Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 11, Number 4, May 2008
Contents
Integrating Chemists Preferences for Shape-Similarity
Clustering of Series Pp. 266-282
Laurent A. Baumes, Remi Gaudin, Pedro Serna,
Nicolas Nicoloyannis and Avelino Corma
[Abstract]
High-Content Screening and Mechanism-Based Evaluation
of Estrogenic Botanical Extracts Pp. 283-293
Cassia R. Overk, Ping Yao, Shaonong Chen, Shixing
Deng, Ayano Imai, Matthew Main, Andreas Schinkovitz, Norman
R. Farnsworth, Guido F. Pauli and Judy L. Bolton
[Abstract]
Alkylsquarates as Key Intermediates for the Rapid
Preparation of Original Drug-Inspired Compounds Pp.
294-303
Julie Charton, Lise Charruault, Rebecca Deprez-Poulain
and Benoit Deprez
[Abstract]
Pharmacological Characterization of Ligands at
Recombinant NMDA Receptor Subtypes by Electrophysiological
Recordings and Intracellular Calcium Measurements
Pp. 304-315
Kasper B. Hansen, Hans Bräuner-Osborne and
Jan Egebjerg
[Abstract]
Functional Human 5-HT6 Receptor Assay
for High Throughput Screening of Chemical Ligands and Binding
Proteins Pp. 316-324
Hyun-Ji Kim, Hyung-Mun Yun, Taehyun Kim, Ghilsoo
Nam, Eun Joo Roh, Evi Kostenis, Hea-Young Park Choo and Ae
Nim Pae and Hyewhon Rhim
[Abstract]
Immunoassays: Biological Tools for High Throughput
Screening and Characterisation of Combinatorial Libraries
Pp. 325-335
M. Ângela Taipa
[Abstract]
Abstracts
[Back to top]
Integrating Chemists Preferences for Shape-Similarity
Clustering of Series
Laurent A. Baumes, Remi Gaudin, Pedro Serna,
Nicolas Nicoloyannis and Avelino Corma
This study shows how chemistry knowledge and reasoning are
taken into account for building a new methodology that aims
at automatically grouping data having a chronological structure.
We consider combinatorial catalytic experiments where the
evolution of a reaction (e.g., conversion) over time
is expected to be analyzed. The mathematical tool has been
developed to compare and group curves taking into account
their shape. The strategy, which consists on combining a hierarchical
clustering with the k-means algorithm, is described and compared
with both algorithms used separately. The hybridization is
shown to be of great interest. Then, a second application
mode of the proposed methodology is presented. Once meaningful
clusters according to chemist’s preferences and goals
are successfully achieved, the induced model may be used in
order to automatically classify new experimental results.
The grouping of the new catalysts tested for the Heck coupling
reaction between styrene and iodobenzene verified the set
of criteria “defined” during the initial clustering
step, and facilitated a quick identification of the catalytic
behaviors following user’s preferences.
[Back to top]
High-Content Screening and Mechanism-Based Evaluation
of Estrogenic Botanical Extracts
Cassia R. Overk, Ping Yao, Shaonong Chen, Shixing
Deng, Ayano Imai, Matthew Main, Andreas Schinkovitz, Norman
R. Farnsworth, Guido F. Pauli and Judy L. Bolton
Symptoms associated with menopause can greatly affect the
quality of life for women. Botanical dietary supplements have
been viewed by the public as safe and effective despite a
lack of evidence indicating a urgent necessity to standardize
these supplements chemically and biologically. Seventeen plants
were evaluated for estrogenic biological activity using standard
assays: competitive estrogen receptor (ER) binding assay for
both alpha and beta subtypes, transient transfection of the
estrogen response element luciferase plasmid into MCF-7 cells
expressing either ER alpha or ER beta, and the Ishikawa alkaline
phosphatase induction assay for both estrogenic and antiestrogenic
activities. Based on the combination of data pooled from these
assays, the following was determined: a) a high rate of false
positive activity for the competitive binding assays, b) some
extracts had estrogenic activity despite a lack of ability
to bind the ER, c) one extract exhibited selective estrogen
receptor modulator (SERM) activity, and d) several extracts
show additive/synergistic activity. Taken together, these
data indicate a need to reprioritize the order in which the
bioassays are performed for maximal efficiency of programs
involving bioassay-guided fractionation. In addition, possible
explanations for the conflicts in the literature over the
estrogenicity of Cimicifuga racemosa (black cohosh)
are suggested.
