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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 7, August 2006
Contents
Serum-Stable RNA Aptamers to an Invariant Surface Domain of
Live African Trypanosomes Pp. 491-499
Matthias Homann, Mihaela Lorger, Markus Engstler, Martin Zacharias
and H. Ulrich Göringer
[Abstract]
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Whole Cell Strategies Based on lux Genes
for High Throughput Applications Toward New Antimicrobials
Pp. 501-514
Lorenzo Galluzzi and Matti Karp
[Abstract] [Purchase
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Design and Characterization of a Functional Library
for NMR Screening Against Novel Protein Targets Pp.
515-534
Kelly A. Mercier, Katherine Germer and Robert Powers
[Abstract] [Purchase
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Synthesis of Metallothionein-Mimic Decapeptides
with Heavy Atom Signaling Properties Pp. 535-544
Ignacio A. Rivero, Tania Gonzalez and Marta E. Diaz-Garcia
[Abstract] [Purchase
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Exploring Structure-Activity Relationships of
Tricyclic Farnesyltransferase Inhibitors Using ECLiPS®
Libraries Pp. 545-558
Laura L. Rokosz, Chia-Yu Huang, John C. Reader, Tara M.
Stauffer, Eileen C. Southwick, Ge Li, Daniel Chelsky and John
J. Baldwin
[Abstract] [Purchase
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A Gradient Descent Algorithm for Minimizing Amino
Acid Coupling Reactions when Synthesizing Cyclic-Peptide Libraries
Pp. 559-563
Paul J. Darwen, Tran T. Tran, Gregory T. Bourne, Jonathon
L. Nielson and Mark L. Smythe
[Abstract] [Purchase
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A Directly Labeled TR-FRET Assay for Monitoring
Phosphoinositide-3-Kinase Activity Pp. 565-570
Xiaoke Yang, Puyao Li, Larry Feldberg, Steven C. Kim,
Michael Bowman, Irwin Hollander, Robert Mallon and Stanley
F. Wolf
[Abstract] [Purchase
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Abstracts
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Serum-Stable RNA Aptamers to an Invariant Surface
Domain of Live African Trypanosomes
Matthias Homann, Mihaela Lorger, Markus Engstler, Martin Zacharias
and H. Ulrich Göringer
African trypanosomes are extracellular blood parasites that
cause sleeping sickness in humans and Nagana in cattle. The
therapeutics used to control and treat these diseases are
very ineffective and thus, the development of new drugs is
urgently needed. We have previously suggested to use trypanosome-specific
RNA aptamers as tools for the development of novel trypanocidal
compounds. Here, we report the selection of a 2’-NH2-modified
RNA aptamer that binds to live trypanosomes with an affinity
of 70 ± 15 nM. The aptamer adopts a stable G-quartet
structure and has a half-life in human serum of ≥ 30
h. RNA binding is restricted to the flagellar attachment zone,
located between the cell body and the flagellum of the parasite.
We demonstrate that antigen-tagged preparations of the aptamer
can bind to live trypanosomes and that they can be used to
re-direct immunoglobulins to the parasite surface.
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Whole Cell Strategies Based on lux Genes
for High Throughput Applications Toward New Antimicrobials
Lorenzo Galluzzi and Matti Karp
The discovery/development of novel drug candidates has
witnessed dramatic changes over the last two decades. Old
methods to identify lead compounds are not suitable to screen
wide libraries generated by combinatorial chemistry techniques.
High throughput screening (HTS) has become irreplaceable and
hundreds of different approaches have been described. Assays
based on purified components are flanked by whole cell-based
assays, in which reporter genes are used to monitor, directly
or indirectly, the influence of a chemical over the metabolism
of living cells. The most convenient and widely used reporters
for real-time measurements are luciferases, light emitting
enzymes from evolutionarily distant organisms. Autofluorescent
proteins have been also extensively employed, but proved to
be more suitable for end-point measurements, in situ
applications – such as the localization of fusion proteins
in specific subcellular compartments – or environmental
studies on microbial populations. The trend toward miniaturization
and the technical advances in detection and liquid handling
systems will allow to reach an ultra high throughput screening
(uHTS), with 100,000 of compounds routinely screened each
day. Here we show how similar approaches may be applied also
to the search for new and potent antimicrobial agents.
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Design and Characterization of a Functional Library
for NMR Screening Against Novel Protein Targets
Kelly A. Mercier, Katherine Germer and Robert Powers
In the past few years, NMR has been extensively utilized
as a screening tool for drug discovery using various types
of compound libraries. The designs of NMR specific chemical
libraries that utilize a fragment-based approach based on
drug-like characteristics have been previously reported. In
this article, a new type of compound library will be described
that focuses on aiding in the functional annotation of novel
proteins that have been identified from various ongoing genomics
efforts. The NMR functional chemical library is comprised
of small molecules with known biological activity such as:
co-factors, inhibitors, metabolites and substrates. This functional
library was developed through an extensive manual effort of
mining several databases based on known ligand interactions
with protein systems. In order to increase the efficiency
of screening the NMR functional library, the compounds are
screened as mixtures of 3-4 compounds that avoids the need
to deconvolute positive hits by maintaining a unique NMR resonance
and function for each compound in the mixture. The functional
library has been used in the identification of general biological
function of hypothetical proteins identified from the Protein
Structure Initiative.
