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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 1, January 2006
Contents
Editorial Pp. 1
Fast Mass Spectrometry-Based Methodologies for
Pharmaceutical Analyses Pp. 3-8
Yunsheng Hsieh, Elaine Fukuda, Julie Wingate and Walter A.
Korfmacher
[Abstract]
Development of a Novel High-Throughput Assay for the Investigation
of GlyT-1b Neurotransmitter Transporter Function
Pp. 9-14
Lynda Allan, J. Lianne Leith, Marianthi Papakosta, John A.Morrow,
Nicholas G. Irving, Brian W. McFerran and Alan G. Clark
[Abstract]
Straightforward Three-Component Palladium-Catalysed
Synthesis of Pyridazin-3(2H)-one Libraries Pp. 15
-19
Alberto Coelho and Eddy Sotelo
[Abstract]
Development of a Fluorescence Peptide Chip for
the Detection of Caspase Activity Pp. 21-25
Aishan Han, Tatsuhiko Sonoda, Jeong-Hun Kang, Masaharu
Murata, Takuro NIiidome and Yoshiki Katayama
[Abstract]
Anti-Endotoxin Agents. 3. Rapid Identification of High-Affinity
Lipopolysaccharide-Binding Compounds in a Substituted Polyamine
Library Pp. 27-36
Stewart J. Wood, Kelly A. Miller, Gerald H. Lushington,
Mark R. Burns and Sunil A. David
[Abstract]
Comparison of G-Protein Coupled Receptor Desensitization-Related
β-Arrestin Redistribution Using Confocal and Non-Confocal
Imaging Pp. 37-47
Dorothea Haasen, Michael Wolff, Martin J. Valler and Ralf
Heilker
[Abstract]
Recognition of Protein Coding Genes in the Yeast Genome
Based on the Relative-Entropy of DNA Pp. 49-54
Chun Li, Nadia Helal and Jun Wang
[Abstract]
A High-Throughput Phage Display Screening Method Using
a Combination of Real-Time PCR and Affinity Chromatography
Pp. 55-61
Kengo Morohashi, Tsuyoshi Arai, Seiich Saito, Madoka Watanabe,
Kengo Sakaguchi and Fumio Sugawara
[Abstract]
Oligosaccharide Synthesis and Library Assembly by
One-Pot Sequential Glycosylation Strategy Pp.
63-75
Yuhang Wang, Li-He Zhang and Xin-Shan Ye
[Abstract]
Abstracts
[Back to top]
Editorial
With this issue Combinatorial Chemistry &
High Throughput Screening begins its ninth year of publication.
CCHTS continues to publish review articles and original
research papers in all areas of combinatorial chemistry and
high throughput screening. This issue, for example, contains
both review articles (see the review by Hsieh, et al.,
of fast mass spectrometric assays that are being used to enhance
the throughput of drug metabolism and pharmacokinetics studies)
and research papers (see the paper by Allan, et al.,
describing the development of a new fluorescence-based HTS
assay for inhibitors of glycine transporters). Synthetic organic
chemistry papers featuring new routes to combinatorial libraries
will continue to appear in CCHTS, such as the research
paper by Coelho and Sotelo in this issue that describes palladium
catalyzed multicomponent reactions leading to the synthesis
of pyridazinone libraries. In addition, the development of
new HTS assays will be covered; see for example the paper
by Han, et al., describing a new peptide chip screening
approach for caspase activity. As always, the use of screening
assays for applicants such as drug discovery will continue
to appear in CCHTS, such as the paper by Wood, et
al., that concerns the use of HTS to discover anti-endotoxin
agents.
As in the past, regular issues will be alternated with special
issues focusing on a single topic of current interest. Some
of these special issues will be organized by members of our
Editorial Board whereas others will be organized by guest
editors. For example, the second issue of CCHTS to
be published in 2006 has been organized by Dr. Donald Kyle,
who is a member of the Editorial Board. Dr. Kyle’s special
issue will address how to increase the productivity of drug
discovery programs, and topics to be covered will include
library synthesis strategies based on computational predictions
of biological activity, utilization of corporate databases,
and comparison of approaches for sequential library screening.
Whether appearing in a special issue addressing a hot topic
or in a regular issue, all papers published in CCHTS
will continue to be peer-reviewed.
In 2005, eight issues of CCHTS were published, and
this frequency of publication was the highest of any journal
in the field of combinatorial chemistry or high throughput
screening. Due to the large number of manuscripts in the publication
queue, the number of issues of CCHTS planned for
2006 will be increased to ten. Therefore, CCHTS will
set a new standard this year as it continues to be the publication
leader in this field.
Papers published in CCHTS are abstracted and indexed
by the major services including Chemical Abstracts, BIOSIS,
CAB Abstracts, EMBASE, BIOBASE, Science Citation Index-Expanded,
MEDLINE, and Current Contents (Life Sciences). The homepage
of our journal and abstracts of articles may be found at the
following Internet address: http://www.bentham.org/cchts.
Information for authors may also be found at our website.
