Combinatorial Chemistry & High Throughput
Screening, Vol. 8, No. 2, 2005
Contents
Screening
for Proteins and Inhibitors
Guest
Editor: Stefan Rose-John
Editorial
Stefan
Rose-John
[Abstract] Considerations
on Antibody-Phage Display Methodology Pp.117-126 Udo
Conrad and Jurgen Scheller Screening
of Molecular Repertoires by Microbial Surface Display Pp.127-133 Thomas
Jostock and Stefan Dubel Synthetic
Peptide Arrays and Peptide Combinatorial Libraries for the Exploration of
Protein-Ligand Interactions and the Design of Protein Inhibitors Pp.135-143 Jutta
Eichler Phage
Display and PEGylation of Therapeutic Proteins Pp.145-152 Yohei
Mukai, Yasuo Yoshioka, and Yasuo
Tsutsumi Combinatorial
Chemistry in Glycobiology Pp.153-159 Obadiah
J. Plante Metalloproteinase
Inhibitors for the Disintegrin-Like Metalloproteinases ADAM10 and ADAM17 that
Differentially Block Constitutive and Phorbol Ester-Inducible Shedding of Cell
Surface Molecules Pp.161-171 Andreas
Ludwig, Christian Hundhausen, Millard H. Lambert, Neil Broadway, Robert C.
Andrews, D. Mark Bickett, M. Anthony Leesnitzer and J. David Becherer Designing
Cytokine Variants by Phage-Display Pp.173-179 Heidi
Schooltink and Stefan Rose-John High
Throughput Screening for Protein Kinase Inhibitors Pp.181-195 Holger
Wesche, Shou-Hua Xiao and Steve W. Young Screening
for Protease Substrate by Polyvalent Phage Display Pp.197-203 Radislav
Sedlacek and Emily Chen Abstracts [Back to top] Stefan
Rose-John High throughput screening
approaches have found their way into many if not all pharmaceutical companies and
have made important contributions to basic science as well as to drug
development. An important aspect is that the
combinatorial principle has not only been applied in molecular biology
techniques, where randomized oligonucleotides have been incorporated into genes
coding for phage and bacteria surface proteins. In the last ten years we have
also seen fundamental progress in glycobiology, chemical protease inhibitor
development and synthetic peptide library development. A further important point, which
can be seen in nearly all reviews in this special issue, is the fundamental
influence of structural biology. Most if not all biotechnological and
therapeutic interventions at one point or the other rely on the interaction of
drugs with proteins. Therefore, the availability of protein structural data is
often pivotal to the understanding of the mechanism of action of a drug.
Furthermore, many inhibitors of enzymes have been refined using structural data
of their respective target proteins. The idea of this special issue
was to assemble reviews of research fields which are strongly influenced by
combinatorial approaches, although they would prima vista be judged to
be scientifically unrelated. There are, however, many points where similar
techniques were used or similar problems arose. The aim of this special issue
has been to demonstrate the interdisciplinary aspect of combinatorial
approaches. This special issue of Combinatorial
Chemistry & High Throughput Screening on the screening for proteins and
inhibitors brings together leading scientists from diverse fields involved in
protein and inhibitor research such as biochemistry, structural biology, signal
transduction, combinatorial chemistry and recombinant antibody technology. This
issue provides the reader with important breakthroughs of the last years and
demonstrates that combinatorial approaches in chemistry and molecular biology
are possible, necessary and successful. [Back to top] For almost 15 years phage display
is being used for the selection of specific antigen-binders from artificial
libraries of single chain antibodies. Filamentous phages have been developed in
a way to express foreign proteins on the surface and at the same time carrying
the genetic information of the surface expressed molecule within the phage
capsid. This property guarantees the coupling of phenotype and genotype during
phage amplification and affinity selection. The possibility to generate large
antibody libraries and the simplified antibody-backbone of a single chain
antibody has made antibody-phage display to a powerful tool for the development
of new therapeutics against various human diseases. In this review we discuss
the general principles and latest developments and applications in antibody
phage display technology. [Back to top] Thomas
Jostock and Stefan Dubel A multitude of systems for the
presentation of foreign peptides or proteins on the surface of microorganisms
has been developed within the past two decades. However, the majority of the
bacterial surface display systems are devoted to the presentation of
heterologous antigens to the immune system (vaccine generation). Bacteria are
the preferable hosts for the generation of vast genetic repertoires, and their
genetic manipulation and cultivation is easy. As a consequence, they provide
promising systems for large-scale functional screenings, e.g. for enzyme
activity or protein-protein interactions. This review will focus on examples of
microbial surface display used for the screening of combinatorial repertoires.
