Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 12, Number 3, March 2009
Contents
Screening of Antioxidant Phenolic Compounds Produced by In
Vitro Shoots of Brassica oleracea L. var.
costata DC Pp. 230-240
Federico Ferreres, Carla Sousa, David M.
Pereira, Patrícia Valentão, Marcos Taveira,
Anabela Martins, José A. Pereira, Rosa M. Seabra and
Paula B. Andrade
[Abstract]
[Purchase
Article]
[PMID:
19275528 PubMed - indexed for MEDLINE]
The Four Human γ-Aminobutyric
Acid (GABA) Transporters: Pharmacological Characterization
and Validation of a Highly Efficient Screening Assay Pp.
241-249
Trine Kvist, Bolette Christiansen, Anders
A. Jensen and Hans Bräuner Osborne
[Abstract]
[Purchase
Article]
[PMID:
19275529 PubMed - indexed for MEDLINE]
Simultaneous Determination of LogD, LogP,
and pKa of Drugs by Using
a Reverse Phase HPLC Coupled with a 96-Well Plate Auto Injector
Pp. 250-257
Po-Chang Chiang and Yiding Hu
[Abstract]
[Purchase
Article]
[PMID:
19275530 PubMed - indexed for MEDLINE]
Development and Validation of a High-Density
Fluorescence Polarization-Based Assay for the Trypanosoma
RNA Triphosphatase TbCet1 Pp. 258-268
Christophe Antczak, David Shum, Constantin
Radu, Venkatraman E. Seshan and Hakim Djaballah
[Abstract]
[Purchase
Article]
[PMID:
19275531 PubMed - indexed for MEDLINE]
Narrowing Laccase Substrate Specificity
Using Active Site Saturation Mutagenesis Pp.
269-274
Nirupama Gupta and Edgardo T. Farinas
[Abstract]
[Purchase
Article]
[PMID:
19275532 PubMed - indexed for MEDLINE]
Design and Combinatorial Synthesis of
a Library of Methylenesulfonamides and Related Compounds as
Potential Kinase Inhibitors Pp. 275-284
Jane E. Torr, Jonathan M. Large and
Edward McDonald
[Abstract]
[Purchase
Article]
[PMID:
19275533 PubMed - indexed for MEDLINE]
A Multi-Step NMR Screen for the Identification
and Evaluation of Chemical Leads for Drug Discovery Pp.
285-295
Kelly A. Mercier, Matthew D. Shortridge
and Robert Powers
[Abstract]
[Purchase
Article]
[PMID:
19275534 PubMed - indexed for MEDLINE]
High Throughput Screening Assay of α1G
T-type Ca2+ Channels and
Comparison with Patch-Clamp Studies Pp.
296-302
Yoonjee Kim, Soonja Kang, Jae Yeol Lee and
Hyewhon Rhim
[Abstract]
[Purchase
Article]
[PMID:
19275535 PubMed - indexed for MEDLINE]
Docking-Based Virtual Screening: Recent
Developments Pp. 303-314
Tiziano Tuccinardi
[Abstract]
[Purchase
Article]
[PMID:
19275536 PubMed - indexed for MEDLINE]
High Throughput Screening of Physicochemical
Properties and In Vitro ADME Profiling in Drug Discovery
Pp. 315-329
Hong Wan and Anders G. Holmén
[Abstract]
[Purchase
Article]
[PMID:
19275537 PubMed - indexed for MEDLINE]
Abstracts
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[Purchase
Article] [PMID:
19275528 PubMed - indexed for MEDLINE]
Screening of Antioxidant Phenolic Compounds
Produced by In Vitro Shoots of Brassica oleracea
L. var. costata DC
Federico Ferreres, Carla Sousa, David M.
Pereira, Patrícia Valentão, Marcos Taveira,
Anabela Martins, José A. Pereira, Rosa M. Seabra and
Paula B. Andrade
The phenolic compounds produced by in vitro shoots
of Brassica oleracea L. var. costata DC
were screened by HPLC-DAD-MS/MS. Thirty seven compounds were
characterized, which included chlorogenic acids, flavonoids
(the majority of them were hydroxycinnamic acid esters of
kaempferol and quercetin glycosides) and hydroxycinnamic acyl
glycosides (with predominance of synapoyl gentiobiosides).
