Combinatorial Chemistry & High Throughput Screening
ISSN: 1386-2073
OPEN ACCESS PLUS
Contents
Two Panels of Steroid Receptor Luciferase Reporter
Cell Lines for Compound Profiling, 2011, 14, 248-266
David Sedlák, Aileen Paguio and
Petr Bartůněk
[Abstract] [Full
Text Article]
Building a Tiered Approach to In Vitro Predictive
Toxicity Screening: A Focus on Assays with In Vivo
Relevance, 2010, 13, 188-206
James M. McKim Jr.
[Abstract] [Full
Text Article]
Generating ‘Omic Knowledge’: The Role of Informatics
in High Content Screening, 2009, 12, 917-925
Mark A. Collins
[Abstract] [Full
Text Article]
Applications of High Content Screening in Life Science
Research, 2009, 12, 870-876
Joseph M. Zock
[Abstract] [Full
Text Article]
Use of Planar Array Electrophysiology for the Development
of Robust Ion Channel Cell Lines, 2009, 12, 96-106
Jeffrey J. Clare, Mao Xiang Chen, David
L. Downie, Derek J. Trezise and Andrew J. Powell
[Abstract] [Full
Text Article]
QPatch: The Missing Link Between HTS and Ion Channel
Drug Discovery, 2009, 12, 78-95
Chris Mathes, Søren Friis, Michael
Finley and Yi Liu
[Abstract] [Full
Text Article]
Combinatorics of Reaction-Network Posets,
2008, 11, 723-733
Douglas J. Klein, Teodora Ivanciuc, Anton Ryzhov
and Ovidiu Ivanciuc
[Abstract] [Full
Text Article]
Machine Learning for In Silico
Virtual Screening and Chemical Genomics: New Strategies,
2008, 11, 677-685
Jean-Philippe Vert and Laurent
Jacob
[Abstract] [Full
Text Article]
Fluorescent Probes for Cellular Assays,
2008, 11, 505-513
George T. Hanson and Bonnie
J. Hanson
[Abstract] [Full
Text Article]
Label-Free Cell-Based Assays for GPCR Screening,
2008, 11, 357-369
Ye Fang, Anthony G. Frutos and Ronald
Verklereen
[Abstract] [Full
Text Article]
Pharmacological Characterization of Ligands at Recombinant
NMDA Receptor Subtypes by Electrophysiological Recordings
and Intracellular Calcium Measurements, 2008, 11,
304-315
Kasper B. Hansen, Hans Bräuner-Osborne and
Jan Egebjerg
[Abstract] [Full
Text Article]
Back to Basics: Label-Free Technologies for Small Molecule
Screening, 2008, 11, 231-237
Andrew K. Shiau, Mark E. Massari and Can C. Ozbal
[Abstract] [Full
Text Article]
Discovery of Novel Targets with High Throughput RNA Interference
Screening, 2008, 11, 175-184
Paul D. Kassner
[Abstract] [Full
TextArticle]
High Throughput Screening of Gene Functions in Mammalian Cells
Using Reversely Transfected Cell Arrays: Review And Protocol,
2008, 11, 159-172
Michael Stürzl, Andreas Konrad, Gaby Sander, Effi
Wies, Frankeipel, Elisabeth Naschberger, Simone Reipschläger,
Nathalie Gonin-Laurent, Raymund E. Horch, Ulrich Kneser,
Werner Hohenberger, Holger Erfle and Mathias
Thurau
[Abstract] [Full
Text Article]
Yeast Genomics and Drug Target Identification,
2007, 10, 618-634
Nikë Bharucha and Anuj Kumar
[Abstract] [Full
Text Article]
Abstracts
[Back to top]
Two
Panels of Steroid Receptor Luciferase Reporter Cell Lines
for Compound Profiling
David Sedlák, Aileen Paguio and
Petr Bartůněk
[Full
Text Article]
Steroid hormone receptors represent a major target in
drug discovery. As ligand inducible transcription factors,
their activity can be modulated by small lipophilic molecules.
