Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 11, Number 1, January 2008
Contents
Development of a Screening Assay for Ligands
to the Estrogen Receptor Based on Magnetic Microparticles
and LC-MS Pp. 1-6
Yongsoo Choi and Richard B. van Breemen
[Abstract]
Anti-Cancer Natural Product Library from Traditional
Chinese Medicine Pp. 7-15
V. Badirenaath Konkimalla and Thomas Efferth
[Abstract]
Post-SELEX Chemical Optimization of a Trypanosome
Specific RNA Aptamer Pp. 16-23
Annette Adler, Nicole Forster, Matthias Homann
and H. Ulrich Göringer
[Abstract]
A High-Complexity, Multiplexed Solution-Phase Assay
for Profiling Protease Activity on Microarrays
Pp. 24-35
Igor A. Kozlov, Peter C. Melnyk, John P. Hachmann, Anu Srinivasan,
Melissa Shults, Chanfeng Zhao, Joseph Musmacker, Nicholas
Nelson, David L. Barker and Michal Lebl
[Abstract]
The Combinatorial Synthesis of Organophosphorus
Compounds Pp. 36-61
Bernhard Lesch, Douglas W. Thomson and Stephen D. Lindell
[Abstract]
Targeted Therapy of the Insulin-Like Growth Factor-1
Receptor in Cancer Pp. 62-69
Keren Paz and Yaron R. Hadari
[Abstract]
A Chemical Genetics Approach for Specific Differentiation
of Stem Cells to Somatic Cells: A New Promising Therapeutical
Approach Pp. 70-82
Agapios Sachinidis, Isaia Sotiriadou, Bianca Seelig, Albrecht
Berkessel and Jürgen Hescheler
[Abstract]
Abstracts
[Back to top]
Development of a Screening Assay for Ligands
to the Estrogen Receptor Based on Magnetic Microparticles
and LC-MS
Yongsoo Choi and Richard B. van Breemen
A high throughput screening assay for the identification of
ligands to pharmacologically significant receptors was developed
based on magnetic particles containing immobilized receptors
followed by liquid chromatography–mass spectrometry
(LC-MS). This assay is suitable for the screening of complex
mixtures such as botanical extracts. For proof-of-principle,
estrogen receptor-α
(ER-α)
and ER-β
were immobilized on magnetic particles functionalized with
aldehyde or carboxylic acid groups. Alternatively, biotinylated
ER was immobilized onto streptavidin-derivatized magnetic
particles. The ER that was immobilized using the streptavidin-biotin
chemistry showed higher activity than that immobilized on
aldehyde or carboxylic acid functionalized magnetic particles.
Immobilized ER was incubated with extracts of Tri-folium
pratense L. (red clover) or Humulus lupulus
L. (hops). As a control for non-specific binding, each botanical
extract was incubated with magnetic particles containing no
ER. After magnetic separation of the particles containing
bound ligands from the unbound components in the extract,
the particles were washed, ligands were released using methanol,
and then the ligands were identified using LC-MS. The estrogens
genistein and daidzein were identified in the red clover extract,
and the estrogen 8-prenylnaringenin was identified in the
hop extract. These screening results are consistent with those
obtained using previous screening approaches.
[Back to top]
Anti-Cancer Natural Product Library from Traditional
Chinese Medicine
V. Badirenaath Konkimalla and Thomas Efferth
The cure rates in cancer chemotherapy are affected by the
development of drug resistance and severe side effects. Due
to these limitations, there is an urgent need for improved
therapeutics. Bioactive compounds from medicinal plants represent
a valuable resource for novel anticancer drugs. To gain a
systematic approach, we established a library of 531 cytotoxic
natural products derived from traditional Chinese medicine.
Cellular and pharmacogenomic profiling was performed for the
10 most cytotoxic natural products. One of these compounds,
helebrin, was analyzed in more detail. The IC50
values for hellebrin of 60 NCI cell lines were associated
with the microarray-based expression of 9,706 genes. By hierarchical
cluster analyses, candidate genes were identified which significantly
predicted sensitivity or resistance of cell lines to hellebrin.
[Back to top]
Post-SELEX Chemical Optimization of a Trypanosome
Specific RNA Aptamer
Annette Adler, Nicole Forster, Matthias Homann
and H. Ulrich Göringer
African trypanosomes are the causative agent of sleeping sickness.
The therapeutics used to control and treat the disease are
very ineffective and thus, the development of improved drugs
is urgently needed. Recently, new strategies for the design
of novel trypanocidals have been put forward. Among them are
techniques that rely on parasite-specific RNA aptamers. One
approach involves the aptamer-directed transport of lytic
compounds to the lysosome of the parasite. The aptamer has
been termed 2-16 RNA and here we report the optimization of
the RNA for its applications in vivo. To convert
aptamer 2-16 into a serum-stable reagent 2'-deoxy-2'-F- and/or
2'-deoxy-2'-NH2-uridine-
and cytidine-substituted RNAs were generated. While 2'-NH2-dC/dU-modified
RNAs were RNase-resistant, they were functionally inactive.
