shown to increase the
efficacy of EGFR and MEK-1 kinase inhibitors, possible implications for
overcoming or preventing drug resistance, lowering effective drug doses, and
providing new strategies for interrogating cellular signaling pathways were all
possible outcomes. This example nicely illustrates the ability of fluorescent
reporter genes to aid in practical, clinically relevant experiments.
MULTIPLEXING
The key to successful multiplexing with fluorescent probes is to be able to
distinguish the multiple parameters that are being studied. This could include
looking at the same event in different populations of cells or different events
occurring within the same cells. This can be done either through the use of
fluorophores with different excitation and/or emission parameters, by individually
tagging specific cells or populations of cells within a complex mixture, or by
temporal or spatial differences in the signals being observed. We will look at
several different versions of multiplexed assays within this review.
We can start by looking at localization assays. With these assays, specific cellular
compartments or proteins are labeled with two different fluorophores. This can be
done by using antibodies to specific proteins labeled with different fluorescent
dyes, by using organic fluorophores known to concentrate in certain
compartments (such as the MitoTracker
®
probes from Life T echnologies), or by
using fluorescent proteins to tag cellular compartments or specific proteins.
Although antibodies allow for very specific labeling of a protein of interest, unless
the protein is on the cell surface, live cell labeling is a challenge. With fluorescent
proteins on the other hand, fusions of the protein of interest can be made with a
fluorescent protein allowing the localization of that protein to be tracked in live
cells. Fluorescent proteins now exist in a wide range of colors [37] enabling the
differential labeling of multiple proteins or compartments without the need for
antibodies. Life Technologies has commercialized fluorescent proteins that label
78 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Hanson and Hanson
specific cellular compartments with their series of Organelle Lights™ reagents
[38]. These are baculovirus constructs which express in mammalian cell lines that
deliver different colors of fluorescent proteins to various subcellular
compartments to enable localization experiments.
A variation of localization type experiments comes with tracking populations of
cells or cell lineages. For these types of experiments, different populations of cells
are labeled with different fluorophores. The event of interest is then studied within
specific populations of cells as shown by Kr utzik et al. [39]. Reading the re sults
of these types of multiplexed assays requires the use of a flow cytometer or an
imaging device that scans for unique spectral signatures as well as for the
fluorescence, luminescence or other labels that re sult from the assay of interest.
Qdot
®
nanocrystals have been used for this application as they are extremely
photostable for several generations and are not transferred to adjacent cells in the
population. Qdots
®
are nanocrystals which can be excited at UV wavelengths and
will emit at various wavelengths depending upon the size on the nanocrystal.
Thus, different populations of cells can be labeled with dif ferent size Qdots
®
and
different fluorescence emission wavelengths will be observed f or each popul ation
while exciting with a single UV wavelength. Qtracker
®
products are being
commercialized by Life Technologies for these types of applications.
Variations of this type of assay can also be made by attaching different
biomolecules to the Qdot
®
nanocrystal or microsphere. T he presence of different
proteins or analytes within a complex mixture can then be studied. The Luminex
assay is another example of this type of assay. With the Luminex assay,
microspheres containing different ratios of red and infrared fluorophores are used
to give the microsphere a unique spectral signature. Antibodies to proteins of
interest can then be attached to the microspheres or nanocrystals, the
microspheres/nanocrystals can be incubated with a sample such as a cell lysate or
serum/plasma, a detection antibody can be added, and the sample can be analyzed
via flow cytometry or a specialized imaging device.
Other types of multiplexed assays can be used with plate readers without the need
for a flow cytometer or imaging device. These types of assays are generally
looking at two different cellular events within the same population of cells and are
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Fluorescent Probes Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 79
dependent upon having two independent signals that have a large enough assay
window to be distinguished within the population of cells. An example of this
type of assay would be the ToxBLAzer™ assay from Life Technologies [40].
This assay simultaneously looks at the viability of cells using a red fluorescence
marker and a GeneBLAzer
®
reporter gene assay that reports the results of a
particular pathway of interest with a ratiometric blue and green fluorescent
readout. Using the ToxBLAzer™ substrate, one can determine whether a
compound activates or inhibits a receptor or pathway of interest and whether or
not the compound is toxic to the cells. Another example would be the
multiplexing of a membrane potential assay with a calcium mobilization assay as
reported by Cassutt [41].
Another way to multiplex assays it to have the two signals of interest be separated
temporally. An example of this would be the multiplexing of a Ca
2+
mobilization
assay with a reporter gene assay for GPCRs [42]. For Gq coupled GPCRs,
activation of the receptor will lead to the release of calcium from intracellular
stores and will also activate the NFAT transcription factor. With calcium
mobilization assays, the calcium sensitive dye is loaded into the cells, and
compounds are added to the cells while the fluorescence emission is
simultaneously observed. An increase in the fluorescence emission spikes within
one minute of compound addition but will return to baseline values within several
minutes. The NFAT reporter gene on the other hand is dependent upon
transcription and translation of the reporter gene and generally takes several
hours. Therefore, it is possible to load cells with a calcium sensitive dye, add
compounds and record the results from the calcium dye, return the cells to the
incubator to allow the reporter gene to be activated, then detect the results from
the reporter gene assay. In this way two different cellular assays can be performed
on the same cells with the combined data having fewer false positives than either
assay format individually.
CONCLUSION
Fluorescent cell-based assays have been developed for major drug targets such as
kinases, GPCRs, ion channels, and cell signaling pathways. In many cases, the
fluorescent assays replace time consuming radioactivity assays and have many
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