Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 11, Number 2, February 2008
Contents
High Throughput Screening Using Eukaryotic Platform
Organisms
Guest Editors: Hildegard Büning and Christian J.
Buchholz
Editorial from Editor-in-Chief
Pp. 83
Editorial from Guest Editors Pp.
84
The Baculovirus Display Technology - An Evolving Instrument
for Molecular Screening and Drug Delivery Pp. 86-98
Anna R. Mäkelä and Christian Oker-Blom
[Abstract]
Retroviral Display and High Throughput Screening
Pp. 99-110
Christian J. Buchholz, Lydia J. Duerner, Sabrina Funke
and Irene C. Schneider
[Abstract]
Library Selection Approaches to Engineering Enhanced
Retroviral and Lentiviral Vectors Pp. 111-117
Kwang-il Lim and David V. Schaffer
[Abstract]
Artificial Evolution with Adeno-Associated Viral
Libraries Pp. 118-126
Luca Perabo, Anke Huber, Stephan Märsch, Michael
Hallek and Hildegard Büning
[Abstract]
A Decade of Yeast Surface Display Technology:
Where Are We Now? Pp. 127-134
Lauren R. Pepper, Yong Ku Cho, Eric T. Boder and Eric
V. Shusta
[Abstract]
Peptide Aptamer Libraries Pp. 135-145
Corina Borghouts, Christian Kunz and Bernd Groner
[Abstract]
Rational Design, Selection and Specificity of
Artificial Transcription Factors (ATFs): The Influence of
Chromatin in Target Gene Regulation Pp. 146-158
Pilar Blancafort and Adriana S. Beltran
[Abstract]
High Throughput Screening of Gene Functions in Mammalian
Cells Using Reversely Transfected Cell Arrays: Review And
Protocol Pp. 159-172
Michael Stürzl, Andreas Konrad, Gaby Sander, Effi
Wies, Frankeipel, Elisabeth Naschberger, Simone Reipschläger,
Nathalie Gonin-Laurent, Raymund E. Horch, Ulrich Kneser,
Werner Hohenberger, Holger Erfle and Mathias Thurau
[Abstract]
Meet
the Guest Editors Pp. 173
Abstracts
[Back to top]
Editorial from Editor-in-Chief
Throughout its first ten years of publication, Combinatorial
Chemistry & High Throughput Screening has occupied
a unique position among the peer reviewed literature by specializing
in the publication of review articles and original research
papers in combinatorial chemistry, high throughput screening,
and the interface of these related fields. Papers published
in CCHTS are highly visible and are abstracted and
indexed by the major services including Current Contents/Life
Sciences, Biological Abstracts, BIOBASE, BIOSIS, BIOSIS Previews,
CAB Abstracts, Chemical Abstracts, Chemistry Citation Index,
EMBASE, Google, Google Scholar, JCR/Science Edition, MEDLINE,
Reaction Citation Index, Science Citation Index Expanded,
and SCI Expanded. During the last year, CCHTS reached
another milestone with its highest impact ever of 2.55 (according
to the ISI Web of Knowledge, Journal Citation Reports, produced
by the Thomson Corporation; Philadelphia, PA). In addition,
CCHTS is ranked 4th
out of 58 journals by Journal Citation Reports in the field
of Applied Chemistry.
After ten years of outstanding service to CCHTS,
our Regional Editor for Asia, Prof. Takenori Kusumi of the
Faculty of Pharmaceutical Sciences at the University of Tokushima,
Japan, has decided to retire from this role at CCHTS.
I would like to recognize Prof. Kusumi for his help when CCHTS
was launched as a new journal ten years ago and then for his
tireless commitment to make it a continuing success. When
CCHTS began publication in 1998, combinatorial chemistry
was so new that few manuscripts were being submitted from
researchers in Asia. Thanks at least in part to the efforts
of Prof. Kusumi, numerous manuscripts are now being submitted
each year from authors in Asia. Taking his place as the new
Regional Editor for Asia is one of the original members of
the CCHTS Editorial Board, Prof. Ikuo Fujii of Osaka
Prefecture University. Since Prof. Fujii brings ten years
of experience on our Editorial Board to this position, the
transition between Regional Editors for Asia should go unnoticed
by authors awaiting referees’ reviews and editorial
decisions from this office.
