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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 5, June 2006
Contents
Analysing the Output from Primary Screening Pp.
331-37
Dawn Nowlin, Patrick Bingham, Andrew Berridge, Philip Gribbon,
Philip Laflin and Andreas Sewing
[Abstract]
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Automated High Content Screening for Phosphoinositide 3 Kinase
Inhibition Using an AKT1 Redistribution Assay Pp.
339-350
Michael Wolff, Dorothea Haasen, Susanne Merk, Margareta Kroner,
Udo Maier, Sandra Bordel, Jörg Wiedenmann, Gerd Ulrich
Nienhaus,Martin Valler and Ralf Heilker
[Abstract] [Purchase
Article]
Successful Screening of Large Encoded Combinatorial
Libraries Leading to the Discovery of Novel p38 MAP Kinase
Inhibitors Pp. 351-358
Axel Metzger, David J. Diller, Tsung H. Lin, Ian Henderson
and Maria L. Webb
[Abstract] [Purchase
Article]
A Pseudo-Ligand Approach to Virtual Screening
Pp. 359-364
Andreas Schüller, Uli Fechner, Steffen Renner, Lutz
Franke, Lutz Weber and Gisbert Schneider
[Abstract] [Purchase
Article]
Microarray Technology as a Universal Tool for High-Throughput
Analysis of Biological Systems Pp. 365-380
Jens Sobek, Kerstin Bartscherer, Anette Jacob, Jvrg D. Hoheisel
and Philipp Angenendt
[Abstract] [Purchase
Article]
Application of Chemical Arrays in Screening Elastase
Inhibitors Pp. 381-388
Feng Gao and Guan-Hua Du
[Abstract] [Purchase
Article]
Microarray and Nanotechnology Applications of Functional
Nanoparticles Pp.389-397
Seidy Pedroso and Isabel Alicia Guillen
[Abstract] [Purchase
Article]
Nitrogen-Containing Macrocycles as Host Molecules
for the Recognition of Undissociated Phenol Derivatives: Mechanism
of Potentiometric Signal Generation Pp. 399-406
Jerzy Radecki and Wim Dehaen
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Article]
Abstracts
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Analysing the Output from Primary Screening
Dawn Nowlin, Patrick Bingham, Andrew Berridge, Philip Gribbon,
Philip Laflin and Andreas Sewing
From a perspective of process knowledge and enhancement,
the analysis of the results of biological screening should
not be limited to the outcome of specific projects, but additionally
encompass a process centric view. Summarising outcomes across
multiple projects is a powerful tool to gain a greater understanding
of biological screening that will also enable optimisation
of the strategy for specific projects or target classes. We
have analysed a set of 73651 compounds with reproducible (confirmed)
results from 63 high-throughput screening (HTS) campaigns
to reveal the underlying trends in the population of active
compounds. We have focused on the overall physico-chemical
profile of compound populations derived from biological screening
since the in vivo activity of drug molecules is the
result of physico-chemical and structural properties of the
compound.
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Automated High Content Screening for Phosphoinositide
3 Kinase Inhibition Using an AKT1 Redistribution Assay
Michael Wolff, Dorothea Haasen, Susanne Merk, Margareta Kroner,
Udo Maier, Sandra Bordel, Jörg Wiedenmann, Gerd Ulrich
Nienhaus,Martin Valler and Ralf Heilker
High Content Screening (HCS), a combination of fluorescence
microscopic imaging and automated image analysis, has become
a frequently applied tool to study test compound effects in
cellular disease-modelling systems. In this work, we established
a medium to high throughput HCS assay in the 384-well format
to measure cellular type I phosphoinositide 3 kinase (PI3K)
activity. Type I PI3K is involved in several intracellular
pathways such as cell survival, growth and differentiation
as well as immunological responses. As a cellular model system
we used Chinese Hamster Ovary (CHO) cells that had been stably
transfected with human insulin receptor (hIR) and an AKT1-enhanced
green fluorescent protein (EGFP) fusion construct. Upon stimulation
of the hIR with insulin-like growth factor-1 (IGF-1), PI3K
was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate
at the 3-position, resulting in the recruitment of AKT1-EGFP
to the plasma membrane.
The AKT1-EGFP redistribution assay was robust and displayed
little day-to-day variability, the quantification of the fluorescence
intensity associated with plasma membrane spots delivered
good Z’ statistics. A novel format of compound dose-response
testing was employed using serial dilutions of test compounds
across consecutive microtiter plates (MTPs). The dose response
testing of a PI3K inhibitor series provided reproducible IC50
values. The profiling of the redistribution assay with isoform-selective
inhibitors indicates that PI3Kα
is the main isoform activated in the CHO host cells after
IGF-1 stimulation. Toxic compound side effects could be determined
using automated image analysis.
We conclude that the AKT1-EGFP redistribution assay represents
a solid medium/high throughput screening (MTS/HTS) format
to determine the cellular activity of PI3K inhibitors under
conditions of growth factor stimulation.
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Successful Screening of Large Encoded Combinatorial
Libraries Leading to the Discovery of Novel p38 MAP Kinase
Inhibitors
Axel Metzger, David J. Diller, Tsung H. Lin, Ian Henderson
and Maria L. Webb
Screening of more than 2 million compounds comprising
41 distinct encoded combinatorial libraries revealed a novel
structural class of p38 mitogen-activated protein (MAP) kinase
inhibitors. The methodology used for screening large encoded
combinatorial libraries combined with the statistical interpretation
of screening results is described. A strong preference for
a particular triaminotriazine aniline amide was discovered
based on biological activity observed in the screening campaign.