Here we describe two panels of potent and selective luciferase
reporter cell lines based on cells with low endogenous steroid
receptor activity (U2OS). The panels contain reporter cell
lines for estrogen receptors α
and β,
androgen, glucocorticoid, mineralocorticoid, and progesterone
receptors. In the first panel, the activation of either synthetic,
steroid response elements containing promoter or viral promoter
is mediated by full-length steroid receptors. The second panel
is based on the expression of the chimeric receptor, which
was created by the replacement of the N-terminal part of the
molecule by Gal4 DBD and that binds to multiple UAS sites
in the reporter promoter. Both panels were extensively characterized
by profiling 28 ligands in dose response manner in agonist
and antagonist mode. We have analyzed and compared the responses
to tested ligands from both panels and concluded that in general
both systems generated similar qualitative response in terms
of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic
profiles and the rank of potencies was well conserved between
both panels. However, we have also identified some artifacts
introduced by the Gal4/LBD reporter assays in contrast to
their full-length receptor reporter counterparts. Keeping
in mind the advantages and drawbacks of each reporter format,
these cell lines represent powerful and selective tools for
profiling large compound libraries (HTS) and for detailed
study of mechanisms by which compounds exert their biological
effects.
[Back to top]
Building a Tiered Approach to In Vitro Predictive
Toxicity Screening: A Focus on Assays with In Vivo
Relevance
James M. McKim Jr.
[Full
Text Article]
One of the greatest challenges facing the pharmaceutical industry
today is the failure of promising new drug candidates due
to unanticipated adverse effects discovered during preclinical
animal safety studies and clinical trials. Late stage attrition
increases the time required to bring a new drug to market,
inflates development costs, and represents a major source
of inefficiency in the drug discovery/development process.
It is generally recognized that early evaluation of new drug
candidates is necessary to improve the process. Building in
vitro data sets that can accurately predict adverse effects
in vivo would allow compounds with high risk profiles
to be deprioritized, while those that possess the requisite
drug attributes and a lower risk profile are brought forward.
In vitro cytotoxicity assays have been used for decades as
a tool to understand hypotheses driven questions regarding
mechanisms of toxicity. However, when used in a prospective
manner, they have not been highly predictive of in vivo
toxicity. Therefore, the issue may not be how to collect in
vitro toxicity data, but rather how to translate in vitro
toxicity data into meaningful in vivo effects. This
review will focus on the development of an in vitro toxicity
screening strategy that is based on a tiered approach to data
collection combined with data interpretation.
[Back to top]
Generating ‘Omic Knowledge’: The Role
of Informatics in High Content Screening
Mark A. Collins
[Full
Text Article]
High Content Screening (HCS) and High Content Analysis (HCA)
have emerged over the past 10 years as a powerful technology
for both drug discovery and systems biology. Founded on the
automated, quantitative image analysis of fluorescently labeled
cells or engineered cell lines, HCS provides unparalleled
levels of multi-parameter data on cellular events and is being
widely adopted, with great benefits, in many aspects of life
science from gaining a better understanding of disease processes,
through better models of toxicity, to generating systems views
of cellular processes. This paper looks at the role of informatics
and bioinformatics in both enabling and driving HCS to further
our understanding of both the genome and the cellome and looks
into the future to see where such deep knowledge could take
us.
[Back to top]
Applications of High Content Screening in Life Science
Research
Joseph M. Zock
[Full
Text Article]
Over the last decade, imaging as a detection mode for cell
based assays has opened a new world of opportunities to measure
“phenotypic endpoints” in both current and developing
biological models. These “high content” methods
combine multiple measurements of cell physiology, whether
it comes from sub-cellular compartments, multicellular structures,
or model organisms. The resulting multifaceted data can be
used to derive new insights into complex phenomena from cell
differentiation to compound pharmacology and toxicity. Exploring
the major application areas through review of the growing
compendium of literature provides evidence that this technology
is having a tangible impact on drug discovery and the life
sciences.
