Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 11, Number 7, August 2008
Contents
Cell-Based Screening
Guest Editor: Guido J.R. Zaman
Editorial Pp. 494
Functional Cell-Based Assays in Microliter Volumes
for Ultra High Throughput Screening Pp. 495-504
Frank Wunder, Bernd Kalthof, Thomas Müller
and Jörg Hüser
[Abstract]
Fluorescent Probes for Cellular Assays
Pp. 505-513
George T. Hanson and Bonnie J. Hanson
[Abstract]
Ion Channel Screening Pp. 514-522
John Dunlop, Mark Bowlby, Ravikumar Peri, Gregory
Tawa, James LaRocque, Veronica Soloveva and John
Morin
[Abstract]
High-Content Analysis of Kinase Activity in Cells
Pp. 523-536
Fabio Gasparri, Francesco Sola, Tiziano Bandiera,
Jürgen Moll and Arturo Galvani
[Abstract]
Profiling of Multiple Signal Pathway Activities
by Multiplexing Antibody and GFP-Based Translocation Assays
Pp. 537-544
Ulla Henriksen, Jacob Fog, Frosty Loechel
and Morten Præstegaard
[Abstract]
A Miniaturized Glucocorticoid Receptor Translocation
Assay Using Enzymatic Fragment Complementation Evaluated with
qHTS Pp. 545-559
Ping Jun Zhu, Wei Zheng, Douglas S. Auld, Ajit
Jadhav, Ryan MacArthur, Keith R. Olson, Kun Peng, Hyna Dotimas,
Christopher P. Austin and James Inglese
[Abstract]
The Use of Immortalized Cell Lines in GPCR Screening:
The Good, Bad and Ugly Pp. 560-565
Richard M. Eglen, Annette Gilchrist and
Terry Reisine
[Abstract]
An Overview of Drug Screening Using Primary and Embryonic
Stem Cells Pp. 566-572
Richard M. Eglen, Annette Gilchrist and
Terry Reisine
[Abstract]
Label-Free Cell-Based Functional Assays Pp.
573-580
Lisa K. Minor
[Abstract]
Meet
the Guest Editors Pp . 581
Abstracts
[Back to top]
Editorial
Although in vitro biochemical assays, such
as enzyme activity and receptor binding, have been used extensively
in the past to discover new drugs, there is a rapid increase
in the use of assays based on living cells. Cell-based assays
provide a target in a more physiologically relevant environment
than biochemical assays. According to a worldwide survey involving
more than 50 pharmaceutical and biotech companies, more than
50% of all primary screens are currently cell-based [1]. Due
to the use of cryopreserved and division-arrested cells, cell-based
screening also has become more efficient and more flexible
[2]. This special issue of Combinatorial Chemistry &
High Throughput Screening is devoted to cell-based screening.
Review articles were collected on screening in live cells
of all major drug target classes, i.e. receptors,
ion channels and protein kinases. New technology developments
are discussed, such as high content analysis, label-free assays
and the use primary and embryonic stem cells.
In the first article, Jörg Hüser and colleagues
from Bayer HealthCare AG (Wuppertal, Germany) review the technological
approaches used for ultra-High Throughput Screening (ultra-HTS).
‘Ultra’ means automated screening of more than
100,000 data points per day. To differentiate target hits
from non-specifically acting compounds, integral reference
signals are incorporated into the ultra-HTS assays. George
Hanson and Bonnie Hanson from Invitrogen Discovery Sciences
(Madison, WI) give an overview of fluorescent probes and types
of fluorescent assays that are applied in cellular assays
for a number of pharmaceutically relevant target classes,
including protein kinases and ion channels. They also discuss
cellular pathway analysis and the combining of multiple read-outs
in one assay (multiplexing). With recent technological advances
in fluorescent probes, the search for novel therapeutics targeting
ion channels is accelerating. John Dunlop and colleagues of
Wyeth Research (Princeton, NJ) review the various techniques
used in screening ion channel targets. Real-life examples
of the screening of a ligand-gated ion channel and a voltage-gated
channel are presented to demonstrate the utility of fluorescence-based
screening.
‘High content analysis’ refers to techniques involving
the multiplexed analysis of fluorescent markers to measure
multiple cellular responses to biological stimuli or drug
treatment at the single-cell level. High content analysis
is usually based on automated microscopy and provides multiparametric
information on single cells within a population. Fabio Gasparri
and colleagues from Nerviano Medical Sciences S.r.l. (Nerviano,
Italy) review the concepts and techniques of high content
analysis of protein kinases, with an emphasis on kinases implicated
in oncology. The assay methods are illustrated with data from
the author’s laboratory of research on cell cycle kinase
inhibitors. Morten Præstegaard and colleagues of Thermo
Fisher Scientific Inc. (Søborg, Denmark) describe different
strategies of multiplexing green fluorescent protein-based
and immunofluorescence translocation assays. The authors differentiate
between multiplexing of readouts in the same signal transduction
pathway (vertical multiplexing) and of readouts across different
signal transduction pathways (horizontal multiplexing). Examples
are shown of multiplexing assays in the p38 MAPK pathway and
of the activation and internalization of G protein-coupled
receptors.