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Synthesis of Metallothionein-Mimic Decapeptides
with Heavy Atom Signaling Properties
Ignacio A. Rivero, Tania Gonzalez and Marta E. Diaz-Garcia
The automated parallel solid-phase synthesis of a 17-member
library of metallothionein-mimic decapeptides carrying a Lariat
ether group is described. The peptides were synthesized in
good yield and the identity and quality of each product were
performed by mass spectrometry and IR. Subsequently, in the
presence of europium(III) ions as fluorescent reporter, each
compound was screened, both attached to the resin and cleaved
off, for their sensing behavior towards metal ions (Cd2+,
Hg2+, Cu2+, Mg2+ and Ca2+)
using fluorimetric techniques. Several of these Cys-enriched
synthetic peptides showed surprisingly low detection limits
for Cd2+ and Hg2+. The analytical potential
of these metallothioneinmimic decapeptides as metal ion recognition
materials for sensor development is outlined. Finally, the
sensing response mechanism, based on an energy transfer process
and a metal ion allosteric interaction, is proposed.
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Exploring Structure-Activity Relationships
of Tricyclic Farnesyltransferase Inhibitors Using ECLiPS®
Libraries
Laura L. Rokosz, Chia-Yu Huang, John C. Reader, Tara M.
Stauffer, Eileen C. Southwick, Ge Li, Daniel Chelsky and John
J. Baldwin
The development of structure-activity relationships (SARs)
relating to the function of a biological protein is often
a long and protracted undertaking when using an iterative
medicinal chemistry approach. High throughput screening of
ECLiPS® (Encoded Combinatorial Libraries on
Polymeric Support) libraries can be used to simplify this
process. In this paper we illustrate how a large ECLiPS library
of 26,908 compounds, based on a tricyclic core structure,
was used to define a multitude of SARs for the oncogenic target,
farnesyltransferase (FTase). This library, FT-2, was prepared
using a split-and-pool approach in which small molecules are
constructed on resin that contains tag/linker constructs to
track the synthetic process [1-5]. Highly defined SARs were
produced from this screen that enhanced our understanding
of FTase binding site interactions. The pivotal compounds
culled from this library were potent in both cell-free and
cell-based FTase assays, selective over the closely related
enzyme, geranylgeranyltransferase I (GGTase I), and inhibited
the adherent-independent growth of a transformed cell line.
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A Gradient Descent Algorithm for Minimizing Amino
Acid Coupling Reactions when Synthesizing Cyclic-Peptide Libraries
Paul J. Darwen, Tran T. Tran, Gregory T. Bourne, Jonathon
L. Nielson and Mark L. Smythe
Combinatorial chemistry has become an invaluable
tool in medicinal chemistry for the identification of new
drug leads. For example, libraries of predetermined sequences
and head-to-tail cyclized peptides are routinely synthesized
in our laboratory using the IRORI approach. Such libraries
are used as molecular toolkits that enable the development
of pharmacophores that define activity and specificity at
receptor targets. These libraries can be quite large and difficult
to handle, due to physical and chemical constraints imposed
by their size. Therefore, smaller sub-libraries are often
targeted for synthesis. The number of coupling reactions required
can be greatly reduced if the peptides having common amino
acids are grouped into the same sub-library (batching). This
paper describes a schedule optimizer to minimize the number
of coupling reactions by rotating and aligning sequences while
simultaneously batching. The gradient descent method thereby
reduces the number of coupling reactions required for synthesizing
cyclic peptide libraries. We show that the algorithm results
in a 75% reduction in the number of coupling reactions for
a typical cyclic peptide library.
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A Directly Labeled TR-FRET Assay for
Monitoring Phosphoinositide-3-Kinase Activity
Xiaoke Yang, Puyao Li, Larry Feldberg, Steven C. Kim,
Michael Bowman, Irwin Hollander, Robert Mallon and Stanley
F. Wolf
Phosphoinositide 3-kinases (PI3Ks) comprise a family of kinases
that transfer the terminal phosphate of adenosine triphosphate
to phosphoinositides at the 3-hydroxyl of the inositol ring
to form phosphoinositide (3,4,5) triphosphate (PIP3). The
PI3Ks have been shown to play key roles in cell growth, motility,
morphology, and survival and thus are of interest as targets
in anti-inflammatory and anti-oncogenic drug development.
To facilitate identification of novel and selective inhibitors
of PI3Ks, we have developed a TR-FRET assay that uses directly
labeled reagents. The assay makes use of the high affinity
binding of phosphoinositides to a Pleckstrin homology (PH)
domain in the general receptor for phosphoinositides 1 (Grp1)
protein. It monitors PIP3 produced from the enzymatic reaction
by measuring its competition with Bodipy®-FL-labeled
PIP3 for binding to Terbium chelate-labeled Grp1. By using
directly labeled reagents, this assay configuration offers
higher sensitivity and faster binding/dissociation kinetics
than existing non-radioactive assays, which are critical for
competitive assay formats. The assay is homogenous, robust
(Z’ = 0.88), and simple and, thus, compatible with high
throughput screening (HTS) processes.
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