Further, we discuss future opportunities and promising candidate proteins not
yet employed for that task. [Back to top]
Jutta
Eichler Synthetic peptides have a long
tradition as molecular tools in biomedical research and drug discovery. The
introduction of high-throughput synthesis and screening technologies for
synthetic peptides, such as arrays and combinatorial libraries, enabled the
large-scale and detailed exploration of protein-ligand interactions, as well as
the discovery of novel biologically active peptides. This review summarizes
currently available synthetic peptide array and library technologies, in
particular mixture-based peptide libraries, which are illustrated by numerous
applications in various fields of biomedical research. [Back to top] Yohei
Mukai, Yasuo Yoshioka and Yasuo
Tsutsumi With the success of the human
genome project, the focus of life science research has shifted to the
functional and structural analyses of proteins, such as disease proteomics and
structural genomics. These novel approaches to the analyses of proteins,
including newly identified ones, are expected to help in the identification and
development of protein therapies for various diseases. Thus, disease
proteomic-based drug discovery has a very high profile. Nevertheless, the use
of bioactive proteins in the clinical setting is not straightforward because, in
vivo, these proteins have a low stability and a pleiotropic action. To
promote disease proteomic-based drug discovery and development, we have
attempted to establish a system for creating functional mutant proteins
(muteins) with the desired properties, and also to develop a site-specific
polymer-conjugation system for further improving their therapeutic potency.
These innovative protein-drug systems are discussed in this review. [Back to top] Obadiah
J. Plante The application of combinatorial
chemistry to glycobiology historically has proven challenging due to numerous
synthetic hurdles. The advent of novel methodologies has enabled the production
of natural as well as mimetic analogues for proof-of-concept experiments and
SAR. This review highlights some of the recent synthetic advances in
combinatorial carbohydrate synthesis. The application of carbohydrate libraries
in glycobiology is also discussed. [Back to top] Andreas
Ludwig, Christian Hundhausen, Millard H. Lambert, Neil Broadway, Robert C.
Andrews, D. Mark Bickett, M. Anthony Leesnitzer and J. David Becherer The transmembrane
metzinkin-proteases of the ADAM (a disintegrin and a metalloproteinase)-family
ADAM10 and ADAM 17 are both implicated in the ectodomain shedding of various
cell surface molecules including the IL6-receptor and the transmembrane
chemokines CX3CL1 and CXCL16. These molecules are constitutively released from
cultured cells, a process that can be rapidly enhanced by cell stimulation with
phorbol esters such as PMA. Recent research supports the view that the
constitutive cleavage predominantly involves ADAM10 while the inducible one is
mediated to a large extent by ADAM17. We here describe the discovery of
hydroxamate compounds with different potency against ADAM10 and ADAM17 and
different ability to block constitutive and inducible cleavage of IL6R, CX3CL1
and CXCL16 by the two proteases. By screening a number of hydroxamate
inhibitors for the inhibition of recombinant metalloproteinases, a compound was
found inhibiting ADAM10 with more than 100-fold higher potency than ADAM17,
which may be explained by an improved fit of the compound to the S1’ specificity
pocket of ADAM10 as compared to that of ADAM17. In cell-based cleavage
experiments this compound (GI254023X) potently blocked the constitutive release
of IL6R, CX3CL1 and CXCL16, which was in line with the reported involvement of
ADAM10 but not ADAM17 in this process. By contrast, the compound did not affect
the PMA-induced shedding, which was only blocked by GW280264X, a potent
inhibitor of ADAM17. As expected, GI254023X did not further decrease the
residual release of CX3CL1 and CXCL16 in ADAM10-deficient cells verifying that
the compound’s effect on the constitutive shedding of these molecules was
exclusively due to the inhibition of ADAM10. Thus, GI254023X may by of use as a
preferential inhibitor of constitutive shedding events without effecting the
inducible shedding in response to agonists acting similar to PMA. [Back to top] Heidi
Schooltink and Stefan Rose-John Cytokines are important mediators of many cellular functions
including coordination of the immune system and regulation of regenerative
processes. Therefore, cytokines can be exploited for therapeutic strategies.
Cytokines can be altered in a way that their biologic activity is enhanced or
antagonized. This can be accomplished by changing the interaction of cytokines
with their cognate cytokine receptor complexes. Therefore, many research groups
tried to design cytokines, which bind with higher affinity to their receptors.