Additional screening of a focused follow-up combinatorial
library yielded data expanding the unique combinatorial SAR
and emphasizing an extraordinary preference for this particular
building block and structural class. The preference is further
highlighted when the p38 inhibitor data set is compared to
data obtained for a panel of other kinases.
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A Pseudo-Ligand Approach to Virtual Screening
Andreas Schüller, Uli Fechner, Steffen Renner, Lutz
Franke, Lutz Weber and Gisbert Schneider
A virtual screening method is presented that is grounded
on a receptor-derived pharmacophore model termed “virtual
ligand” or “pseudo-ligand”. The model represents
an idealized constellation of potential ligand sites that
interact with residues of the binding pocket. For rapid virtual
screening of compound libraries the potential pharmacophore
points of the virtual ligand are encoded as an alignment-free
correlation vector, avoiding spatial alignment of pharmacophore
features between the pharmacophore query (i.e., the virtual
ligand) and the candidate molecule. The method was successfully
applied to retrieving factor Xa inhibitors from a Ugi three-component
combinatorial library, and yielded high enrichment of actives
in a retrospective search for cyclooxygenase-2 (COX-2) inhibitors.
The approach provides a concept for “de-orphanizing”
potential drug targets and identifying ligands for hitherto
unexplored or allosteric binding pockets.
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Microarray Technology as a Universal Tool for High-Throughput
Analysis of Biological Systems
Jens Sobek, Kerstin Bartscherer, Anette Jacob, Jvrg D. Hoheisel
and Philipp Angenendt
Over the last years microarray technology has become
one of the principal platform technologies for the high-throughput
analysis of biological systems. Starting with the construction
of first DNA microarrays in the 1990s, microarray technology
has flourished in the last years and many different new formats
have been developed. Peptide and protein microarrays are now
applied for the elucidation of interaction partners, modification
sites and enzyme substrates. Antibody microarrays are envisaged
to be of high importance for the high-throughput determination
of protein abundances in translational profiling approaches.
First cell microarrays have been constructed to transform
microarray technology from an in vitro technology
to an in vivo functional analysis tool. All of these
approaches share a common prerequisite: the solid support
on which they are generated. The demands on this solid support
are thereby as manifold as the applications themselves. This
review is aimed to display the recent developments in surface
chemistry and derivatization, and to summarize the latest
developments in the different application areas of microarray
technology.
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Application of Chemical Arrays in Screening Elastase
Inhibitors
Feng Gao and Guan-Hua Du
Protein chip technology provides a new and useful tool for
high-throughput screening of drugs because of its high performance
and low sample consumption. In order to screen elastase inhibitors
on a large scale, we designed a composite microarray integrating
enzyme chip containing chemical arrays on glass slides to
screen for enzymatic inhibitors. The composite microarray
includes an active proteinase film, screened chemical arrays
distributed on the film, and substrate microarrays to demonstrate
change of color. The detection principle is that elastase
hydrolyzes synthetic colorless substrates and turns them into
yellow products. Because yellow is difficult to detect, bromochlorophenol
blue (BPB) was added into substrate solutions to facilitate
the detection process. After the enzyme had catalyzed reactions
for 2 h, effects of samples on enzymatic activity could be
determined by detecting color change of the spots. When chemical
samples inhibited enzymatic activity, substrates were blue
instead of yellow products. If the enzyme retained its activity,
the yellow color of the products combined with blue of BPB
to make the spots green. Chromogenic differences demonstrated
whether chemicals inhibited enzymatic activity or not. In
this assay, 11,680 compounds were screened, and two valuable
chemical hits were identified, which demonstrates that this
assay is effective, sensitive and applicable for high-throughput
screening (HTS).
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Microarray and Nanotechnology Applications of Functional
Nanoparticles
Seidy Pedroso and Isabel Alicia Guillen
Microarrays are a sensitive, specific, miniaturized devices
that may be used to detect selected DNA sequences and proteins,
or mutated genes associated with human diseases. Several methods
have been developed to detect the binding of complementary
molecules to microarrays by generating an optical signal.
One of the most commonly used molecular labeling methods at
present is fluorescence, but its application is expensive
due to sophisticated equipment required to design the platform,
hybridize it, and interpret the images derived from microarray-based
studies. This is a drawback for its use in laboratories and
clinical services. Another less expensive procedure having
similar sensitivity and specificity is DNA and protein functional
nanoparticles (FNP). Nanoparticles are sphere-like biocompatible
materials made of inert silica, metal or crystals of a nanometer
in size, which are generally coated with a thin gold layer.
They may be used as hybridization probes in single nucleotide
polymorphism (SNP) screening and to detect biological markers
for cancer, infection, and cardiovascular diseases.
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Nitrogen-Containing Macrocycles as Host Molecules
for the Recognition of Undissociated Phenol Derivatives: Mechanism
of Potentiometric Signal Generation
Jerzy Radecki and Wim Dehaen
The mechanism of the generation of potentiometric signals
of corrole- and calix[4]pyrrole-containing liquid membrane
electrodes was elucidated and compared in the presence of
the neutral forms of phenol derivatives. In addition, the
influence of lipophilic, anionic or cationic salts on this
phenomenon was explored. Finally, the relationship between
the acid-base properties of the phenolic guests and the hydrogen
bond donor activity of the calix[4]pyrrole and corrole hosts,
and the influence on the molecular recognition phenomenon
occurring at the organic/aqueous interface are discussed.
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