[Back to top]
Use of Planar Array Electrophysiology for the Development
of Robust Ion Channel Cell Lines
Jeffrey J. Clare, Mao Xiang Chen, David
L. Downie, Derek J. Trezise and Andrew J. Powell
[Full
Text Article]
The tractability of ion channels as drug targets has been
significantly improved by the advent of planar array electrophysiology
platforms which have dramatically increased the capacity for
electrophysiological profiling of lead series compounds. However,
the data quality and throughput obtained with these platforms
is critically dependent on the robustness of the expression
reagent being used. The generation of high quality, recombinant
cell lines is therefore a key step in the early phase of ion
channel drug discovery and this can present significant challenges
due to the diversity and organisational complexity of many
channel types. This article focuses on several complex and
difficult to express ion channels and illustrates how improved
stable cell lines can be obtained by integration of planar
array electrophysiology systems into the cell line generation
process per se. By embedding this approach at multiple
stages (e.g., during development of the expression strategy,
during screening and validation of clonal lines, and during
characterisation of the final cell line), the cycle time and
success rate in obtaining robust expression of complex multi-subunit
channels can be significantly improved. We also review how
recent advances in this technology (e.g., population patch
clamp) have further widened the versatility and applicability
of this approach.
[Back to top]
QPatch: The Missing Link Between HTS and Ion Channel
Drug Discovery
Chris Mathes, Søren Friis, Michael
Finley and Yi Liu
[Full
Text Article]
The conventional patch clamp has long been considered the
best approach for studying ion channel function and pharmacology.
However, its low throughput has been a major hurdle to overcome
for ion channel drug discovery. The recent emergence of higher
throughput, automated patch clamp technology begins to break
this bottleneck by providing medicinal chemists with high-quality,
information-rich data in a more timely fashion. As such, these
technologies have the potential to bridge a critical missing
link between high-throughput primary screening and meaningful
ion channel drug discovery programs. One of these technologies,
the QPatch automated patch clamp system developed by Sophion
Bioscience, records whole-cell ion channel currents from 16
or 48 individual cells in a parallel fashion. Here, we review
the general applicability of the QPatch to studying a wide
variety of ion channel types (voltage-/ligand-gated cationic/anionic
channels) in various expression systems. The success rate
of gigaseals, formation of the whole-cell configuration and
usable cells ranged from 40-80%, depending on a number of
factors including the cell line used, ion channel expressed,
assay development or optimization time and expression level
in these studies. We present detailed analyses of the QPatch
features and results in case studies in which secondary screening
assays were successfully developed for a voltage-gated calcium
channel and a ligand-gated TRP channel. The increase in throughput
compared to conventional patch clamp with the same cells was
approximately 10-fold. We conclude that the QPatch, combining
high data quality and speed with user friendliness and suitability
for a wide array of ion channels, resides on the cutting edge
of automated patch clamp technology and plays a pivotal role
in expediting ion channel drug discovery.
[Back to top]
Combinatorics of Reaction-Network Posets
Douglas J. Klein, Teodora Ivanciuc, Anton Ryzhov
and Ovidiu Ivanciuc
[Full
Text Article]
Reaction networks are viewed as derived from ordinary molecular
structures related in reactant-product pairs so as to manifest
a chemical super-structure. Such super-structures
then are candidates for applications in a general combinatoric
chemistry. Notable additional characterization of a reaction
super-structure occurs when such reaction graphs are directed,
as for example when there is progressive substitution (or
addition) on a fixed molecular skeleton. Such a set of partially
ordered entities is in mathematics termed a poset,
which further manifests a number of special properties, as
then might be utilized in different applications.
Focus on the overall "super-structural" poset goes
beyond ordinary molecular structure in attending to how a
structure fits into a (reaction) network, and thereby brings
an extra "dimension" to conventional stereochemical
theory. The possibility that different molecular properties
vary smoothly along chains of interconnections in such a super-structure
is a natural assumption for a novel approach to molecular
property and bioactivity correlations. Different manners to
interpolate/extrapolate on a poset network yield quantitative
super-structure/activity relationships (QSSARs),
with some numerical fits, e.g., for properties of polychlorinated
biphenyls (PCBs) seemingly being quite reasonable. There seems
to be promise for combinatoric posetic ideas.