The antioxidant capacity of the shoots was assessed against
DPPH radical and two reactive oxygen species (superoxide radical
and hypochlorous acid). A strong concentration-dependent antioxidative
capacity was verified in the DPPH and superoxide radicals
assays, but a reduced effect was noticed against hypochlorous
acid. The results obtained indicate that the in vitro
production of B. oleracea var. costata
shoots can become important in the obtention of a noticeable
dietary source of compounds with health protective potential.
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[Purchase
Article] [PMID:
19275529 PubMed - indexed for MEDLINE]
The Four Human γ-Aminobutyric
Acid (GABA) Transporters: Pharmacological Characterization
and Validation of a Highly Efficient Screening Assay
Trine Kvist, Bolette Christiansen, Anders
A. Jensen and Hans Bräuner Osborne
The neurotransmission mediated by γ-aminobutyric
acid (GABA) in the mammalian brain is terminated by a family
of four GABA transporters (GATs). Inhibition of GATs is currently
used in the treatment of epilepsy and these proteins are generally
considered as important drug targets. In this study, we perform
the first elaborate pharmacological characterization of all
four human GAT subtypes. We conduct the experiments in parallel
in a [3H]GABA uptake assay
using 14 standard GAT substrates and inhibitors. This setup
enables direct comparison of the absolute values of inhibitory
activities of the compounds between the different GAT subtypes.
The results are overall in agreement with data reported by
other groups for the orthologous murine GATs. However, there
do seem to be some minor variations among species. In contrast
to the several subtype selective ligands identified for the
GAT-1 subtype, no subtype selective ligands have been reported
for the three remaining GATs. Given the potential therapeutic
relevance of the individual GAT subtypes, a search for novel
structures displaying selectivities for specific GAT subtypes
is important. In this study, we validate our [3H]GABA
uptake assay for use in high throughput screening. We find
that the assay is categorized by high Z’-factors (Z’
> 0.5)
for all four GAT subtypes, demonstrating that the assay is
excellent for a high throughput screen. This [3H]GABA
uptake assay therefore enables future high throughput screening
of compound libraries at the four human GATs.
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[Purchase
Article] [PMID:
19275530 PubMed - indexed for MEDLINE]
Simultaneous Determination of LogD, LogP, and pKa
of Drugs by Using a Reverse Phase HPLC Coupled with a 96-Well
Plate Auto Injector
Po-Chang Chiang and Yiding Hu
For years, the physicochemical properties of drug candidates
have been used to predict their in vivo pharmacokinetic
behaviors. Several theories and empirical correlations have
been established by various researchers with the overall goal
of expediting the drug candidate selection process, with greater
confidence and faster turnaround. This study describes a 96-well
reverse phase HPLC method, simultaneously determining LogD,
LogP, and pKa values of drugs
in a throughput mode. The LogD and LogP values of each compound
were determined, based on the octanol-aqueous partitioning
behavior of the charged and non-charged species under different
pH values. The pKa value
was determined by using the Polynomial fit between LogP and
LogD and the equation LogD (pKa)
≈
LogP – 0.301. The advantages of this method are: low
sample consumption, suitability for low solubility compounds,
less restriction on compound purity, potential for higher
throughput, precise data, and multiple determinations in one
assay.
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[Purchase
Article] [PMID:
19275531 PubMed - indexed for MEDLINE]
Development and Validation of a High-Density Fluorescence
Polarization-Based Assay for the Trypanosoma RNA Triphosphatase
TbCet1
Christophe Antczak, David Shum, Constantin
Radu, Venkatraman E. Seshan and Hakim Djaballah
RNA triphosphatases are attractive and mostly unexplored
therapeutic targets for the development of broad spectrum
antiprotozoal, antiviral and antifungal agents. The use of
malachite green as a readout for phosphatases is well characterized
and widely employed. However, the reaction depends on high
quantities of inorganic phosphate to be generated, which makes
this assay not easily amenable to screening in 1536-well format.