Jim Inglese and colleagues of the Chemical Genomics Center
of the National Institutes of Health (Bethesda, MD) present
a case study of the screening of the glucocorticoid receptor
with a translocation assay based on enzyme fragment complementation.
A ‘quantitative’ screening approach was used,
which means that compounds were screened at multiple concentrations
in the primary run. Quantitative HTS increases the information
content of HTS.
Richard Eglen of PerkinElmer (Waltham, MA), Annette Gilchrist
of Caden Biosciences (Madison, WI) and Terry Reisine review
the disparities that have been found in the pharmacology of
compounds acting on G protein-coupled receptors in recombinant
cells used for screening and in natural tissues. The efficiency
of identifying effective therapeutics may increase when natural
tissues are used more in the drug discovery process. In the
following article, Eglen et al. review the use of
primary and embryonic stem cells for screening. Human stem
cells offer unique opportunities in that they can be directed
to specific phenotypes, providing a framework to identify
tissue-selective agents.
In the last article, Lisa Minor from Johnson & Johnson
Pharmaceutical Research Institute (Spring House, PA) reviews
the application of electrical impedance and refractive index
to measure cell-based functional response. These label-free
technologies are rapidly generating interest because they
allow measuring phenotypic responses without the addition
of exogenous labels or extraction of the cells.
I would like to thank the authors for contributing to this
special issue.
REFERENCES
[1] Fox, S.; Farr-Jones, S.; Sopchak, L.; Boggs, A.; Wang
Nicely, H.; Khoury, R.; Biros, M. J. Biomol. Screen.,
2006, 11, 864.
[2] Zaman, G.J.R.; de Roos, J.A.D.M.; Blomenröhr, M.;
van Koppen, C.J.; Oosterom, J. Drug Discov. Today,
2007, 12, 521.
Guido J.R. Zaman
N.V. Organon
(A Part of Schering-Plough Corporation)
P.O. Box 20
5340 BH
Oss
The Netherlands
E-mail: guido.zaman@organon.com
[Back to top]
Functional Cell-Based Assays in Microliter Volumes for Ultra
High Throughput Screening
Frank Wunder, Bernd Kalthof, Thomas Müller
and Jörg Hüser
Functional cell-based assays have gained increasing importance
for microplate-based high throughput screening (HTS). The
use of high-density microplates, most prominently 1536-well
plates, and miniaturized assay formats allow screening of
comprehensive compound collections with more than 1 million
compounds at ultra-high throughput, i.e. in excess of 100,000
samples per day. uHTS operations with numerous campaigns per
year should generally support this throughput at all different
steps of the process, including the underlying compound logistics,
the (automated) testing of the corporate compound collection
in the bioassay, and the subsequent follow-up studies for
hit confirmation and characterization.
A growing number of reports document the general feasibility
of cell-based uHTS in microliter volumes. In addition, full
automation with integrated robotic systems allows the realization
of also complex assay protocols with multiple liquid handling
and signal detection steps. For this review, cell-based assays
are categorized based on the kinetics of the cellular response
to be quantified in the test and the readout method employed.
Thus, assays measuring fast cellular responses with high temporal
resolution, e.g., receptor mediated calcium signals or changes
in membrane potential, are at one end of this spectrum, while
tests quantifying cellular transcriptional responses mark
the opposite end. Trends for cell-based uHTS assays developed
at Bayer-Schering Pharma are, first, to incorporate assay
integral reference signals allowing the experimental differentiation
of target hits from non-specifically acting compounds, and
second, to make use of kinetic, real-time readouts providing
additional information on the mode-of-action of test compounds.
[Back to top]
Fluorescent Probes for Cellular Assays
George T. Hanson and Bonnie J. Hanson
A fluorescent probe is a fluorophore designed to localize
within a specific region of a biological specimen or to respond
to a specific stimulus. Fluorescent probes have been used
for nearly a century to study cellular processes due to their
exquisite sensitivity and selectivity. Fluorescent probes
have also gained in popularity as safety and environmental
concerns over the use of radioactive probes have grown. At
the same time, cellular assays are being more widely used
now than ever before. This review will give a broad overview
of types of fluorescent probes, types of fluorescent assays,
and their application in cellular assays for a number of pharmaceutically
relevant target classes.