[Back to top]
Machine Learning for In Silico Virtual Screening
and Chemical Genomics: New Strategies
Jean-Philippe Vert and Laurent
Jacob
[Full
Text Article]
Support vector machines and kernel methods belong to the same
class of machine learning algorithms that has recently become
prominent in both computational biology and chemistry, although
both fields have largely ignored each other. These methods
are based on a sound mathematical and computationally efficient
framework that implicitly embeds the data of interest, respectively
proteins and small molecules, in high-dimensional feature
spaces where various classification or regression tasks can
be performed with linear algorithms. In this review, we present
the main ideas underlying these approaches, survey how both
the “biological” and the “chemical”
spaces have been separately constructed using the same mathematical
framework and tricks, and suggest different avenues to unify
both spaces for the purpose of in silico chemogenomics.
[Back to top]
Fluorescent Probes for Cellular Assays
George T. Hanson and Bonnie
J. Hanson
[Full Text Article]
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]
Label-Free Cell-Based Assays for GPCR Screening
Ye Fang, Anthony G. Frutos and Ronald
Verklereen
[Full Text Article]
G protein-coupled receptors (GPCRs) have been
proven to be the largest family of druggable targets in the
human genome. Given the importance of GPCRs as drug targets
and the de-orphanization of novel targets, GPCRs are likely
to remain the frequent targets of many drug discovery programs.
With recent advances in instrumentation and understanding
of cellular mechanisms for the signals measured, biosensor-centered
label-free cell assay technologies become a very active area
for GPCR screening. This article reviews the principles and
potential of current label-free cell assay technologies in
GPCR drug discovery.
[Back to top]
Pharmacological Characterization of Ligands at Recombinant
NMDA Receptor Subtypes by Electrophysiological Recordings
and Intracellular Calcium Measurements
Kasper B. Hansen, Hans Bräuner-Osborne and
Jan Egebjerg
[Full
Text Article]
Generation of in vitro cellular assays using
fluorescence measurements at heterologously expressed NMDA
receptors would speed up the process of ligand characterization
and enable high-throughput screening. The major drawback to
the development of such assays is the cytotoxicity caused
by Ca2+-flux into the cell
via NMDA receptors upon prolonged activation by agonists
present in the culture medium. In the present study, we established
four cell lines with stable expression of NMDA receptor subtypes
NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D in BHK-21 cells.
To assess the usefulness of the stable cell lines in conjunction
with intracellular calcium ([Ca2+]i)
measurements for evaluation of NMDA receptor pharmacology,
several ligands were characterized using this method. The
results were compared to parallel data obtained by electrophysiological
recordings at NMDA receptors expressed in Xenopus oocytes.
This comparison showed that agonist potencies determined by
[Ca2+]i measurements
and electrophysiological recordings correlated well, meaning
that the stable cell lines in conjunction with [Ca2+]i
measurements provide a useful tool for characterization of
NMDA receptor ligands. The agonist series of conformationally
constrained glutamate analogues (2S,3R,4S)-α
(carboxycyclopropyl)glycine (CCG), 1-aminocyclobutane-r-1,cis-3-dicarboxylic
acid (trans-ACBD), and (±)-1-
aminocyclopentane-r-1,cis-3-dicarboxylic acid (cis-ACPD),
as well as the highly potent agonist tetrazolylglycine were
among the characterized ligands that were assessed with respect
to subtype selectivity at NMDA receptors. However, none of
the characterized agonists displays more than 2-3 fold selectivity
towards a specific NMDA receptor subtype. Thus, the present
study provides a broad pharmacological characterization of
structurally diverse ligands at recombinant NMDA receptor
subtypes.
[Back to top]
Back to Basics: Label-Free Technologies for Small Molecule
Screening
Andrew K. Shiau, Mark E. Massari and Can C. Ozbal
[Full Text Article]
Small molecule high-throughput screening in drug
discovery today is dominated by techniques which are dependent
upon artificial labels or reporter systems. While effective,
these approaches can be affected by certain experimental limitations,
such as conformational restrictions imposed by the selected
label or compound fluorescence/quenching. Label-free approaches
potentially address many of these issues by allowing researchers
to investigate more native systems without fluorescence or
luminescence-based readouts. However, due to throughput and
expense constraints, label-free methods have been largely
relegated to a supporting role as the basis of secondary assays.