In addition to a new Regional Editor for Asia, there are several
new members joining our Editorial Board as well as others
who are retiring after many years of service. In particular,
I would like to thank the retiring members of the Editorial
Board of CCHTS for ten years of peer review of manuscripts
submitted for publication and support of the journal. These
distinguished Editorial Board members include Dr. Koshi Arai
(Taisho Pharmaceutical; Saitama), Dr. Nobuyuki Okajima (Japan
Tobacco; Osaka), Prof. Shigeki Sasaki (Kyushu University;
Fukuoka), and Dr. Jun-ya Tamura (Yamanouchi Pharmaceutical;
Ibaraki). For a complete list of the current Editorial Board
members and Regional Editors of CCHTS, please visit
our web site at the following Internet address: http://www.bentham.org/cchts.
Information for authors, an index of papers published in CCHTS
and free access to sample issues may also be found at our
website.
As we begin our 11th year
of publication, Combinatorial Chemistry & High Throughput
Screening remains a unique and essential scientific journal
defining the intersection of both combinatorial chemistry
and screening. We will continue to publish 10 issues per year,
which is the most frequent publication rate in this field.
This effort is made possible by the distinguished members
of the Editorial Board, the Regional Editors, the production
staff and management of Bentham Science publishers, the guest
editors of special issues, the authors, and of course you,
the readers. Thank you for ten years (and counting) of support
for CCHTS.
Richard B. van Breemen
(Editor-in-Chief)
University of Illinois
College of Pharmacy
833 S. Wood Street
Chicago
IL 60612
USA
E-mail: breemen@uic.edu
[Back to top]
Editorial from Guest Editors
High throughput screening (HTS) has become an indispensable
tool in modern drug development and has been extended from
the screening of chemical libraries to the screening of biological
molecules or even whole organisms. Libraries of proteins and
peptides can be screened for diverse applications including
enzyme optimisation, functional genomics, or therapeutic purposes
like drug development or targeted delivery. Mainly for technical
difficulties in generating libraries covering large repertoires
of polypeptide variants platform organisms were previously
restricted to the prokaryotic kingdom. The scope of this edition
is to review recent developments in display technologies beyond
the well known and often used phage display technology. During
the past years more and more library and screening systems
have been described that are based on viruses and eukaryotic
cells as platforms. This way, many sophisticated screening
systems have been established. In this issue of Combinatorial
Chemistry & High Throughput Screening, we have assembled
a collection of articles describing the state of the art for
different eukaryotic display platforms and for specific screening
strategies performed in eukaryotic expression systems.
The first two reviews focus on the use of two enveloped viruses
as screening platform, baculovirus and retrovirus (reviews
one and two). Both are used in many laboratories as vectors
to produce large amounts of recombinant protein in a eukaryotic
cell system or to genetically modify mammalian cells. Several
unique properties of these viruses make them an ideal platform
also for protein display and screening. Small peptides and
also large polypeptides can be displayed on the viral surface
or, using the virus as vector, on the cell membrane. In each
case full processing and glycosylation of the displayed protein
is possible. Screening strategies using retroviruses and baculoviruses
include, but are not limited to, the identification of single-chain
antibodies, protein interaction partners, antigenic epitopes
or protease substrate peptides. Besides screening, display
of polypeptides can also be applied for other purposes such
as evoking an efficient immune response, identifying ligand
receptor interaction partners, or modifying the tropism of
viral vectors.
HTS is also being used to improve viral gene transfer vectors.
Libraries of vial variants of retroviruses and adeno-associated
viruses are being generated by random peptide insertions,
error prone PCR, DNA shuffling or transposon-based approaches
(reviews three and four). Vectors based on the natural occurring
virions have been used in many clinical gene therapy trials
worldwide. However, to become more efficient in gene delivery
in patients, properties like particle stability, nuclear entry
into quiescent cells, escape from an inactivating immune response,
or receptor choice and thus biodistribution are being improved
by library screening. Yeast cells have become increasingly
popular as display platform during the past years. Large repertoires
can be covered and screening can be facilitated through fluorescent
cell sorting techniques. Yeast based display libraries have
been screened for different purpose including epitope mapping,
the identification of interacting protein partners, or single-chain
antibodies recognising specific molecules on the surface of
human cells and tissue (review five). In peptide aptamers
and Zn-finger domains, which are in the focus of review six
and seven, amino acid residues available for random diversification
and library generation are well defined. Libraries of peptide
aptamers are often screened in eukaryotic cell systems to
identify peptides that interfere with certain cellular pathways
by binding to a target protein whereas Zn-finger domains can
be engineered to bind selectively to specific sequences of
DNA. By combining them with effector domains of transcription
factors, designer transcription factors can be generated.
Effective “designer” transcription factors can
be obtained through library screening for optimal binding
and activation of a defined DNA sequence or a gene of interest.
We conclude this issue with a review describing a novel approach
that allows the screening for gene functions based on reversely
transfected cell arrays. Effects on the cell phenotype resulting
from the transfection of cDNA or siRNA can be screened in
multi-well dishes. Applications of this technique in functional
genomics are described and a laboratory protocol is provided.