The overly long reading times required also prohibit its use
to screen large chemical libraries. To overcome these limitations,
we sought to develop a fluorescence polarization (FP) -based
assay for triphosphatases, compatible with miniaturization
and fast readouts. For this purpose, we took advantage of
the nucleoside triphosphatase activity of this class of enzyme
to successfully adapt the Transcreener™
ADP assay based on the detection of generated ADP by immunocompetition
fluorescence polarization to the RNA triphosphatase TbCet1
in 1536-well format. We also tested the performance of this
newly developed assay in a pilot screen of 3,000 compounds
and we confirmed the activity of the obtained hits. We present
and discuss our findings and their importance for the discovery
of novel drugs by high throughput screening.
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Article]
[PMID:
19275532 PubMed - indexed for MEDLINE]
Narrowing Laccase Substrate Specificity Using Active Site
Saturation Mutagenesis
Nirupama Gupta and Edgardo T. Farinas
The laccase CotA from Bacillus subtilis was
converted from a generalist, an enzyme with broad specificity,
to a specialist, an enzyme with narrowed specificity. Laccases
are members of the multicopper oxidase family and have many
applications in biotechnology. To date, it has not been demonstrated
that substrate specificity can be tapered for a laccase. Wild-type
CotA oxidizes ABTS (ABTS = diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)
and SGZ (SGZ = 4-hydroxy-3,5- dimethoxy-benzaldehyde azine),
and it was engineered for increased specificity for ABTS by
combining rational and directed evolution approaches. The
wild-type was evolved by simultaneously randomizing 19 amino
acids found in the substrate-binding pocket. A mutant was
identified that had a catalytic efficiency, (kcat/KM)ATBS
/ (kcat/KM)SGZ,
7.0 times greater when compared to the wild-type after one
round of screening. This illustrates that the substrate-binding
pocket is highly evolvable for specificity.
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Article]
[PMID:
19275533 PubMed - indexed for MEDLINE]
Design and Combinatorial Synthesis of a Library of Methylenesulfonamides
and Related Compounds as Potential Kinase Inhibitors
Jane E. Torr, Jonathan M. Large and
Edward McDonald
An effective parallel solid phase route to methylenesulfonamides
and amides bearing a wide variety of substituents is described.
The three key reaction steps were reductive amination of a
haloheteroaromatic aldehyde onto a benzhydrylamine type polystyrene
resin, sulfonamide or amide formation and palladium catalysed
transformation of the remaining heteroaromatic halogen. A
process of virtual library design and filtering, together
with solution and solid phase optimisations, aided the preparation
of several novel drug-like product classes in high purities
and should allow access to a variety of further useful analogues.
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[PMID:
19275534 PubMed - indexed for MEDLINE]
A Multi-Step NMR Screen for the Identification and Evaluation
of Chemical Leads for Drug Discovery
Kelly A. Mercier, Matthew D. Shortridge
and Robert Powers
A multi-step NMR based screening assay is described for
identifying and evaluating chemical leads for their ability
to bind a target protein. The multi-step NMR assay provides
structure-related information while being an integral part
of a structure based drug discovery and design program. The
fundamental principle of the multi-step NMR assay is to combine
distinct 1D and 2D NMR techniques, in such a manner, that
the inherent strengths and weakness associated with each technique
is complementary to each other in the screen. By taking advantage
of the combined strengths of 1D and 2D NMR experiments, it
is possible to minimize protein requirements and experiment
time and differentiate between non-specific and stoichiometric
binders while being able to verify ligand binding, determine
a semi-quantitative dissociation constant, identify the ligand
binding site and rapidly determine a protein-ligand co-structure.
Furthermore, the quality and physical behavior of the ligand
is readily evaluated to determine its appropriateness as a
chemical lead. The utility of the multi-step NMR assay is
demonstrated with the use of PrgI from Salmonella typhimurium
and human serum albumin (HSA) as target proteins.
