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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 10, December 2006
Contents
Combinatorial Saturation Mutagenesis by
In Vivo Overlap Extension for the Engineering of
Fungal Laccases Pp. 719-727
Miguel Alcalde, Miren Zumarraga, Julio Polaina, Antonio
Ballesteros and Francisco J. Plou
[Abstract]
A New Library of C-16 Modified Artemisinin Analogs
and Evaluation of Their Anti-Parasitic Activities
Pp. 729-741
Rani B. Menon, Muraleedharan M. Kannoth, Babu L. Tekwani,
Jiri Gut, Phillip J. Rosenthal and Mitchell A. Avery
[Abstract]
Novel Synthesis of N4-Substituted 1,4-Benzodiazepine-2,5
diones Using Poly(Ethylene Glycol) as Soluble Polymer Support
Pp. 743-746
Zhen Rao, Hongdan Peng, Guichun Yang and Zuxing Chen
[Abstract]
Selective Molecularly Imprinted Polymer Obtained from
a Combinatorial Library for the Extraction of Bisphenol A
Pp. 747-751
Antonio Martin-Esteban and Jose Luis Tadeo
[Abstract]
Comparative Molecular Surface Analysis (CoMSA) for
Virtual Combinatorial Library Screening of Styrylquinoline
HIV-1 Blocking Pp. 753-770
Agents Halina Niedbala, Jaroslaw Polanski, Rafal Gieleciak,
Robert Musiol, Dominik Tabak, Barbara Podeszwa, Andrzej Bak,
Anna Palka, Jean-Francois Mouscadet, Johann Gasteiger and
Marc Le Bret
[Abstract]
Synthesis of Tetrahydrobenzimidazo[1,2-b]quinazolin-1(2H)-one
and Tetrahydro-1,2,4-triazolo[5,1-b]quinazolin-8(4H)-one
Ring Systems Under Solvent-Free Conditions
Pp. 771-776
Ahmad Shaabani, Elham Farhangi and Abbas Rahmati
[Abstract]
A Screening Platform for Compounds with Potential
Immuno-Regulatory Activities Using Human Cord Blood Mononuclear
Cells Pp. 777-784
Chia-Jung Chen, Chia Che Tsai, Jung-Feng Hsieh, Chichen
Michael Chien, Tzu-Hua Wu and Shui-Tein Chen
[Abstract]
Combinatorial Dispensing as a Fast and Efficient Means
to Create Complex Screens Pp. 785-790
Bart Hazes
[Abstract]
Abstracts
[Back to top]
Combinatorial Saturation Mutagenesis by In Vivo Overlap
Extension for the Engineering of Fungal Laccases
Miguel Alcalde, Miren Zumarraga, Julio Polaina, Antonio
Ballesteros and Francisco J. Plou
Combinatorial saturation mutagenesis -CSM-
is a valuable tool for improving enzymatic properties from
hot-spot residues discovered by directed enzyme evolution
or performing semi-rational studies. CSM coupled to a reliable
high-throughput screening assay –coefficient of variance
below 10%- has been used to enhance turnover rates in the
fungal laccase variant T2 from Myceliophthora thermophila.
The influence of the highly conserved pentapeptide 509-513
on the redox potential of blue-copper containing enzymes is
well described. We focused combinatorial saturation mutagenesis
in residues Ser510 and Leu513. Libraries were constructed
in Saccharomyces cerevisiae by in vivo overlap
extension -IVOE- of the PCR products. This methodology provides
a simple manner to build CSM libraries avoiding extra PCR
reactions, by-products formation and in vitro ligation
steps. After exploring more than 1,700 clones, mutant (7E1)
with ~
3-fold higher kinetics than parent type was found. 7E1 showed
one synonymous mutation (L513L, CGT/TTG) and one beneficial
mutation S510G (TCG/GGG) that can not be achieved by conventional
error-prone PCR techniques. Mutation S510G seems to affect
the C-terminal plug, which modulates the transit of water
and oxygen to the trinuclear copper cluster.