Alternatively, cytokine variants have been created which do bind to their
receptors but do not elicit a signal. Such strategies have been followed using
high throughput techniques like error-prone polymerase chain reaction and phage
display. Designer cytokines can be used to specifically inhibit cytokine
functions. Moreover, peptides have been generated with the help of phage
display techniques, which exhibit cytokine activity. Surprisingly, such mimetic
peptides do not show any sequence similarity to the parental cytokines. Such
peptide mimetics can be used as lead structures for the generation of
non-peptidic chemical compounds with cytokine activity. [Back to top] Holger
Wesche, Shou-Hua Xiao and Steve W. Young The pivotal role of kinases in
signal transduction and cellular regulation has lent them considerable appeal
as pharmacological targets across a broad spectrum of pathologies. Since the discovery that the
v-Src oncogene encoded a protein kinase in 1978, kinases have remained a focus
of research for pharmaceutical laboratories and academic groups alike. Many
have sought to develop orally available low molecular weight synthetic kinase
modulators (predominantly inhibitors) and thus capitalize on the links between
aberrant regulation and disease. This interest in kinases as drug
targets was fueled in recent years by the success of several kinase inhibitors
in the clinic, primarily Gleevec for the treatment of chronic myelogenous
leukemia and Iressa for the treatment of advanced non-small cell lung cancer. This review focuses on the
development of small molecule drugs, most of them binding in or close to the
ATP binding pocket. After some general considerations regarding the selection
of a particular kinase for drug discovery, we will discuss the encouraging
lessons learned from some of the kinase inhibitors currently in various stages
of development. The majority of this review is dedicated to a detailed
description and discussion of the various assay formats currently being
employed for high throughput screening. [Back to top] Radislav
Sedlacek and Emily Chen Proteases are key regulators of
many physiological and pathological processes [1,2], and are recognized as
important and tractable drug candidates. Consequently, knowledge of protease
substrate recognition and specificity promotes identification of biologically
relevant substrates, helps elucidating a protease’s biological function, and
the design of specific inhibitors. Traditional methods for establishing
substrate recognition profiles involve the identification of the scissile bond
within a given protein substrate by proteomic methods such as Edman degradation.
Then, synthetic peptide variants of this sequence can be screened in an
iterative fashion to arrive at more optimized substrates. Even though it can be
fruitful, this iterative strategy is biased toward the original substrate
sequence and it is also tremendously cumbersome. Furthermore, it is not
amenable to high throughput analysis. In 1993, Matthew & Wells
presented a method for the use of monovalent “substrate phage” libraries for
discovering peptide substrates for proteases, in which more than 107
potential substrates can be tested concurrently [3]. A library of fusion
proteins was constructed containing randomized substrate sequences placed
between a binding domain and the gene III coat protein of the filamentous
phage, M13, which displays the fusion protein and packages the gene coding for
it inside. Each fusion protein was displayed as a single copy on filamentous
phagemid particles (substrate phage). This method allows one to rapidly survey
the substrate recognition and specificity of individual or closely related
members of proteases. Over the past decade, substrate phage screening has shown
terrific utility in rapidly determining protease specificity and
characterization of substrate recognition profile of proteases. In some cases,
the structural insights of the catalytic domain were obtained from comparison
of substrate specificity among closely related family of proteases [4-6]. The
number of proteases (from various classes) characterized by this approach
testifies to its power. Since the initial development of substrate phage
library, different versions of the substrate phage cloning vectors have been
constructed to further improve the utility of substrate phage display. This
review will provide an overview of the construction of substrate phage display
libraries, screening of substrate phage libraries, examples of application,
summary and future directions.
Editorial
Considerations on Antibody-Phage Display Methodology Udo Conrad and Jurgen Scheller
Screening of Molecular Repertoires by Microbial Surface Display
Synthetic Peptide Arrays and Peptide Combinatorial Libraries for the Exploration
of Protein-Ligand Interactions and the Design of Protein Inhibitors
Phage Display and PEGylation of Therapeutic Proteins
Combinatorial Chemistry in Glycobiology
Metalloproteinase Inhibitors for the Disintegrin-Like Metalloproteinases ADAM10
and ADAM17 that Differentially Block Constitutive and Phorbol Ester-Inducible
Shedding of Cell Surface Molecules
Designing Cytokine Variants by Phage-Display
High Throughput Screening for Protein Kinase Inhibitors
Screening for Protease Substrate by Polyvalent Phage Display