By contrast, 2'-F-dC/dU-substituted 2-16 RNA retained its
ability to bind to live trypanosomes (Kd=45
nM) and was routed to the lysosome identically to unmodified
RNA. 2'-F-dC/dU-substituted 2-16 RNA is thermostable (Tm=750C)
and has a serum half-life of 3.4 days. Furthermore, aptamer
2-16 was site-specifically PEGylated to increase its serum
retention time. Conjugation with PEG polymers ≤10
kDa only marginally impacted the binding characteristics of
the RNA, while the addition of higher molecular mass PEG molecules
resulted in non-functional aptamers. Together, the data provide
optimized conjugation chemistries for the large-scale production
of substituted aptamer 2-16 preparations with improved in
vivo functionality.
[Back to top]
A High-Complexity, Multiplexed Solution-Phase Assay
for Profiling Protease Activity on Microarrays
Igor A. Kozlov, Peter C. Melnyk, John P. Hachmann, Anu Srinivasan,
Melissa Shults, Chanfeng Zhao, Joseph Musmacker, Nicholas
Nelson, David L. Barker and Michal Lebl
We have developed a miniaturized and multiplexed solution
assay for the measurement of protease activity in complex
samples. This technology can accelerate research in functional
proteomics and enable biologists to carry out multiplexed
protease inhibitor screens on a large scale. The assay readout
is based on Illumina’s universal Sentrix®
BeadArrays. The peptide sequences that serve as protease substrates
are conjugated to oligonucleotide sequences complementary
to the oligo tags on randomly assembled and decoded bead arrays.
The peptide portion is C-terminally labeled with a biotin
residue and contains a sequence of five histidine residues
on the amino terminus. The unique oligonucleotide part of
each oligonucleotide-peptide conjugate is attached to amino
terminus of the peptide sequence. Upon protease cleavage,
the biotin residue is cleaved from the oligonucleotide-peptide
conjugate. Following the reaction, all biotin-containing species
are captured and removed by incubation with streptavidin beads.
The cleaved conjugates that remain in solution are captured
by hybridization of their oligo sequence to Sentrix BeadArrays
and detected using a labeled antibody against pentahistidine
tag of the conjugate or by an antibody sandwich assay. We
have generated multiple sets of oligonucleotide tagged peptide
substrates of varying complexity (100 to 1000 substrates in
a mixture) and show that the response of individual substrate
is independent of the complexity of the mixture. Our initial
results demonstrate the feasibility of assaying proteases
in a multiplexed environment with high sensitivity.
[Back to top]
The Combinatorial Synthesis of Organophosphorus
Compounds
Bernhard Lesch, Douglas W. Thomson and Stephen D. Lindell
The primary literature concerning the combinatorial synthesis
of organophosphorus compounds is reviewed and discussed. The
subject matter is divided into three main sections describing
the solid phase, solution phase and solvent-free synthesis
of phosphorus containing organic molecules. The review covers
the synthesis of compounds in which the final products contain
phosphorus-carbon bonds, primarily phosphonates, phosphinates,
phosphine oxides and phosphines.
[Back to top]
Targeted Therapy of the Insulin-Like Growth Factor-1
Receptor in Cancer
Keren Paz and Yaron R. Hadari
Recently, significant progress has been made towards
understanding the pathogenesis of cancer from the molecular
standpoint. To this end, a growing number of approaches are
being exploited for the identification and validation of new
therapeutic targets suitable for potent and specific intervention.
The type 1 insulin-like growth factor receptor (IGF-1R) system
has recently become the focus of major attention in the arena
of cancer research. The involvement of the receptor and its
downstream signaling cascades in the carcinogenesis process
makes this system an excellent target for potential cancer
therapy. Indeed, advances in the understanding of the molecular
mechanisms behind IGF-1R activation have led to the discovery
of agents designed selectively for targeting IGF-1R. The potential
application of these inhibitors is currently under intense
clinical investigation. This review describes the biology
of IGF-1R particularly from a cancer perspective. The attempts
to develop effective IGF-1R antagonists are discussed comprehensively
with special emphasis on antibodies and small tyrosine kinase
inhibitors.
[Back to top]
A Chemical Genetics Approach for Specific Differentiation
of Stem Cells to Somatic Cells: A New Promising Therapeutical
Approach
Agapios Sachinidis, Isaia Sotiriadou, Bianca Seelig, Albrecht
Berkessel and Jürgen Hescheler
Cell replacement therapy of severe degenerative diseases such
as diabetes, myocardial infarction and Parkinson’s disease
through transplantation of somatic cells generated from embryonic
stem (ES) cells is currently receiving considerable attention
for the therapeutic applications. ES cells harvested from
the inner cell mass (ICM) of the early embryo, can proliferate
indefinitely in vitro while retaining the ability
to differentiate into all somatic cells thereby providing
an unlimited renewable source of somatic cells. In this context,
identifying soluble factors, in particular chemically synthesized
small molecules, and signal cascades involved in specific
differentiation processes toward a defined tissue specific
cell type are crucial for optimizing the generation of somatic
cells in vitro for therapeutic approaches. However,
experimental models are required allowing rapid and “easy-to-handle”
parallel screening of chemical libraries to achieve this goal.
Recently, the forward chemical genetic screening strategy
has been postulated to screen small molecules in cellular
systems for a specific desired phenotypic effect. The current
review is focused on the progress of ES cell research in the
context of the chemical genetics to identify small molecules
promoting specific differentiation of ES cells to desired
cell phenotype. Chemical genetics in the context of the cell
ES-based cell replacement therapy remains a challenge for
the near future for several scientific fields including chemistry,
molecular biology, medicinal physics and robotic technologies.
|
