Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 13, Number 4, May 2010
Contents
Directed Evolution Toward Improved Production of L-Ribose from Ribitol Pp. 302-308
Trevor N. Christ, Kara A. Deweese and Ryan D. Woodyer
[Abstract] [Purchase
Article] [PMID:
20156148 PubMed - indexed for MEDLINE]
High Throughput Bioassays Using Nanoparticles Pp. 309-317
Agustina Gómez-Hens, Juan M. Fernández-Romero and
Maria P. Aguilar-Caballos
[Abstract] [Purchase
Article] [PMID:
20156145 PubMed - indexed for MEDLINE]
High Throughput Discovery of Families of High Activity WGS Catalysts:
Part I - History and Methodology Pp. 318-330
Karin Yaccato, Ray Carhart, Alfred Hagemeyer, Michael Herrmann,
Andreas Lesik, Peter Strasser, Anthony Volpe, Howard Turner,
Henry Weinberg, Robert K. Grasselli, Christopher J. Brooks and John M. Pigos
[Abstract] [Purchase
Article] [PMID:
20156149 PubMed - indexed for MEDLINE]
High Throughput Calorimetry for Evaluating Enzymatic Reactions
Generating Phosphate Pp. 331-336
Lieve Hoflack, Manu De Groeve, Tom Desmet, Peter Van Gerwen and
Wim Soetaert
[Abstract] [Purchase
Article] [PMID:
20015015 PubMed - indexed for MEDLINE]
Design, Synthesis and Biological Evaluation of a Library of Thiocarbazates
and Their Activity as Cysteine Protease Inhibitors Pp. 337-351
Zhuqing Liu, Michael C. Myers, Parag P. Shah, Mary P. Beavers,
Phillip A. Benedetti, Scott L. Diamond, Amos B. Smith, III and Donna M. Huryn
[Abstract] [Purchase
Article]
[PMID:
20438448 PubMed - indexed for MEDLINE]
Coupling the Inhibition of Viral Transduction with a Positive Fluorescence
Signal Pp. 352-357
Jenifer Clausell-Tormos, Andrew D. Griffiths and Christoph A. Merten
[Abstract] [Purchase
Article] [PMID:
20156147 PubMed - indexed for MEDLINE]
Fluorescent Substrates Useful as High Throughput Screening Tools for
ADAM9 Pp. 358-365
Marcia L. Moss, Fred H. Rasmussen, Raphael Nudelman, Peter J. Dempsey and Jason Williams
[Abstract] [Purchase
Article] [PMID:
20015014 PubMed - indexed for MEDLINE]
MoStBioDat – Molecular and Structural Bioinformatics Database Pp. 366-374
Andrzej Bak, Jaroslaw Polanski, Thomas Stockner and Agata Kurczyk
[Abstract] [Purchase
Article]
[PMID:
20438449 PubMed - indexed for MEDLINE]
Abstracts
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20156148 PubMed - indexed for MEDLINE]
Directed Evolution Toward Improved Production of L-Ribose from Ribitol
Trevor N. Christ, Kara A. Deweese and Ryan D. Woodyer
Improvement of the one-step production of L-ribose from ribitol using a recombinant Escherichia coli is
described. The gene encoding the enzyme mannitol-1-dehydrogenase (MDH) from Apium graveolens has previously been
codon-optimized, cloned into the constitutive pZuc10 vector, and expressed in E. coli. This MDH catalyzes the NADdependent
conversion of mannitol to D-mannose and has the ability to convert several polyols to their L-sugar
counterparts, including ribitol to L-ribose. Here, three rounds of directed evolution using libraries generated through errorprone
PCR and screened using a dinitrosalicylate reagent. Mutants were selected for improved conversion of L-ribose, and
the best mutant was isolated by combining two round 2 mutations. Libraries were also selected for thermal stability and
screened at increasingly higher temperatures with each round of mutagenesis. An overall 19.2-fold improvement was
observed with a final conversion of 46.6 ± 1.7% and a productivity of 3.88 ± 0.14 gL-1d-1 in 50 mL shaken flasks at 34°C.
Further characterization of the mutants suggests that increased enzyme thermal stability and expression are responsible for
the increase in L-ribose production. The mutant E. coli production strain isolated represents an improved system for largescale
production of L-ribose.
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High Throughput Bioassays Using Nanoparticles
Agustina Gómez-Hens, Juan M. Fernández-Romero and
Maria P. Aguilar-Caballos
An overview of the usefulness of different nanoparticles to improve the features of high throughput separation
and individual and multiplexed detection bioassays is presented. Although the development of microarray and
microfluidic systems has expanded the capabilities of these high throughput assays, the combined use of NPs and these
devices has provided them with new applications in drug discovery, proteomic and genomic studies, and clinical
diagnosis. This article reviews the wide application field of magnetic, gold, silver, semiconductor and other nanoparticles
in high throughput bioassays. Also, the versatility of the detection systems described shows that NPs are useful
alternatives to fluorescent dyes, which are often used in these assays.
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20156149 PubMed - indexed for MEDLINE]
High Throughput Discovery of Families of High Activity WGS Catalysts:
Part I - History and Methodology
Karin Yaccato, Ray Carhart, Alfred Hagemeyer, Michael Herrmann,
Andreas Lesik, Peter Strasser, Anthony Volpe, Howard Turner,
Henry Weinberg, Robert K. Grasselli, Christopher J. Brooks and John M. Pigos
State-of-art water gas shift catalysts (FeCr for high temperature shift and CuZn for low temperature shift) are
not active enough to be used in fuel processors for the production of hydrogen from hydrocarbon fuels for fuel cells. The
need for drastically lower catalyst volumes has triggered a search for novel WGS catalysts that are an order of magnitude
more active than current systems. Novel catalytic materials for the high, medium and low temperature water gas shift
reactions have been discovered by application of combinatorial methodologies. Catalyst libraries were synthesized on
4 inch wafers in 16x16 arrays and screened in a high throughput scanning mass spectrometer in the temperature range
200°C to 400°C. More than 200 wafers were screened under various conditions and more than 250,000 experiments were
conducted to comprehensively examine catalyst performance for various binary, ternary and higher-order compositions.
