|
Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 8, September 2006
Contents
Combinatorial Approaches to Iminosugars
as Glycosidase and Glycosyltransferase Inhibitors
Pp. 571-582
Laura Cipolla, Barbara La Ferla and Maria Gregori
[Abstract]
Combinatorial Synthesis and Screening of Novel
Odorants Such as Polyfunctional Thiols Pp. 583-590
Catherine Vermeulen and Sonia Collin
[Abstract]
LNA-Modified Oligodeoxynucleotide Hybridization
with DNA Microarrays Printed on Nanoporous Membrane Slides
Pp. 591-597
Jian-Ping Liu, Anna Guerasimova, Regine Schwartz, Matthias
Lange, Hans Lehrach, Lajos Nyársik and Michal Janitz
[Abstract]
Inhibition of Lethal Endotoxin Shock with an L-Pyridylalanine
Containing Metalloproteinase Inhibitor Selected by High Throughput
Screening of a New Peptide Library Pp. 599-611
Jialiang Hu, Véronique Dubois, Patrick Chaltin,
Pierre Fiten, Christiane Dillen, Philippe E. Van den Steen
and Ghislain Opdenakker
[Abstract]
Development and Optimization of a Non-Radioactive
JNK3 Assay Pp. 613-618
Christian Peifer, Sabine Luik, Sabine Thuma, Yvonne Herweh
and Stefan Laufer
[Abstract]
DNA and RNA Aptamers: From Tools for Basic Research
Towards Therapeutic Applications Pp. 619-632
Henning Ulrich, Cleber A. Trujillo, Arthur A. Nery, Janaina
M. Alves, Paromita Majumder, Rodrigo R. Resende and Antonio
H. Martins
[Abstract]
Modifications to Increase the Efficiency of the
Fluorometric Cycling Assay for Cyclic ADP-Ribose Pp.
633-637
Genevieve S. Young and James B. Kirkland
[Abstract]
Abstracts
[Back to top]
Combinatorial Approaches to Iminosugars as Glycosidase
and Glycosyltransferase Inhibitors
Laura Cipolla, Barbara La Ferla and Maria Gregori
Oligosaccharide processing enzymes such as glycosidases
and glycosyltransferases are important classes of biocatalysts
involved in synthesising specific oligosaccharide structures
on proteins and lipids. These enzymes are known to be involved
in a wide range of important biological processes, such as
intestinal digestion, post-translational processing of glycoproteins,
lysosomal catabolism of glycoconjugates and inter-cellular
recognition events. Inhibition of these enzymes can disrupt
biosynthesis of oligosaccharides, thus interfering in all
of these processes. Hence, “glyco-enzyme” inhibitors
might have enormous therapeutic potential in many diseases
such as viral infection, cancer and diabetes. This very important
prospect has led to increasing interest and demand for these
compounds. Interference in oligosaccharide processing is the
basis for the anti-influenza neuraminidase inhibitors that
have recently been marketed and also for the potential use
of glycosidase inhibitors against HIV, Gaucher’s disease,
hepatitis, and cancer. Since a rational design and synthesis
of inhibitors are often extremely difficult due to the limited
information regarding the structure of the active site, combinatorial
approaches are particularly promising. This review will focus
on synthetic efforts for the preparation of combinatorial
libraries of glyco-enzyme inhibitors.
[Back to top]
Combinatorial Synthesis and Screening of Novel
Odorants Such as Polyfunctional Thiols
Catherine Vermeulen and Sonia Collin
Combinatorial chemistry was shown to be an efficient
tool for the preparation of new aroma-impact compounds. In
this case, polyfunctional thiols were synthesized quickly
using halide reagents or Bunte salt intermediates. They were
separated by gas chromatography and then characterized using
low resolution EI and CI mass spectrometry. The individual
sensorial properties of the thiol products (i.e. odor and
perception threshold) were determined by GC-O (olfactometry)
which uses the human nose as detector. The thiols were characterized
based on their particular odors. 3-Methyl-2-buten-1-thiol,
a relevant flavor naturally present in beer and coffee, emerged
as the most powerful of the thiol library.
[Back to top]
LNA-Modified Oligodeoxynucleotide Hybridization
with DNA Microarrays Printed on Nanoporous Membrane Slides
Jian-Ping Liu, Anna Guerasimova, Regine Schwartz, Matthias
Lange, Hans Lehrach, Lajos Nyársik and Michal Janitz
We report a robust method for the detection of hybridization
events using a microarray-based assay on a nanoporous membrane
platform. The technique is characterized by a hybridization
time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide
probes modified with locked nucleic acid (LNA) nucleotides.
We show that the volume of the DNA spotted onto a nanomembrane
can be reduced to ~4 nL with detectable signal intensity.
Moreover, the amount of the DNA target could be reduced to
4 fmol. The described approach could dramatically increase
the throughput of techniques based on sequencing by hybridization,
such as oligofingerprinting, by decreasing the total number
of probes that are needed for analysis of large clone sets
and reduction of the sample/reagent consumption. The method
is particularly advantageous when numerous hybridization-based
assays must be performed for characterization of sample sets
of 100,000 or more.