[Back to top]
Alkylsquarates as Key Intermediates for the Rapid
Preparation of Original Drug-Inspired Compounds
Julie Charton, Lise Charruault, Rebecca Deprez-Poulain
and Benoit Deprez
Many natural privileged scaffolds contain a basic nitrogen
atom, which often is a key element of pharmacophore and a
chemically reactive centre as well. In our ongoing research
program devoted to the design of targeted libraries based
on acidic templates, we developed methods to convert privileged
basic compounds -like natural alkaloids or drugs into acidic
compounds. This conversion led to a profound alteration of
the pharmacophore, without changing the overall shape and
lipophilicity of the molecule. We expect such modifications
to generate unexpected biological activities. Recently, we
focused on derivatives of squaric acid, a vinylogous carboxylic
acid. Two series were studied. First we describe a new, selective
parallel synthesis of squaramic acids from a dissymmetric
diester (3-tert-butoxy-4-ethoxycyclobut-3-en-1,2-dione). This
efficient procedure avoids the synthesis of the undesired
squaramides. Secondly we describe a microplate parallel synthesis
(15 µmol-scale) of squaric acid hydroxamate amides from
a squaric hydroxamate ester.
[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
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]
Functional Human 5-HT6 Receptor Assay for
High Throughput Screening of Chemical Ligands and Binding
Proteins
Hyun-Ji Kim, Hyung-Mun Yun, Taehyun Kim, Ghilsoo
Nam,
Eun Joo Roh, Evi Kostenis, Hea-Young Park Choo and Ae Nim
Pae and Hyewhon Rhim
Continuous identification and validation of novel drug
targets require the development of rapid, reliable, and sensitive
cell-based high-throughput screening (HTS) methods for proposed
targets. Recently, the 5-HT6
receptor (5-HT6R), a member
of the class of recently discovered 5-HT receptors, has received
considerable attention for its possible implications in depression,
cognition, and anxiety. However, the cellular signaling mechanisms
of 5-HT6R are poorly understood
due to the lack of selective 5-HT6R
ligands. In the present study, we examined functional coupling
of the human 5-HT6R, 5-HT7AR,
or 5-HT7BR with various Gα-proteins
(Gα15,
Gα qs5,
or Gα qG66Ds5)
to develop a reliable cell-based HTS method for 5-HT receptors.
Among variable couplings between 5-HT receptors and G-proteins,
we found that functional coupling of human 5-HT6R
with GαqG66Ds5
produced the highest levels of Ca2+
signaling in HEK293 cells as measured by the fluorescence-based
HTS plate reader, FDSS6000. After validation of this new 5-HT6R
HTS system (Z´-factor = 0.56) in 96-well plates and
characterization of the pharmacological profile of the 5-HT6R,
we screened ~500 synthetic chemical compounds including butanamide
and benzenesulfonamide derivatives. Based on this preliminary
screening, we found that the butanamide derivative LSG11104
produced an IC50
value of 6.3 μM.
This compound will serve as a lead structure for further chemical
modification to develop novel 5-HT6R
ligands. Furthermore, we demonstrated that this HTS method
can be utilized to identify proteins that modulate 5-HT6R
function and present Fyn tyrosine kinase as an example, which
is already known as a 5-HT6R
interacting protein. Taken together, these results suggest
that the 5-HT6R/GαqG66Ds5
FDSS6000 system can be utilized to screen for selective 5-HT6R
ligands and to examine any functional relationships between
5-HT6R and its binding proteins.
[Back to top]
Immunoassays: Biological Tools for High Throughput
Screening and Characterisation of Combinatorial Libraries
M. Ângela Taipa
In the demanding field of proteomics, there is an urgent need
for affinity-catcher molecules to implement effective and
high throughput methods for analysing the human proteome or
parts of it. Antibodies have an essential role in this endeavour,
and selection, isolation and characterisation of specific
antibodies represent a key issue to meet success. Alternatively,
it is expected that new, well-characterised affinity reagents
generated in rapid and cost-effective manners will also be
used to facilitate the deciphering of the function, location
and interactions of the high number of encoded protein products.
Combinatorial approaches combined with high throughput screening
(HTS) technologies have become essential for the generation
and identification of robust affinity reagents from biological
combinatorial libraries and the lead discovery of active/mimic
molecules in large chemical libraries. Phage and yeast display
provide the means for engineering a multitude of antibody-like
molecules against any desired antigen. The construction of
peptide libraries is commonly used for the identification
and characterisation of ligand-receptor specific interactions,
and the search for novel ligands for pro-tein purification.
Further improvement of chemical and biological resistance
of affinity ligands encouraged the “intelligent”
design and synthesis of chemical libraries of low-molecular-weight
bio-inspired mimic compounds. No matter what the ligand source,
selection and characterisation of leads is a most relevant
task. Immunological assays, in microtiter plates, biosensors
or microarrays, are a biological tool of inestimable value
for the iterative screening of combinatorial ligand libraries
for tailored specificities, and improved affinities. Particularly,
enzyme-linked immunosorbent assays are frequently the method
of choice in a large number of screening strategies, for both
biological and chemical libraries.
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