[Back to top]
A New Library of C-16 Modified
Artemisinin Analogs and Evaluation of Their Anti-Parasitic
Activities
Rani B. Menon, Muraleedharan M. Kannoth, Babu
L. Tekwani, Jiri Gut, Phillip J. Rosenthal and Mitchell A.
Avery
A library of C-16 modified artemisinin analogs
was prepared and their antimalarial as well as antileishmanial
activities were evaluated. Synthesis of these compounds involved
the conversion of artemisinin to its phenol derivatives 7
and 12, and subsequent parallel derivatization
by introducing new chemical groups through ester, carbamate,
sulfate, phosphate and isourea linkages. Comparison of in
vitro antimalarial activities showed that C9-β
artemisinin analogs (8a-f) are more potent
than the corresponding C9-α
diastereomers (9a-f); however, their antileishmanial
activities were in the same range. Many of the 10-deoxoartemisinin
analogs studied here showed promising antiparasitic activities.
For example, compounds 13a-e are approximately
three times more active against drug resistant W2 strain of
P. falciparum, compared to artemisinin (IC50,
~0.2 – 0.6 nM; cf. artemisinin = 1.6 nM). Further, a
number of compounds in this series were notably leishmanicidal,
with activities comparable to or better than pentamidine (e.g.,
13g and 13j). Detailed in
vivo studies involving these active compounds are underway
to identify lead candidates for further development.
[Back to top]
Novel Synthesis of N4-Substituted
1,4-Benzodiazepine-2,5 diones Using Poly(Ethylene Glycol)
as Soluble Polymer Support
Zhen Rao, Hongdan Peng, Guichun Yang and Zuxing
Chen
An efficient method for the liquid-phase combinatorial
synthesis of N4-substituted 1,4-benzodiazepine-2,5-diones
has been developed. Poly(ethylene glycol) (PEG) stepwise reacted
with bromoacetyl bromide, a primary amine and 2-azidobenzoic
acid to give a potential PEG-bound dipeptide, which was reduced
by NaI / acetic acid, along with concurrent cyclization and
cleavage of the seven-membered heterocycle from the PEG support.
[Back to top]
Selective Molecularly Imprinted Polymer
Obtained from a Combinatorial Library for the Extraction of
Bisphenol A
Antonio Martin-Esteban and Jose Luis Tadeo
In the present work, an analytical methodology
based on molecularly imprinted solid-phase extraction (MISPE)
has been developed for the determination of bisphenol A (BPA)
in environmental and food samples. In order to select the
optimum material, a combinatorial library of molecularly imprinted
polymers in small-scale (mini-MIPs) was prepared using BPA
as template. Different monomers (methacrylic acid or 4-vinylpyridine),
crosslinkers (ethylene glycol dimethacrylate or trimethylolpropane
trimethacrylate) and porogens (methanol, acetonitrile or toluene)
were used leading to 24 different polymerisation mixtures.
After BPA removal, the ability of mini-MIPs to recognise BPA
was evaluated by equilibrium rebinding-elution experiments.
The copolymer of 4-vinylpyridine (4-VP) and trimethylolpropane
trimethacrylate (TRIM) prepared in toluene showed the higher
affinity for the template. Subsequently, a scaled-up version
of the optimum polymer was prepared and used in the development
of MISPE procedures for the extraction of BPA. The optimised
MISPE protocols were successfully applied to the selective
extraction of BPA from soils and aqueous canned peas samples.
[Back to top]
Comparative Molecular Surface Analysis
(CoMSA) for Virtual Combinatorial Library Screening of Styrylquinoline
HIV-1 Blocking
Agents Halina Niedbala, Jaroslaw Polanski,
Rafal Gieleciak, Robert Musiol, Dominik Tabak, Barbara Podeszwa,
Andrzej Bak, Anna Palka, Jean-Francois Mouscadet, Johann Gasteiger
and Marc Le Bret
We used comparative molecular surface analysis
to design molecules for the synthesis as part of the search
for new HIV-1 integrase inhibitors. We analyzed the virtual
combinatorial library (VCL) constituted from various moieties
of styrylquinoline and styrylquinazoline inhibitors. Since
imines can be applied in a strategy of dynamic combinatorial
chemistry (DCC), we also tested similar compounds in which
the -C=N- or -N=C- linker connected the heteroaromatic and
aromatic moieties. We then used principal component analysis
(PCA) or self-organizing maps (SOM), namely, the Kohonen neural
networks to obtain a clustering plot analyzing the diversity
of the VCL formed. Previously synthesized compounds of known
activity, used as molecular probes, were projected onto this
plot, which provided a set of promising virtual drugs. Moreover,
we further modified the above mentioned VCL to include the
single bond linker –C-N- or –N-C-. This allowed
increasing compound stability but expanded also the diversity
between the available molecular probes and virtual targets.