[Back to top] [Purchase
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20015015 PubMed - indexed for MEDLINE]
High Throughput Calorimetry for Evaluating Enzymatic Reactions
Generating Phosphate
Lieve Hoflack, Manu De Groeve, Tom Desmet, Peter Van Gerwen and Wim Soetaert
A calorimetric assay is described for the high throughput screening of enzymes that produce inorganic
phosphate. In the current example, cellobiose phosphorylase (EC 2.4.1.20) is tested for its ability to synthesize rare
disaccharides. The generated phosphate is measured in a high throughput calorimeter by coupling the reaction to pyruvate
oxidase and catalase. This procedure allows for the simultaneous analysis of 48 reactions in microtiter plate format and
has been validated by comparison with a colorimetric phosphate assay. The proposed assay has a coefficient of variation
of 3.14% and is useful for screening enzyme libraries for enhanced activity and substrate libraries for enzyme
promiscuity.
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20438448 PubMed - indexed for MEDLINE]
Design, Synthesis and Biological Evaluation of a Library of Thiocarbazates
and Their Activity as Cysteine Protease Inhibitors
Zhuqing Liu, Michael C. Myers, Parag P. Shah, Mary P. Beavers,
Phillip A. Benedetti, Scott L. Diamond, Amos B. Smith, III and Donna M. Huryn
Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead.
Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of
thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity
against a panel of proteases indicated a significant preference for members of the papain family of cysteine proteases over
serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several potent inhibitors of cathepsin L and S
were identified. The SAR data were employed in docking studies in an effort to understand the structural elements
required for cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of
the papain family of cysteine proteases.
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20156147 PubMed - indexed for MEDLINE]
Coupling the Inhibition of Viral Transduction with a Positive Fluorescence
Signal
Jenifer Clausell-Tormos, Andrew D. Griffiths and Christoph A. Merten
Cell-based assays for the inhibition of viral infections most commonly couple a positive signal (e.g., an
increase in fluorescence) to the infection itself and not to its inhibition. When performing drug screens, compounds
decreasing the signal are therefore considered as putative inhibitors. However, this approach can cause the selection of
many false positives, since, for example, both killing of the host cell and inhibiting viral cell-entry results in the same
signal. Using a model system based on murine leukemia virus (MLV) particles pseudotyped with the G-protein of
vesicular stomatitis virus (VSV-G), we have developed generic assays coupling a positive readout to the inhibition of viral
transduction. Consequently, the system favors drug candidates (and concentrations thereof) that do not harm human cells
and significantly decreases the probability for selecting false positives. The assay allows Z-factors of ~0.9, takes cytotoxic
side effects into account and could in theory be adapted for high-throughput screening of inhibitors against further viral
species.
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Article] [PMID:
20015014 PubMed - indexed for MEDLINE]
Fluorescent Substrates Useful as High Throughput Screening Tools for
ADAM9
Marcia L. Moss, Fred H. Rasmussen, Raphael Nudelman, Peter J. Dempsey and
Jason Williams
Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The
donor/quencher pair used were 5-carboxyfluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since
they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro-
Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)-NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)-NH2, and
C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)-NH2 cleavage sites were
effectively processed by ADAM9 with turnover numbers of 100 ± 20 x 10-2 min-1, 20 ± 10 x 10-2 min-1, and 10 ± 3 x 10-2
min-1. In addition, a peptide based on the 33kDa cleavage site of the low affinity receptor for IgE, CD23, Dabcyl-Leu-
Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)- NH2 was processed as well but with less efficiency. A more selective
substrate for ADAM9 was found based on the betacellulin cleavage site. However, the valine containing precursor TNFalpha
based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was
processed most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1, with an IC50 of 2.1 ±
0.3 nM. In addition, GI254023, previously identified as a selective inhibitor of ADAM10, also inhibited ADAM9 with an
IC50 of 280 ± 110 nM. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in
high throughput screening assays for ADAM9.
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[Purchase
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20438449 PubMed - indexed for MEDLINE]
MoStBioDat – Molecular and Structural Bioinformatics Database
Andrzej Bak, Jaroslaw Polanski, Thomas Stockner and Agata Kurczyk
Computer simulations play a crucial role in contemporary chemical investigations, generating enormous
amounts of data. The constraint of sharing data and results is regarded as a major impediment in drug discovery. Among
the steepest barriers to overcome in the high throughput screening studies is the limited number of suitable, freely
accessible repositories for storing drug and drug target data. By offering a uniform data storage and retrieval mechanism,
various data might be compared and exchanged easily. This paper presents the stages of the MoStBioDat software
platform development, originally designed for the efficient storage, management and access of SDF and PDB data. The
detailed architecture and software implementation of this project are described, indicating also the disadvantages of the
solutions chosen. The current implementation of the first prototype is written in Python, an open-source, high-level,
object-oriented scripting language. The modular architecture of the package enables future extension with the necessary
functionalities. The main objective of the MoStBioDat is to serve as an alternative, extensible open-source database
derived partly from SDF and PDB files.
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