[Back to top]
Inhibition of Lethal Endotoxin Shock with an
L-Pyridylalanine Containing Metalloproteinase Inhibitor Selected
by High Throughput Screening of a New Peptide Library
Jialiang Hu, Véronique Dubois, Patrick Chaltin,
Pierre Fiten, Christiane Dillen, Philippe E. Van den Steen
and Ghislain Opdenakker
Gelatinase B/MMP-9 fulfills crucial regulator or effector
functions in disease states and may be pharmacologically targeted
by specific inhibitors. The characteristics of cleavages of
a natural gelatinase B substrate were simulated and amino
acids with zinc-ion chelating side-groups were employed to
design a library of peptide-based inhibitors. Here, we extend
previous findings of combinatorial chemical synthesis and
subsequent library deconvolution with a recently established
high-throughput technology. This enabled us to study MMP inhibitors
with two zinc-binding groups and to identify a new L-pyridylalanine-containing
gelatinase B inhibitor. The peptide analog was found to inhibit,
almost to the same degree, the neutrophil enzymes collagenase
2/ MMP-8 and MMP-9 and the monocytic tumor necrosis factor-α
(TNF-α
) converting enzyme (TACE/ADAM-17) in vitro
and to protect mice against lethal endotoxinemia in vivo.
These data illustrate the usefulness of the screening platform
for protective inhibitor discovery and complement recent insights
in the pathogenesis and treatment of shock syndromes.
[Back to top]
Development and Optimization of a Non-Radioactive
JNK3 Assay
Christian Peifer, Sabine Luik, Sabine Thuma, Yvonne Herweh
and Stefan Laufer
In light of emerging interest in the relevance of c-Jun
NH2-terminal protein kinase 3 (JNK3) as a promising drug target,
we describe here an advanced non-radioactive immunosorbent
JNK3 activity assay that is applicable for routine screening
of small molecule ATP-competitive enzyme inhibitors. We modified
and established a JNK3/ATF-2 protocol based on our previously
described p38 MAPK method [1] for a substrate-bound non-radioactive
procedure that represents a convenient alternative to conventional
radioactive protein kinase assays. The objective of the present
study was to validate these conditions by using the reference
compounds SP600125 and SB203580 to achieve comparable IC50
results to published data. Furthermore, an IC50 for staurosporine
was determined. The protocol we describe here represents an
accessible and robust screening assay for JNK3 inhibitors.
[Back to top]
DNA and RNA Aptamers: From Tools for Basic Research
Towards Therapeutic Applications
Henning Ulrich, Cleber A. Trujillo, Arthur A. Nery, Janaina
M. Alves, Paromita Majumder, Rodrigo R. Resende and Antonio
H. Martins
The systematic evolution of ligands by exponential enrichment
(SELEX) is a combinatorial oligonucleotide library-based in
vitro selection approach in which DNA or RNA molecules
are selected by their ability to bind their targets with high
affinity and specificity, comparable to those of antibodies.
Nucleic acids with high affinity for their targets have been
selected against a wide variety of compounds, from small molecules,
such as ATP, to membrane proteins and even whole organisms.
Recently, the use of the SELEX technique was extended to isolate
oligonucleotide ligands, also known as aptamers, for a wide
range of proteins of importance for therapy and diagnostics,
such as growth factors and cell surface antigens. The number
of aptamers generated as inhibitors of various target proteins
has increased following automatization of the SELEX process.
Their diagnostic and therapeutic efficacy can be enhanced
by introducing chemical modifications into the oligonucleotides
to provide resistance against enzymatic degradation in body
fluids. Several aptamers are currently being tested in preclinical
and clinical trials, and aptamers are in the process of becoming
a new class of therapeutic agents. Recently, the anti-VEGF
aptamer pegaptanib received FDA approval for treatment of
human ocular vascular disease.
[Back to top]
Modifications to Increase the Efficiency of the
Fluorometric Cycling Assay for Cyclic ADP-Ribose
Genevieve S. Young and James B. Kirkland
Cyclic ADP-ribose (cADPR) is an intracellular messenger
that triggers the release of calcium ions from intracellular
stores in a variety of cell types. The fluorometric cycling
assay has become the preferred method for measuring cADPR
due to its high level of sensitivity (in the sub-nanomolar
range) and its use of commercially available reagents. Additionally,
the assay is performed in multiwell plates, making it suitable
for high throughput screening using a fluorescence plate reader.
The findings reported in this paper present several problems
that may be encountered during various stages of the assay,
and provide solutions to these problems. Modifications to
the assay address reduced recovery of sample and cADPR with
removal of perchloric acid (PCA) using organic solvent, reduction
in diaphorase activity with heat treatment, and effects on
resorufin fluorescence by pH range. Using these modifications,
we report an increase of approximately 15% in recovery of
brain cADPR, and show that between-subject variability is
greatly reduced. We hope that these observations will encourage
more widespread application of this valuable assay.
|