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[PMID:
19275535 PubMed - indexed for MEDLINE]
High Throughput Screening Assay of α1G
T-type Ca2+ Channels and
Comparison with Patch-Clamp Studies
Yoonjee Kim, Soonja Kang, Jae Yeol Lee and
Hyewhon Rhim
Whole-cell patch-clamp-based electrophysiological techniques
are powerful tools for examining the biophysical and pharmacological
properties of ion channels. However, the recent validation
of ion channels as novel drug targets necessitates the development
of a faster screening method for ion channels. Therefore,
we have developed a rapid, reliable, and sensitive cell-based
high throughput screening (HTS) assay for T-type Ca2+
channels. We had previously constructed HEK293/α1G/Kir2.1
cell lines that stably expressed α1G
and Kir2.1 subunits [1] and found that α1G
T-type channel-sensitive Ca2+
signals were detected by the application of high concentrations
of KCl under fura-2-based single cell measurements of intracellular
Ca2+ concentration ([Ca2+]i).
In the present study, we applied HEK293/α1G/Kir2.1
cells to the FDSS6000 (Functional Drug Screening System) to
develop a fast and reliable cell-based HTS method for α1G
T-type Ca2+ channels. After
detecting 70 mM KCl-induced [Ca2+]i
increases using the FDSS6000 system, we verified this new
α1G
channel HTS system by examining two T-type Ca2+
channel blockers, Ni2+ and
mibefradil, and measuring the Z´-factor (Z´ factor
= 0.66) in 96-well plates. Furthermore, we assayed selected
3,4-dihydroquinazolin derivatives using this FDSS6000-based
α1G
channel HTS system at the level of IC50
values and compared the results with those obtained from whole-cell
patch-clamp recordings. Taken together, our results suggest
that the FDSS6000-based α1G
channel HTS system is a fast and feasible assay for α1G
T-type Ca2+ channels. This
assay can be utilized as a primary screening method for T-type
Ca2+ channel-targeted chemicals
and for the development of HTS systems for other types of
ion channels.
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[PMID:
19275536 PubMed - indexed for MEDLINE]
Docking-Based Virtual Screening: Recent Developments
Tiziano Tuccinardi
Virtual (database) screening (VS) of molecules promises
to accelerate the discovery of new drugs and reduce costs
by identifying molecules with high probabilities of binding
to a target receptor.
The large amount of available protein X-ray crystal structures,
together with the development of more effective homology modelling
techniques, has led recently to a steep increase in docking-based
VS studies. This approach needs computational fitting of molecules
into a receptor active site using advanced algorithms, followed
by the scoring and ranking of these molecules to identify
potential leads. In this review, the main published docking-based
VS studies developed over the last eight years are investigated,
and details are provided about the software used, the results
achieved and the novel methods employed.
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Article]
[PMID:
19275537 PubMed - indexed for MEDLINE]
High Throughput Screening of Physicochemical Properties and
In Vitro ADME Profiling in Drug Discovery
Hong Wan and Anders G. Holmén
Current advances of new technologies with robotic automated
assays combined with highly selective and sensitive LC-MS
enable high-speed screening of lead series libraries in many
in vitro assays. In this review, we summarize state
of the art high throughput assays for screening of key physicochemical
properties such as solubility, lipophilicity, pKa, drug-plasma
protein binding and brain tissue binding as well as in
vitro ADME profiling. We discuss two primary approaches
for high throughput screening of solubility, i.e. an automated
96-well plate assay integrated with LC-MS and a rapid multi-wavelength
UV plate reader. We address the advantages of newly developed
miniaturized techniques for high throughput pKa screening
by capillary electrophoresis combined with mass spectrometry
(CE-MS) with automated data analysis flow. Several new lipophilicity
approaches other than octanol-water partitioning are critically
reviewed, including rapid liquid chromatographic retention
based approach, immobilized artificial membrane (IAM) partitioning
and liposome, and potential microemulsion electrokinetic chromatography
(MEEKC) for accurate screening of LogP. We high-light the
sample pooling (namely cassette dosing, all-in-one, cocktail)
as an efficient approach for high throughput screening of
physicochemical properties and in vitro ADME profiling
with emphasis on the benefit of on-line quality control. This
cassette dosing approach has been widely adapted in drug discovery
for rapid screening of in vivo pharmacokinetic parameters
with significantly increased capacity and dramatically reduced
animal usage.
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