[Back to top]
Ion Channel Screening
John Dunlop, Mark Bowlby, Ravikumar Peri, Gregory
Tawa, James LaRocque, Veronica Soloveva and John
Morin
Ion channels are attractive targets for drug discovery
with recent estimates indicating that voltage and ligandgated
channels account for the third and fourth largest gene families
represented in company portfolios after the G protein coupled
and nuclear hormone receptor families. A historical limitation
on ion channel targeted drug discovery in the form of the
extremely low throughput nature of the gold standard assay
for assessing functional activity, patch clamp electrophysiology
in mammalian cells, has been overcome by the implementation
of multi-well plate format cell-based screening strategies
for ion channels. These have taken advantage of various approaches
to monitor ion flux or membrane potential using radioactive,
non-radioactive, spectroscopic and fluorescence measurements
and have significantly impacted both high-throughput screening
and lead optimization efforts. In addition, major advances
have been made in the development of automated electrophysiological
platforms to increase capacity for cell-based screening using
formats aimed at recapitulating the gold standard assay. This
review addresses the options available for cell-based screening
of ion channels with examples of their utility and presents
case studies on the successful implementation of high-throughput
screening campaigns for a ligandgated ion channel using a
fluorescent calcium indicator, and a voltage-gated ion channel
using a fluorescent membrane potential sensitive dye.
[Back to top]
High-Content Analysis of Kinase Activity in Cells
Fabio Gasparri, Francesco Sola, Tiziano Bandiera,
Jürgen Moll and Arturo Galvani
High-content analysis (HCA) is a term used to describe
techniques involving multiplexed analysis of fluorescent markers
to measure multiple cellular responses to biological stimuli
or drug treatment. HCA is usually based on automated microscopy
or related technologies, and its value lies in providing multiparametric
information on single cells within a population. During the
last decade, several HCA approaches have been developed and
applied to assess cellular mechanism of action of pharmacologically
relevant compounds identified through biochemical screening
or similar in vitro methods. With automation and
instrument development, these approaches have evolved to the
extent that the technique is now routinely used in screening
applications, including primary HTS on compound collections.
Here, we review the field and discuss in particular the application
of HCA to the discovery of small molecule inhibitors targeting
kinases which are implicated in Oncology.
[Back to top]
Profiling of Multiple Signal Pathway Activities
by Multiplexing Antibody and GFP-Based Translocation Assays
Ulla Henriksen, Jacob Fog, Frosty Loechel
and Morten Præstegaard
Multiplexing of GFP based and immunofluorescence translocation
assays enables easy acquisition of multiple readouts from
the same cell in a single assay run. Immunofluorescence assays
monitor translocation, phosphorylation, and up/down regulation
of endogenous proteins. GFP-based assays monitor translocation
of stably expressed GFP-fusion proteins. Such assays may be
multiplexed along (vertical), across (horizontal), and between
(branch) signal pathways. Examples of these strategies are
presented: 1) The MK2-GFP assay monitors translocation of
MK2-GFP from the nucleus to the cytoplasm in response to stimulation
of the p38 pathway. By applying different immunofluorescent
assays to the MK2 assay, a multiplexed HCA system is created
for deconvolution of p38 pathway activation including assay
readouts for MK2, p38, NFκB,
and c-Jun. 2) A method for evaluating GPCR activation and
internalization in a single assay run has been established
by multiplexing GFP-based internalization assays with immunofluorescence
assays for downstream transducers of GPCR activity: pCREB
(cAMP sensor), NFATc1 (Ca2+ sensor), and ERK (G-protein
activation). Activation of the AT1 receptor is given as an
example. 3) Cell toxicity readouts can be linked to primary
readouts of interest via acquisition of secondary
parameters describing cellular morphology. This approach is
used to flag cytotoxic compounds and deselect false positives.
The ATF6 Redistribution assay is provided as an example. These
multiplex strategies provide a unique opportunity to enhance
HCA data quality and save time during drug discovery. From
a single assay run, several assay readouts are obtained that
help the user to deconvolute the mode of action of test compounds.
