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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 10, Number 6, July 2007
Contents
Supramolecular Devices for the High-Throughput Screening
of Drugs, Nutrients and Metabolites in Postgenomic Era (Part
2)
Guest Editor: Grzegorz Bazylak
Editorial Pp.
397-398
Review Articles
Design Considerations for High Throughput Screening
and In Vitro Diagnostic Assays Pp. 399-412
Komandoor E. Achyuthan and David G. Whitten
[Abstract]
Optical Chemical Biosensors for High Throughput Screening
of Drugs Pp. 413-432
María E. Bosch, Antonio J.R. Sánchez, Fuensanta
S. Rojas and Catalina B. Ojeda
[Abstract]
Optical Biosensors as a Tool for Early Determination
of Absorption and Distribution Parameters of Lead Candidates
and Drugs Pp. 433-440
Carlo Bertucci, Angela Piccoli and Marco Pistolozzi
[Abstract]
Application of Liposome Based Sensors in High Throughput
Screening Systems Pp. 441-450
Magda Przybylo, Tomasz Borowik and Marek Langner
[Abstract]
Research Articles
Fluorescence Sensing and Selective Binding of L- and
D Tryptophan-Modified Permethylated β-Cyclodextrins
for Aliphatic Oligopeptides Pp. 451-458
Yu Liu, Shu Kang, Yong Chen, Jun Shi and Chen-Feng Ke
[Abstract]
Hydroxycamptothecin Deactivation Rates and Binding
to Model Membranes and HSA Determined by Fluorescence Spectra
Analysis Pp. 459-465
Blanka Ziomkowska, Michal Cyrankiewicz and Stefan Kruszewski
[Abstract]
Combinatorial Synthesis and Characterization of New
Asymmetric Porphyrins as Potential Photosensitizers in Photodynamic
Therapy Pp. 466-472
Eugenia Fagadar-Cosma, Liliana Cseh, Valentin Badea, Gheorghe
Fagadar-Cosma and Dana Vlascici
[Abstract]
Preparation and Characterization of Thiacalix[4]arene
Coated Water-Soluble CdSe/ZnS Quantum Dots as a Fluorescent
Probe for Cu2+ Ions
Pp. 473-479
Takashi Jin, Fumihiko Fujii, Eiji Yamada, Yoshinobu Nodasaka
and Masataka Kinjo
[Abstract]
Preparation and Property Screening of the Solid Inclusion
Complex of Norfloxacin with p-Sulfonated Calix[4]arene
Pp. 480-485
Qin Lu, Yunyou Zhou, Junyong Sun, Lian Wu, Huapeng Yu,
Hongwei Xu and Lun Wang
[Abstract]
Determination of Hydroxycamptothecin Affinities to
Albumin and Membranes by Steady-State Fluorescence Anisotropy
Measurements Pp. 486-492
Blanka Ziomkowska, Michal Cyrankiewicz and Stefan Kruszewski
[Abstract]
Abstracts
[Back to top]
Editorial
Instrumentation-based HTS and μHTS
is being improved by introducing nanolitre devices for dispensing
fluids with extraordinary precision and biosensor-chip based
multimode ultra-high sensitive readout protocols both of which
are compatible with high density microwell and multiplate
formats. A wide range of catalytic and affinity-based sensing
elements together with variable fluorescence detection technologies
have been implemented successfully into HTS which shifted
the focus from observing macroscopic intermolecular events
to the nanoscale, supramolecular and even single molecular
level. In this way, HTS assays for drug discovery have developed
information-rich, multiparameter readout systems for diverse
molecular and supramolecular data that can include internal
quality controls such as real-time feedback, self-diagnosis
and adaptive supervising. These capabilities can reduce false-positive
or false-negative rates caused by non-specific binding, degradation,
precipitation, evaporation, poor solubility, or viscosity
related sub-optimal concentration. Some of these emerging
concepts and challenges for HTS are discussed in this issue
of Combinatorial Chemistry & High Throughput Screening,
which is Part 2 of a special issue commemorating the beginning
of supramolecular chemistry in 1967.