In summary, we have collected several articles that discuss
the most important display platforms and screening strategies
in eukaryotic expression systems. Only they enable the screening
for properties, that exclusively manifest in mammalian cells
or that require processing of the displayed polypeptide in
a eukaryotic environment. Moreover, translational problems
as they occur when molecules selected in a prokaryotic expression
system are transferred to mammalian or human cells are avoided.
It is therefore well conceivable that especially for therapeutic
purposes HTS based on eukaryotic expression systems will become
more and more popular in near future.
Hildegard Büning
University of Cologne
Clinic I for Internal Medicine
and Center for Molecular Medicine Cologne
Kerpener Str. 62
D-50937 Cologne
Germany
E-mail:
hildegard.buening@uk-koeln.de
Christian J. Buchholz
Division of Medical Biotechnology
Paul-Ehrlich-Institut
Paul-Ehrlich-Str. 51-53
D-63225 Langen
Germany
E-mail: bucch@pei.de
[Back to top]
The Baculovirus Display Technology - An Evolving Instrument
for Molecular Screening and Drug Delivery
Anna R. Mäkelä and Christian Oker-Blom
High throughput screening is a core technology in drug
discovery. During the past decade, several strategies have
been developed to screen (poly)peptide libraries for diverse
applications including disease diagnosis and profiling, imaging,
as well as therapy. The recently established baculovirus display
vector system (BDVS) represents a eukaryotic screening platform
that combines the positive attributes of both cell and virus-based
display approaches, allowing presentation of complex polypeptides
on cellular and viral surfaces. Compared to microbial display
systems, the BDVS has the advantage of correct protein folding
and post-translational modifications similar to those in mammals,
facilitating expression and analysis of proteins with therapeutic
interest. The applicability of the system is further expanded
by the availability of genetically engineered insect cell
lines capable of performing e.g. mammalianized glycosylation
in combination with high level of expression. In addition
to insect cells, baculovirus can mediate delivery and expression
of heterologous genes in a broad spectrum of primary and established
mammalian cells. Currently, a variety of baculovirus-based
assays aiming at routine high throughput identification of
agents targeting cell surface receptors or studies on ligand-receptor
interactions are under construction. Here, the advancements
and future prospects of the baculovirus display technologies
with emphasis on molecular screening and drug delivery applications
using insect cell display, mammalian cell display, and virion
display are described.
[Back to top]
Retroviral Display and High Throughput Screening
Christian J. Buchholz, Lydia J. Duerner, Sabrina Funke
and Irene C. Schneider
Retroviruses distinguish themselves from all other mammalian
viruses by their abilities to infect and propagate in mammalian
cells without causing a cytopathic effect and to stably integrate
their genetic information into the genome of the host cell.
These unique properties make them an ideal platform for the
display and directed evolution of proteins in a mammalian
cell environment. This review will describe the essentials
about retrovirus biology and then discuss in detail display
and screening strategies that have been developed during the
past 15 years of retroviral display technology.
[Back to top]
Library Selection Approaches to Engineering Enhanced Retroviral
and Lentiviral Vectors
Kwang-il Lim and David V. Schaffer
Retroviral and lentiviral based gene delivery vectors
have been used in numerous pre-clinical studies and clinical
trials due to their advantages, including stable and prolonged
expression of therapeutic transgenes and minimal immune responses
against the vector. Despite such advantages, however, retroviral
vectors also have several limitations for gene therapy applications.
For example, they can suffer from a lack of efficient or targeted
gene delivery to key cell types. In addition, retroviral vector
stability can be compromised by their envelope proteins. This
review briefly describes how such limitations have been overcome
by recently developed library selection approaches that borrow
a lesson from nature: the ability of evolution to generate
biomolecules with novel function. These library selection
approaches are based on the construction of retroviral libraries
where the sequences encoding natural viral components are
partially randomized using a variety of methods in order to
generate diverse libraries that can be selected to create
improved or novel functions. These high throughput, library-based
approaches provide a strong complement to rational engineering
of viral components for the rapid development of efficient
and safe retroviral and lentiviral vector systems for gene
therapy.
[Back to top]
Artificial Evolution with Adeno-Associated Viral Libraries
Luca Perabo, Anke Huber, Stephan Märsch, Michael
Hallek and Hildegard Büning
After attracting the attention of the scientific community
due to a number of favourable characteristics that make it
an attractive vector for human gene therapy [1, 2], AAV has
been thoroughly investigated in the past two decades. Standard
technologies for the manipulation of the viral genome and
for efficient packaging and purification protocols have paved
the road for trial and error manipulation by educated guesses
to study viral infectious biology by reverse genetics and
to generate improved vectors for human gene transfer. However,
despite remarkable progress, our limited knowledge of molecular
mechanisms implicated in virus-cell interactions has been
a limiting factor.