Manuscripts may be submitted in print or electronic format,
and our readers and subscribers will continue to be able to
obtain CCHTS in printed or electronic format. Through
the combination of frequent publication and high visibility,
Combinatorial Chemistry & High Throughput Screening
remains a unique and essential scientific journal defining
the intersection of these two interdependent disciplines.
I would like to thank the distinguished members of the Editorial
Board, the Regional Editors, the guest editors, the authors,
and of course you, the readers, for the continuing success
of our journal.
Richard B. van Breemen
(Editor-in-Chief)
University of Illinois
College of Pharmacy
833 S. Wood Street
Chicago
IL 60612
USA
E-mail: breemen@uic.edu
[Back to top]
Fast Mass Spectrometry-Based Methodologies for Pharmaceutical
Analyses
Yunsheng Hsieh, Elaine Fukuda, Julie Wingate and Walter A.
Korfmacher
Historically, most bioanalytical methods for drug analysis
in pharmaceutical industry were developed using HPLC coupled
with UV or fluorescence detection. However, there is a trend
toward interfacing separation technologies with more sensitive
tandem mass spectrometry (MS/MS)-based systems. MS/MS detection
offers complete resolution of the parent compounds from their
first pass metabolites to avoid extra efforts for separation
and sample clean-up procedures resulting in shorter run times.
With the increasing demand for ever faster screening, there
is a continuing demand for bioanalytical methods possessing
higher sample throughput for both in vitro and in
vivo drug metabolism and pharmacokinetic evaluations
to accelerate the discovery process. This review focuses on
the current approaches for fast MS-based assays (cycle-time
less than 5 min) of pharmaceuticals and their metabolites
that have been reported in the peer-reviewed publications.
[Back to top]
Development of a Novel High-Throughput Assay for the
Investigation of GlyT-1b Neurotransmitter Transporter Function
Lynda Allan, J. Lianne Leith, Marianthi Papakosta, John A.Morrow,
Nicholas G. Irving, Brian W. McFerran and Alan G. Clark
The glycine transporter (GlyT-1b) is a Na+/Cl--dependent
electrogenic transporter which mediates the rapid re-uptake
of glycine from the synaptic cleft. Based on its tissue distribution,
GlyT-1 has been suggested to co-localise with the NMDA receptor
where it may modulate the concentration of glycine at its
co-agonist binding site. This data has led to GlyT-1 inhibitors
being proposed as targets for disorders such as schizophrenia
and cognitive dysfunction. Radiolabelled uptake assays (e.g.
[3H]glycine) have been traditionally used in compound
screening to identify glycine transporter inhibitors. While
such an assay format is useful for testing limited numbers
of compounds, the identification of novel glycine uptake inhibitors
requires a functional assay compatible with high-throughput
screening (HTS) of large compound libraries. Here, the authors
present the development of a novel homogenous cell-based assay
using the FLIPR membrane potential blue dye (Molecular Devices)
and FLEXstation. Pharmacological data for the GlyT-1 inhibitors
Org 24598 and ALX 5407 obtained using this novel electrogenic
assay correlated well with the conventional [3H]-glycine
uptake assay format. Furthermore, the assay has been successfully
miniaturised using FLIPR3 and therefore has the
potential to be used for high-throughput screening.
[Back to top]
Straightforward Three-Component Palladium-Catalysed
Synthesis of Pyridazin-3(2H)-one Libraries
Alberto Coelho and Eddy Sotelo
A simple and straightforward three-component methodology
for the solution phase high-throughput synthesis of 2,5-disubstituted
pyridazin-3(2H)-one libraries has been developed.
The diversity at position 5 of the heterocycle is determined
by the use of Suzuki, Sonogashira, Heck or Stille coupling.
[Back to top]
Development of a Fluorescence Peptide Chip for the
Detection of Caspase Activity
Aishan Han, Tatsuhiko Sonoda, Jeong-Hun Kang, Masaharu
Murata, Takuro NIiidome and Yoshiki Katayama
Proteases play a key role in cell functions, and it is very
important to monitor their activities for drug screening and
diagnosis of diseases. In the present study, a new class of
fluorescence probe, into which a fluorophore and a quencher
have been introduced, was developed and applied to the on-chip
detection of caspase-3 activity. This probe is non fluorescent
in the absence of caspase-3. However, when it is treated with
active caspase-3, the fluorescence intensity increases dependent
on the caspase-3 activity due to the cleavage of the quencher-containing
moiety on a glass slide. This caspase-dependent increase in
the fluorescence intensity was also detected when the glass
slide immobilizing the probe peptide was treated with cell
lysate stimulated by staurosporine (STP), which is an apoptosis-inducing
agent. On the other hand, such an increase was not detected
in the case of control cell lysate without STP-stimulation.
The developed system is a rapid and sensitive method and is
useful for the direct measurement of protease activity on
a glass array.