In the tutorial review by Achyuthan and Whitten opening this
issue, some central principles of statistical data analysis
in the development, optimization, validation and inter-laboratory
comparability and traceability of nanoscale operated HTS as
well as in vitro diagnostic (IVD) assays are reconsidered
or even redefined. Since the availability of statistical and
chemometric data analysis tools today does not always ensure
knowledge of their boundaries, this tutorial should be useful
to researchers in the pharmaceutical and combinatorial chemist
communities. Recent trends, pitfalls and developments in the
use of optical biosensors for different HTS protocols, especially
for direct label free detection of reagents, beads and cells,
are elaborated in the paper by Sanchez Rojas et al.
These considerations are also discussed in the paper by Bertucci
et al., which presents more detailed applications
of optical biosensors in HTS of highly specific drug-protein
interactions and determination of ADMET parameters of drug
candidates. In addition, the paper by Langner et al.,
discusses the application of new concept of fluorescence probes
and other supramolecular aggregate based biosensors for high-throughput
assessment of membrane permeability coefficients and membrane
adsorption of drugs and lead compounds.
In the two research papers by Kruszewski et al.,
the binding affinities of a series of hydroxycamptothecins,
anticancer drugs candidates, to human serum albumin and dimyristoylphosphatidylcholine
liposomes were determined using innovative high-throughput
steady-state fluorescence anisotropy measurements and factorial
analysis of fluorescence excitation spectra. Similarly, Fadagar-Cosma
et al. described the combinatorial synthesis and
fluorescence quantum yields of a series of second generation
asymmetrical meso-tetraphenylporphyrins as potential photosensitizers
in photodynamic therapy. The fluorescence sensing approach
was applied in the mini series of research papers by Liu et
al., Wang et al., and Jin et al., for
the study of molecular recognition and selective supramolecular
binding processes between aliphatic oligopeptides and tryptophan-modified
permethylated-β-cyclodextrins,
fluoroquinolone-type antibiotic norfloxacin and p-sulfonated-calix[4]arene,
and copper(II) cations and thiacalix[4]arene coated CdSe/ZnS
quantum dots, respectively. These papers illustrate the impact
of newly developed supramolecular materials, devices and procedures
on peptidomics, proteomics, solubility enhancing drug encapsulation,
and highly sensitive determination of inorganic impurities
in drugs and pharmaceuticals, thus improving drug safety.
The set of papers in Part 2 of this special issue of Combinatorial
Chemistry & High Throughput Screening indicates clearly
that introduction of biosensing nano-devices and nano-materials
into various HTS platforms enables accurate and high precision
liquid handling of nanolitre volumes of reagents and helps
provide insight into the dynamic characteristics of self-organised
living nanostructures and molecular signaling pathways. This
type of self-sensing in time of rearrangement and self-assembly
(or self-disassembly) of reacting molecular species, such
as drugs and receptors, quite often generates fluorescence
signaling which can be reproducibly detected, amplified, stored,
retrieved, and processed into the pharmacologically valuable
data. This approach opens a new route for establishing the
next generation of HTS procedures focused on exploring variation
and selection rules which promote, obstruct, hamper, or facilitate
desired pharmacological effects. Thus, think what is feasible.
Grzegorz Bazylak
Department of Pharmaco-Bromatology & Molecular Nutrition
Faculty of Pharmacy, Collegium Medicum
Nicolaus Copernicus University
PL-85067 Bydgoszcz
Poland
E-mail: gbazylak@cm.umk.pl
[Back to top]
Design Considerations for High Throughput Screening
and In Vitro Diagnostic Assays
Komandoor E. Achyuthan and David G. Whitten
This paper reviews the several different factors that must
be considered during the development of assays for high throughput
screening (HTS) or in vitro diagnostic (IVD)
applications. The reader is introduced to the terminology
used in assay development as well as the statistical approaches
for evaluating the data. The review is intended to serve as
a tutorial to biotechnology, pharmaceutical and clinical professionals,
the academic researcher, as well as a guide for established
investigators of HTS and IVD. This review is not
a comprehensive treatise in its scope or content, but is meant
to introduce the reader to key concepts of assay development.
Elementary mathematical and statistical tools for designing
robust assays and data management are described. While certain
design concepts overlap HTS and IVDs, others are
more pertinent to one or the other topic. An overview of the
regulatory requirements for IVDs is included in the
context of the United States Food and Drug Administration.
Quality concepts and high content screening are also briefly
described. The review does not focus upon any particular assay
technology nor does it provide detailed laboratory procedures
on specific assays. The references cited are not exhaustive,
but meant to steer the reader toward a general status report
of the various technologies discussed. The information presented
in this review is not intended to replace the judgment of
the experienced laboratory scientist. However, this review
should assist the scientific professional in executing well
designed assays and being aware of design considerations.