Combinatorial engineering and high-throughput selection techniques
hold the potential to boost technological improvement by offering
the possibility to screen large numbers of randomly generated
clones by appropriate selection protocols. These approaches
not only require lesser knowledge of viral biology, but can
also be employed as valuable tools to investigate molecular
mechanisms that drive the infection process.
In this review we recapitulate the rationale for employment
of combinatorial methods in AAV vector development and the
accomplishments achieved so far, discussing current limitations
and interesting developments that are in sight.
[Back to top]
A Decade of Yeast Surface Display Technology: Where Are We
Now?
Lauren R. Pepper, Yong Ku Cho, Eric T. Boder and Eric
V. Shusta
Yeast surface display has become an increasingly popular
tool for protein engineering and library screening applications.
Recent advances have greatly expanded the capability of yeast
surface display, and are highlighted by cell-based selections,
epitope mapping, cDNA library screening, and cell adhesion
engineering. In this review, we discuss the state-of-the-art
yeast display methodologies and the rapidly expanding set
of applications afforded by this technology.
[Back to top]
Peptide Aptamer Libraries
Corina Borghouts, Christian Kunz and Bernd Groner
Peptide aptamers are molecules that bind to protein targets
and are able to interfere with their functions. In the past,
important achievements have been made using such peptide aptamers
in different approaches and for various purposes. Peptide
aptamers are comprised of a variable peptide region of 8 to
20 amino acids in length, which is displayed by a scaffold
protein. An overview of the numerous scaffold proteins that
have been investigated for their suitability to present peptide
aptamers will be given. To identify peptide aptamers efficiently
and specifically binding to a predetermined target, two eukaryotic
systems have been used in multiple studies: a modified version
of the Gal4 yeast-two-hybrid system and the optimized LexA
interaction trap system. The two yeast systems are compared
and the design of high-complexity peptide aptamer libraries
for these systems is described. Although the yeast-two-hybrid
system is based on intracellular interactions mammalian screens,
performed in cell culture experiments, are sometimes preferred
or required. We will give an overview of the mammalian selection
systems available, which are based on the expression of peptide
aptamers in retroviral or lentiviral vectors. We will show
that the isolation and use of peptide aptamers as inhibitors
of individual signaling components represents a new challenge
for drug development.
[Back to top]
Rational Design, Selection and Specificity of Artificial Transcription
Factors (ATFs): The Influence of Chromatin in Target Gene
Regulation
Pilar Blancafort and Adriana S. Beltran
Artificial Transcription Factors (ATFs) are engineered
DNA-binding proteins designed to bind specific sequences of
DNA. ATFs made of Zinc Finger (ZF) domains have been developed
to regulate specific genes and phenotypes both in cells and
whole organisms. Recently, an emerging application of engineered
DNA-binding domains include the specific editing of the genome,
the ability to specifically cut, recombine, modify DNA and
image protein-nucleic acid interactions in living cells. In
this review we will summarize the techniques used for the
rational design, screening and functional selection of ZF
proteins in mammalian cell systems and their applications
in areas of biotechnology, functional genomics and molecular
therapeutics. The in vivo specificity of the engineered
ATFs will be discussed, with particular emphasis on epigenetic
modifications influencing ATF-DNA interactions.
[Back to top]
High Throughput Screening of Gene Functions in Mammalian Cells
Using Reversely Transfected Cell Arrays: Review And Protocol
Michael Stürzl, Andreas Konrad, Gaby Sander, Effi
Wies, Frank Neipel, Elisabeth Naschberger, Simone Reipschläger,
Nathalie Gonin-Laurent, Raymund E. Horch, Ulrich Kneser,
Werner Hohenberger, Holger Erfle and Mathias Thurau
Reversely transfected cell microarrays (RTCM) have been
introduced as a method for parallel high throughput analysis
of gene functions in mammalian cells. Hundreds to thousands
of different recombinant DNA or RNA molecules can be transfected
into different cell clusters at the same time on a single
glass slide with this method. This allows either the simultaneous
overexpression or - by using the recently developed RNA interference
(RNAi) techniques - knockdown of a huge number of target genes.
A growing number of sophisticated detection systems have been
established to determine quantitatively the effects of the
transfected molecules on the cell phenotype. Several different
cell types have been successfully used for this procedure.
This review summarizes the presently available knowledge on
this technique and provides a laboratory protocol.
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