[Back to top]
A Miniaturized Glucocorticoid Receptor Translocation
Assay Using Enzymatic Fragment Complementation Evaluated with
qHTS
Ping Jun Zhu, Wei Zheng, Douglas S. Auld, Ajit
Jadhav, Ryan MacArthur, Keith R. Olson, Kun Peng, Hyna Dotimas,
Christopher P. Austin and James Inglese
Nuclear translocation is an important step in glucocorticoid
receptor (GR) signaling and assays that measure this process
allow the identification of nuclear receptor ligands independent
of subsequent functional effects. To facilitate the identification
of GR-translocation agonists, an enzyme fragment complementation
(EFC) cell-based assay was scaled to a 1536-well plate format
to evaluate 9,920 compounds using a quantitative high throughput
screening (qHTS) strategy where compounds are assayed at multiple
concentrations. In contrast to conventional assays of nuclear
translocation the qHTS assay described here was enabled on
a standard luminescence microplate reader precluding the requirement
for imaging methods. The assay uses β-galactosidase
α complementation
to indirectly detect GR-translocation in CHO-K1 cells. 1536-well
assay miniaturization included the elimination of a media
aspiration step, and the optimized assay displayed a Z' of
0.55. qHTS yielded ECM50
values for all 9,920 compounds and allowed us to retrospectively
examine the dataset as a single concentration-based screen
to estimate the number of false positives and negatives at
typical activity thresholds. For example, at a 9 μM
screening concentration, the assay showed an accuracy that
is comparable to typical cell-based assays as judged by the
occurrence of false positives that we determined to be 1.3%
or 0.3%, for a 3s or 6σ
threshold, respectively. This corresponds to a confirmation
rate of ~30% or ~50%, respectively. The assay was consistent
with glucocorticoid pharmacology as scaffolds with close similarity
to dexamethasone were identified as active, while, for example,
steroids that act as ligands to other nuclear receptors such
as the estrogen receptor were found to be inactive.
[Back to top]
The Use of Immortalized Cell Lines in GPCR Screening:
The Good, Bad and Ugly
Richard M. Eglen, Annette Gilchrist and
Terry Reisine
For most membrane-bound molecular targets, including
G protein linked receptors (GPCRs), the optimal approach in
drug discovery involves the use of cell based high throughput
screening (HTS) technologies to identify compounds that modulate
target activity. Most GPCRs have been cloned and can therefore
be routinely expressed in immortalized cell lines. These cells
can be easily and rapidly grown in unlimited quantities making
them ideal for use in current HTS technologies.
A significant advantage of this approach is that immortalized
recombinant cells provide a homogenous background for expression
of the target which greatly facilitates consistency in screening,
thus allowing for a better understanding of the mechanism
of action of the interacting compound or drug. Nonetheless,
it is now evident that numerous disparities exist between
the physiological environment of screening systems using recombinant
cells and natural tissues. This has lead to a problem in the
validity of the pharmacological data obtained using immortalized
cells in as much as such cells do not always reflect the desired
clinical efficacy and safety of the compounds under examination.
This brief review discusses these issues and describes how
they influence the discovery of drugs using modern HTS.
[Back to top]
An Overview of Drug Screening Using Primary and Embryonic
Stem Cells
Richard M. Eglen, Annette Gilchrist and
Terry Reisine
Cellular technologies are widely used in drug discovery
to treat human diseases. Most studies involve the expression
of recombinant targets in immortalized cells and measure drug
interactions using simple, quantifiable responses. Such cells
are also amenable to high throughput screening (HTS) methods.
However, the cell phenotype employed in HTS is often determined
by the assay technology available, rather than the physiological
relevance of the cell background. They are, therefore, suboptimal
surrogates for cells that accurately reflect human diseases.
Consequently, there is growing interest in adopting primary
and embryonic stem cells in drug discovery. Primary cells
are already used in secondary screening assays in conjunction
with confocal imaging techniques, as well as in target validation
studies employing, for example, gene silencing approaches.
Stem cells can be grown in unlimited quantities and can be
derived from transgenic animals engineered to express disease
causing proteins better coupling the molecular target with
function in vivo. Human stem cells also offer unique
opportunities for drug discovery in that they can be directed
to specific phenotypes thus providing a framework to identify
tissueselective agents. Organizing stem cells into networks
resembling those in native tissues, potentially returns drug
discovery back to the highly successful pharmacological methods
of the past, in which organ and tissue based systems were
used, but with the advantage that they can be utilized using
modern HTS technologies. This emerging area will lead to discovery
of compounds whose effect in vivo is more predictable
thereby increasing the efficiency of drugs that ameliorate
human disease.
[Back to top]
Label-Free Cell-Based Functional Assays
Lisa K. Minor
Label-free technologies based on electrical impedance
or refractive index are new tools for measuring a cell-based
functional response. Although the technologies are relatively
new to high throughput screening cell-based applications,
they are rapidly generating interest in that they are able
to measure a phenotypic response using cells natively expressing
the target protein without using dyes or cellular extracts.
In addition, one can measure the cellular response using a
kinetic mode resulting in an assay potentially rich in content.
This article will describe these technologies and their applications
in measuring cell proliferation, cell attachment and spreading,
cell apoptosis and their application for several receptor
target classes, including receptor tyrosine kinases and G
protein-coupled receptors. The potential utility and drawbacks
of these tools for high throughput screening, directed screening
and compound profiling will also be discussed.
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