[Back to top]
Optical Chemical Biosensors for High Throughput Screening
of Drugs
María E. Bosch, Antonio J.R. Sánchez, Fuensanta
S. Rojas and Catalina B. Ojeda
Optical biosensors have been commercially available since
the early 1990s, and have been used extensively in many areas
of research in the life sciences. Optical biosensors developed
for drug analysis generally exploit the high selectivity of
the antigen-antibody and drug-protein interaction. Optical
biosensors can be made based on optical diffraction or electro-chemiluminescence.
High throughput screening, (HTS) which includes automated
preparation of a large number of samples and then screening
of their properties in multi-well plates, improves the efficiency
of research in many scientific areas, e.g., catalyst screening,
food processing, chemical synthesis, drug discovery, absorption,
distribution, metabolism, and excretion and toxicological
and cell based screening. The three most common detection
techniques used in HTS are UV–VIS absorbance, fluorescence
and luminescence. In this review, we summarize some recent
trends and developments in the construction of optical chemical
biosensors used in high throughput screening of drugs. Also,
we have included environmental, biological and other medical
applications of biosensors.
[Back to top]
Optical Biosensors as a Tool for Early Determination
of Absorption and Distribution Parameters of Lead Candidates
and Drugs
Carlo Bertucci, Angela Piccoli and Marco Pistolozzi
Specific molecular interactions provide a fundamental mechanism
for selectivity in every aspect of biological structure and
function. The ability to measure quantitatively such interaction
properties across a wide range of affinity, size, and purity
is a growing need. A short review on the use of the optical
biosensor techniques is presented, focused on its application
for determining the absorption and distribution parameters
of drugs and lead compounds. The basic biosensor technology
principles are described together with some immobilization
methods commonly used for the preparation of selective and
specific biosensor surfaces for assays. Some relevant research
topics in the field of small molecule recognition phenomena
are presented as examples, including binding to plasma proteins,
and binding to lipid membranes, in the frame of ADME (absorption,
distribution, metabolism and excretion) parameter determinations.
These applications demonstrate the applicability of such techniques
to the study of low mass compounds and illustrates their potential
for the screening of libraries of compounds with regard to
their binding to target bio-molecules as part of drug development.
[Back to top]
Application of Liposome Based Sensors in High Throughput
Screening Systems
Magda Przybylo, Tomasz Borowik and Marek Langner
High throughout screening is an approach based on the concept
which assumes that when sufficiently large library of compounds
are tested, the chance of discovering a new active compound
is increased. In order to meet this expectation, proper testing
criteria need to be devised. Those criteria should be related
to the fate of a compound in the organism to have any predictive
power. Not long ago, the main criteria were based exclusively
on parameters defined by the maximum activity (QSAR). In this
system, the activity criteria have not been included therefore
the compound ability to reach the target is not accounted
for. Considering that, the construction of yet another set
of parameters has been initiated (QSPR). The parameters are
in fact semi-empirical numbers which need to be tested on
real, physical models. Whereas the activity tests are straightforward,
the pharmacokinetic ones are difficult and controversial.
One such parameter describes the critical property of an active
compound, namely its ability to cross biological membranes.
This review describes new concepts in the determination of
the permeability coefficient with the help of methods which
are based on liposome biosensors. Two methods using fluorescence
probes incorporated in the lipid bilayer of liposome are described
in detail and compared to other currently available techniques.
[Back to top]
Fluorescence Sensing and Selective Binding of L- and
D Tryptophan-Modified Permethylated β-Cyclodextrins
for Aliphatic Oligopeptides
Yu Liu, Shu Kang, Yong Chen, Jun Shi and Chen-Feng Ke
Two tryptophan-modified permethylated β-cyclodextrins,
61-L-Trp-61-deoxy-
21,31-di-O-methyl-hexakis(2II-VII,3II-VII,6II-VII-tri-O-methyl)-β-cyclodextrin
(3) and 61-D-Trp-
61-deoxy-21,31-di-O-methyl-hexakis(2II-VII,3II-VII,6II-VII-tri-O-
methyl)-β-cyclodextrin
(4), were synthesized, and their binding
behaviors were investigated with the aliphatic oligopeptides,
Leu-Gly, Gly-Leu, Gly-Pro, Glu-Glu, and Gly-Gly. Fluorescence
spectrophotometric studies indicated that 3
and 4 can act as efficient fluorescence sensors
for aliphatic oligopeptides. Due to their intermolecular co-inclusion
binding mode with substrates, 3 and 4
not only afforded high binding constants of up to 103
−104 M-1
for guest oligopeptides but also good molecular selectivities
of up to ca. 7 for Gly-Gly/Leu-Gly and Glu-Glu/Gly-Gly pairs.