The application of the CoMSA with SOM indicated important
differences between such compounds and active molecular probes.
We synthesized such compounds to verify the computational
predictions.
[Back to top]
Synthesis of Tetrahydrobenzimidazo[1,2-b]quinazolin-1(2H)-one
and Tetrahydro-1,2,4-triazolo[5,1-b]quinazolin-8(4H)-one Ring
Systems Under Solvent-Free Conditions
Ahmad Shaabani, Elham Farhangi and Abbas Rahmati
Tetraheterocyclic benzimidazo[1,2-α]quinazolin-4(1H)-one
and tetrahydro-1,2,4-triazolo[5,1-b]quinazolin-8(4H)-one
were synthesized in relatively high yields by the condensation
reaction of an aldehyde and a cyclic β-diketone
with 2-aminobenzothiazole, 2-aminobenzimidazole or 3-amino-1,2,4-triazole
without using any catalyst under solvent-free conditions.
[Back to top]
A Screening Platform for Compounds
with Potential Immuno-Regulatory Activities Using Human Cord
Blood Mononuclear Cells
Chia-Jung Chen, Chia Che Tsai, Jung-Feng Hsieh,
Chichen Michael Chien, Tzu-Hua Wu and Shui-Tein Chen
A systematic and combinatorial approach was
adopted using human umbilical cord blood mononuclear cells
(hUCB-MNCs) to screen for potential immuno-regulatory compounds.
The hUCB-MNCs contain several types of immunogenic cells,
which are a suitable material to mimic the in vivo
immuno-response after drug treatment. hUCB-MNCs were treated
with various natural products such as quercetin, astaxanthin,
caffeic acid, bilobalide, eugenol, rutin and γ-dodecalactone
(γ-DDL).
Phenotypic expression analysis revealed that the subpopulation
of CD3+ T cells, CD56+ NK cells and
CD1a+ dendritic cells apparently increased after
being treated with γ-DDL
for 6 days. The expression of CD56 reached a maximum at 72
h with a dose-dependent relationship. The NK cells activation
marker (CD69) also elevated following γ-DDL
treatment. These results demonstrated that the γ-DDL
has immuno-regulatory effects to enhance cord blood NK cells
population and bioactivities. Such a high-throughput methodology
using hUCB-MNCs may be an effective platform for systematically
screening potential immuno-regulatory compounds.
[Back to top]
Combinatorial Dispensing as a Fast
and Efficient Means to Create Complex Screens
Bart Hazes
Liquid handling robots carry out tasks from
simple plate filling to complex operations such as creating
reagent cocktails from multiple stock solutions. The latter
task is conceptually a combinatorial process where each cocktail
is created by combining a subset of stock solutions in user-defined
volumes. General-purpose liquid handlers can perform this
task, but their hardware lacks the inherent properties needed
to exploit the combinatorial nature of the problem at hand.
Here we present the use of non-contact dispensing technologies
to create complex screens at low volume and high density.
Our approach is based on the "inkjet printer principle"
where a block of dispensers (print head) travels over a multi-well
plate (paper) to deliver the reagents (inks) in a user-defined
pattern. Impact-induced mixing and the lack of tip contamination
remove the need for extensive tip washing or the use of large
numbers of disposable tips. As an example, protein crystallization
screening is used to demonstrate the technology. This application
requires the creation of complex mixtures from many stock
solutions with a great diversity of viscosities and surface
tensions. In addition, dispense volumes cover a range from
50 nL to 50 μL,
illustrating its utility in low-volume high-density screening.
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