[Back to top]
Anti-Endotoxin Agents. 3. Rapid Identification of
High-Affinity Lipopolysaccharide-Binding Compounds in a Substituted
Polyamine Library
Stewart J. Wood, Kelly A. Miller, Gerald H. Lushington,
Mark R. Burns and Sunil A. David
Lipopolysaccharides (LPS), otherwise termed ‘endotoxins’,
are an integral part of the outer leaflet of the outer-membrane
of Gram-negative bacteria. Lipopolysaccharides play a pivotal
role in the pathogenesis of ‘Septic Shock’, a
major cause of mortality in the critically ill patient, worldwide.
The sequestration of circulatory endotoxin may be a viable
therapeutic strategy for the prophylaxis and treatment of
Gram-negative sepsis. We have earlier shown that the pharmacophore
necessary for small molecules to bind LPS involves two protonatable
cationic functions separated by about 15 Å, permitting
the simultaneous interaction with the negatively charged phosphates
on lipid A, the toxically active center of endotoxin. In this
report, screening of a multi-thousand membered polyamine library
through the combined use of computational and bioassay-guided
screens resulted in the discovery of two novel classes of
LPS-binding agents. These are represented by the 1) spermine
sulfonamides and 2) C-aryl-substituted spermine analogs. We
present the selection approach, screening results, computational
multivariate analyses and initial structure-activity relationship
evaluation herein.
[Back to top]
Comparison of G-Protein Coupled Receptor Desensitization-Related
β-Arrestin Redistribution Using Confocal and Non-Confocal
Imaging
Dorothea Haasen, Michael Wolff, Martin J. 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 compared
a confocal and a non-confocal cellular HCS system, the IN
Cell Analyzers1 3000TM and 1000TM, respectively.
As a cellular model system we used the TransfluorTM 2
technology in the 384-well microtiter plate (MTP) format.
The Transfluor HCS assay for G-protein coupled receptor (GPCR)
activation is based on the recruitment of a green fluorescent
protein-labelled arrestin (ArrGFP) from the cytosol to the
plasma membrane. We investigated two GPCRs, the wild-type
(wt) β2
adrenergic receptor (β2AR)
and the β2AR-enhanced
(E), a C-terminally mutated receptor with a higher affinity
to arrestin. Upon agonist stimulation, the β2AR-wt
induced the redistribution of ArrGFP to coated pits, the β2AR-E
maintained the interaction with ArrGFP down to the formation
of endocytic vesicles. Our findings reveal that the assay
is feasible on both instruments, with sufficiently robust
Z’ statistics. Improved Z’ statistics, though,
are achieved with the confocal system, particularly in case
of weak signals. Moreover, throughput is dramatically higher
for the IN Cell Analyzer 3000. We conclude that, depending
on the needs for throughput and assay biology, either instrument
may fulfil a successful role in the drug discovery process.
Confocal optics, however, provide a better basis for the detection
of smaller subcellular structures with lower fluorescence
intensity.
[Back to top]
Recognition of Protein Coding Genes in the Yeast Genome
Based on the Relative-Entropy of DNA
Chun Li, Nadia Helal and Jun Wang
In this paper, a new protein coding gene-finding algorithm
specific for the yeast genome at 96% accuracy is proposed.
Five-fold cross-validation tests are performed to confirm
the above accuracy. By the new algorithm, the number of protein
coding genes in the yeast genome is re-estimated. The estimate
is based on the assumption that the unknown genes have similar
statistical properties to the known genes. It is found that
the number of protein coding genes in the 16 yeast chromosomes
is less than or equal to 5873, significantly coincident with
the widely accepted range 5800-6000.
[Back to top]
A High-Throughput Phage Display Screening Method Using
a Combination of Real-Time PCR and Affinity Chromatography
Kengo Morohashi, Tsuyoshi Arai, Seiich Saito, Madoka Watanabe,
Kengo Sakaguchi and Fumio Sugawara
Phage display is one of the best methods to identify drug
targets, although technical problems including imprecision
in quantifying phage and false-positive results are common.
To address these difficulties, we propose two methods to more
rapidly identify drug-binding phage particles. First, quantification
of phage using SYBR Green Real-time PCR significantly improved
accuracy and reproducibility. Second, affinity-column chromatography
for selection of drug-binding phage particles concentrated
particles more than a 96-well plate, making a phage amplification
step, which can bias phage distribution, unnecessary. The
methods proposed here should be suitable for high-throughput
phage-display screenings and ultimately lead to more rapid
identification of drug targets.
[Back to top]
Oligosaccharide Synthesis and Library Assembly by
One-Pot Sequential Glycosylation Strategy
Yuhang Wang, Li-He Zhang and Xin-Shan Ye
The oligosaccharide residue in glycoconjugates located in
cell membranes is responsible for intercellular recognition
and interaction: it acts as a receptor for proteins, hormones,
and microorganisms and governs immune reactions. These significant
activities have stimulated great interest in the field of
oligosaccharides and glycoconjugates. Although many advances
have been made in the synthesis of oligosaccharides, more
convenient and efficient methods are still needed. This review
describes one of these new methods—the one-pot sequential
glycosylation approach as a potent tool for oligosaccharide
assembly. The oligosaccharide library construction in a one-pot
fashion is also summarized.
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