[Back to top]
Hydroxycamptothecin Deactivation Rates and Binding
to Model Membranes and HSA Determined by Fluorescence Spectra
Analysis
Blanka Ziomkowska, Michal Cyrankiewicz and Stefan Kruszewski
Camptothecins (CPTs) are fluorescent compounds exhibiting
anticancer activity. They can exist in two forms, a lactone
and a carboxylate. In neutral and base solution, lactone forms
hydrolyse and convert into carboxylates. Only the lactone
forms of CPTs are biologically active. Because of strong affinity
of the carboxylate form of the parent drug camptothecin to
human serum albumin (HSA), this protein promotes the deactivation
of this compound. On the other hand, the lactone forms of
camptothecins do not hydrolyse and are stabilized when bound
to membranes. The following three hydroxycamptothecins, 10
hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin
(SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin
(DB-67) were studied. Factor analysis of a set of fluorescence
excitation spectra recorded during lactone hydrolysis facilitated
the high-throughput determination of the deactivation rates
of camptothecin and each hydroxycamptothecin in phosphate
buffered saline. The fluorescence spectra of hydroxycamptothecins
di-luted in HSA solution or suspended in DMPC liposomes were
recorded, and the association constants of these drugs to
membranes and plasma proteins were calculated. Among the analysed
agents, DB-67 exhibited the most desirable properties including
low affinity of the carboxylate form for albumin and high
affinity of its lactone form for model membranes.
[Back to top]
Combinatorial Synthesis and Characterization of New
Asymmetric Porphyrins as Potential Photosensitizers in Photodynamic
Therapy
Eugenia Fagadar-Cosma, Liliana Cseh, Valentin Badea, Gheorghe
Fagadar-Cosma and Dana Vlascici
Porphyrins play a major role as active photosensitizers in
noninvasive optical photodynamic therapy (PDT). In a modular
approach, this paper presents a short review of the recent
developments of porphyrin structures and materials with improved
photosensitizing properties and then presents the synthesis
and characterization of a series of new second generation
asymmetrical meso-tetraphenylporphyrins varied by
substituent in the meta positions of the phenyl rings
with either -OH or -OCH3
groups, whereas in the para positions only with -OCH3
groups. The new series of differentially functionalized porphyrins
were obtained by a combinatorial multicomponent synthesis
(Adler-Longo method) by simultaneously using two different
aldehydes: 3,4-dimethoxybenzaldehyde and 3-hydroxybenzaldehyde.
The porphyrins were isolated, purified and characterized by
HPLC, TLC, UV-vis, fluorescence, MS, 1H-NMR,
and 13C-NMR analysis, accompanied
by DEPT 135 experiments. Because of the fact that the medium
in cancerous tissues is often more acidic than in normal tissues,
the capacity of these porphyrins to exist simultaneously in
aggregated and protonated forms was also investigated, in
tetrahydrofuran (THF) and acid THF-water systems, underlying
the changes in the photophysical behaviour. The relative fluorescence
quantum yields (Φf
) were calculated in comparison with meso-tetraphenylporphyrin
(TPP), and the values between 0.14-0.26 were found to be promising
for further trials. The series of asymmetrically substituted
tetra-phenylporphyrins, as the new class of supramolecular
materials, are suitable for further functionalization in order
to improve their photophysical properties, and they could
represent interesting potential PDT photosensitizers.