In this review, we describe recent improvements in impedance-based,
optical biosensor-based,automated patch clamp and mass spectrometry
technologies that have enhanced their ease of use and throughput
and,hence, their utility for primary screening of small- to
mediumsized compound libraries. The ultimate maturation of
these techniques will enable drug discovery researchers to
screen large chemical libraries against minimally manipulated
biological systems.
[Back to top]
Discovery of Novel Targets with High Throughput RNA Interference
Screening
Paul D. Kassner
[Full Text Article]
High throughput technologies have the potential
to affect all aspects of drug discovery. Considerable attention
is paid to high throughput screening (HTS) for small molecule
lead compounds. The identification of the targets that enter
those HTS campaigns had been driven by basic research until
the advent of genomics level data acquisition such as sequencing
and gene expression microarrays. Large-scale profiling approaches
(e.g., microarrays, protein analysis by mass spectrometry,
and metabolite profiling) can yield vast quantities of data
and important information. However, these approaches usually
require painstaking in silico analysis and low-throughputbasic
wet-lab research to identify the function of a gene and validate
the gene product as a potential therapeutic drug target.Functional
genomic screening offers the promise of direct identification
of genes involved in phenotypes of interest. In this review,
RNA interference (RNAi) mediated loss-of-functionscreens will
be discussed and as well as their utility in target identification.
Some of the genes identified in these screens should produce
similar phenotypes if their gene products are antagonized
with drugs. With a carefully chosen phenotype, an understanding
of the biology of RNAi and appreciation of the limitations
of RNAi screening, there is great potential for the discovery
of new drug targets.
[Back to top]
High Throughput Screening of Gene Functions in Mammalian Cells
Using Reversely Transfected Cell Arrays: Review And Protocol
Michael Stürzl, Andreas Konrad, Gaby Sander, Effi
Wies, Frankeipel, Elisabeth Naschberger, Simone Reipschläger,
Nathalie Gonin-Laurent, Raymund E. Horch, Ulrich Kneser,
Werner Hohenberger, Holger Erfle and Mathias
Thurau
[Full
Text Article]
Reversely transfected cell microarrays (RTCM) have
been introduced as a method for parallel high throughput analysis
of gene functions in mammalian cells. Hundreds to thousands
of different recombinant DNA or RNA molecules can be transfected
into different cell clusters at the same time on a single
glass slide with this method. This allows either the simultaneous
overexpression or - by using the recently developed RNA interference
(RNAi) techniques - knockdown of a huge number of target genes.
A growing number of sophisticated detection systems have been
established to determine quantitatively the effects of the
transfected molecules on the cell phenotype. Several different
cell types have been successfully used for this procedure.
This review summarizes the presently available knowledge on
this technique and provides a laboratory protocol.
[Back to top]
Yeast Genomics and Drug Target Identification
Nikë Bharucha and Anuj Kumar
[Full
Text Article]
The budding yeast Saccharomyces cerevisiae is well
recognized as a preferred eukaryote for the development of
genomic technologies and approaches. Accordingly, a sizeable
complement of genomic resources has been developed in yeast,
and this genomic foundation is now informing a wide variety
of disciplines. In particular, yeast genomic methodologies
are gaining an expanding foothold in drug development studies,
most notably as a preliminary tool towards drug target identification.
In this review, we highlight many applications of yeast genomics
in the identification of targeted genes and pathways of small
molecules or therapeutic drugs. The applicability of genome-wide
resources of yeast disruption and deletion mutants for drug-sensitivity/resistance
screening is presented here, along with a summary of microarray
technologies for drug-based transcriptional profiling and
synthetic interaction mapping. Applications of protein-interaction
traps for potential drug target identification are also considered.
Collectively, this overview of yeast genomics emphasizes the
growing intersection between high-throughput model organism
biology and medicinal chemistry — an intersection promising
tangible advances for both academic and pharmaceutical fields
alike.
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