[Back to top]
Preparation and Characterization of Thiacalix[4]arene
Coated Water-Soluble CdSe/ZnS Quantum Dots as a Fluorescent
Probe for Cu2+ Ions
Takashi Jin, Fumihiko Fujii, Eiji Yamada, Yoshinobu Nodasaka
and Masataka Kinjo
Highly fluorescent water-soluble CdSe/ZnS (core/shell) quantum
dots (QDs) as a fluorescent Cu2+
ion probe were synthesized using thiacalix[4]arene carboxylic
acid (TCC) as a surface coating agent. Hydrophobic trioctylphosphine
oxide (TOPO) capped CdSe/ZnS QDs were overcoated with TCC
in tetrahydrofuran at room temperature, and deprotonation
of the carboxyl groups of TCC resulted in the formation of
water-soluble QDs. The surface structure of the QDs was characterized
by using transmission electron microscopy (TEM) and fluorescence
correlation spectroscopy (FCS). TEM images showed that TCC-coated
QDs were monodispersed with the particle size (core-shell
moiety) of approximately 5 nm. Hydrodynamic diameter of the
TCC-coated QDs was determined to be 8.9 nm by FCS, showing
that the thickness of the surface organic layer of the QDs
was approximately 2 nm. These results indicate that the surface
layer of TCC-coated QDs forms a bilayer structure consisting
of TOPO and TCC molecules. TCC-coated CdSe/ZnS QDs were highly
fluorescent (quantum yield, 0.21) compared to the QDs surface-modified
with mercaptoacetic acid and mercaptoundecanoic acid. Fluorescence
of the TCC-coated QDs was effectively quenched by Cu2+
ions even in the presence of other transition metal ions such
as Cd2+, Zn2+,
Co2+, Fe2+,
and Fe3+ ions in the same
solution. The Stern-Volmer plot for the fluorescence quenching
by Cu2+ ions showed a linear
relationship up to 30 μM
of Cu2+ ions. The ion selectivity
of TCC-coated QDs was determined by measurements of fluorescence
responses towards biologically important transition metal
ions (50 μM)
including Fe2+, Fe3+,
Co2+> Zn2+,
Cd2+. The fluorescence of
TCC-coated QDs was almost insensitive to other biologically
important ions such as Na+,
K+, Mg2+,
and Ca2+, suggesting that
TCC-coated QDs can be used as a fluorescent Cu2+
ion probe for biological samples. A possible quenching mechanism
by Cu2+ ions was also discussed
on the basis of a Lang-muir-type adsorption isotherm.
[Back to top]
Preparation and Property Screening of the Solid Inclusion
Complex of Norfloxacin with p-Sulfonated Calix[4]arene
Qin Lu, Yunyou Zhou, Junyong Sun, Lian Wu, Huapeng Yu,
Hongwei Xu and Lun Wang
The complexation of norfloxacin (NFLX) by
p-sulfonated calix[4]arene (SC4A)
in aqueous solution has been studied by fluorescence spectroscopy
and 1H NMR spectroscopy.
A 1:1 stoichiometry and a 8086 L mol-1
stability constant of the NFLX-SC4A complex
was obtained by spectrofluorometric titrations. The equimolar
solid state inclusion complex of NFLX-SC4A
was prepared by the co-precipitation method and then characterized
by various techniques, including differential scanning calorimetry
(DSC), X-ray powder diffractometry (XRD), Fourier-transform
infrared analysis (FT-IR) and scanning electron microscopy
(SEM). The experimental results of these chemical property
screenings confirmed that NFLX and SC4A
can form a stable host-guest complex in the solid state, and
SC4A appears to function as a complexing
and solubilizing agent for NFLX.
[Back to top]
Determination of Hydroxycamptothecin Affinities to
Albumin and Membranes by Steady-State Fluorescence Anisotropy
Measurements
Blanka Ziomkowska, Michal Cyrankiewicz and Stefan Kruszewski
Camptothecin (CPT) and its hydroxycamptothecin analogs are
fluorescent compounds exhibiting strong anticancer properties.
They exist in two forms: active lactone and inactive carboxylate.
The deactivation proceeds via hydrolysis in neutral
and base solutions. A serious limitation to the clinical application
of CPT is the strong affinity of its carboxylate form to human
serum albumin (HSA) which destabilizes its active lactone
form. However, binding to membranes in blood improves the
stability of the lactone form of CPT and its analogs. A high-throughput
screening assay based on the steady-state fluorescence anisotropy
method was used to determine the protein- and membrane-binding
properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin
(SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin
(DB-67). The relative affinities of hydroxycamptothecins to
HSA and model membranes in the form of DMPC liposomes were
determined, and DB-67 exhibited the most desirable properties
including the highest affinity to membranes in its lactone
form and low affinity to HSA in its carboxylate form.
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