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Current
Drug Discovery Technologies
ISSN: 1570-1638
Current Drug Discovery Technologies
Volume 5, Number 1, March 2008
Contents
Generation of Fusion Proteins for Selective Occlusion
of Tumor Vessels Pp. 1-8
Torsten Kessler, Christian Schwöppe, Ruediger Liersch,
Christoph Schliemann, Heike Hintelmann, Ralf Bieker, Wolfgang
E. Berdel and Rolf M. Mesters
[Abstract]
Genomic Signatures for Individualized Treatment
of Malignant Tumors Pp. 9-14
Manfred Kunz
[Abstract]
Neurokinin Receptors as Potential Targets in
Breast Cancer Treatment Pp. 15-19
Bobby Y. Reddy, Katarzyna A. Trzaska, Raghav G. Murthy,
Peter Navarro and Pranela Rameshwar
[Abstract]
Biocombinatorial Selection of Carbohydrate Binding
Agents of Therapeutic Significance Pp. 20-28
Sergei A. Svarovsky and Lokesh Joshi
[Abstract]
Targeting Toll-Like Receptor Signaling Pathways
for Design of Novel Immune Therapeutics Pp. 29-38
Elizabeth Hong-Geller, Anu Chaudhary and Sabine Lauer
[Abstract]
Targeted Drug Delivery to Bone: Pharmacokinetic
and Pharmacological Properties of Acidic Oligopeptide-Tagged
Drugs Pp. 39-48
Tatsuo Takahashi-Nishioka, Koichi Yokogawa, Shunji Tomatsu
Masaaki Nomura, Shinjiro Kobayashi and Ken-ichi Miyamoto
[Abstract]
MicroRNAs-Based Therapeutic Strategy for Virally
Induced Diseases Pp. 49-58
Alhousseynou Sall, Zhen Liu, Huifang M. Zhang, Ji Yuan,
Travis Lim, Yue Su and Decheng Yang
[Abstract]
Current In Vitro Kinase Assay Technologies:
The Quest for a Universal Format Pp. 59-69
Yong Jia, Christopher M. Quinn, Silvia Kwak and Robert
V. Talanian
[Abstract]
Emerging the Role of the Structure of Brain Membrane
Targets Recognizing Glutamate Pp. 70-74
Ágnes Simon, Ákos Bencsura, Anna Palló,
László Héja and Julianna Kardos
[Abstract]
Targeting Apoptosis to Treat Multiple Sclerosis
Pp. 75-77
Andrea L.O. Hebb, Craig S. Moore, Virender Bhan and George
S. Robertson
[Abstract]
Controlling Receptor Downregulation by Ubiquitination
and Deubiquitination Pp. 78-84
Masayuki Komada
[Abstract]
Abstracts
[Back to top]
Generation of Fusion Proteins for Selective Occlusion of Tumor
Vessels
Torsten Kessler, Christian Schwöppe, Ruediger Liersch,
Christoph Schliemann, Heike Hintelmann, Ralf Bieker, Wolfgang
E. Berdel and Rolf M. Mesters
Selective activation of blood coagulation in tumor vessels
with subsequent thrombosis and tumor infarction is a promising
strategy in cancer therapy. To this end, different fusion
proteins consisting of the extracellular domain of tissue
factor (truncated tissue factor, tTF) were fused to the peptides
GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or cyclic derivates
of these peptides, which selectively target αv
-integrins or aminopeptidase N (CD13), respectively.
Rationale for this strategy is the fact that these surface
receptors are preferentially expressed on tumor endothelial
cells. The tTF constructs were expressed in Escherichia
coli BL21 (DE3). The integrity of the fusion proteins
was evaluated by SDS-PAGE, immunoblotting and mass spectrometry.
The screening process for the activity contained coagulation
assays as well as purified receptor binding assays. The fusion
proteins which retained their thrombogenic and binding activity
were evaluated further. In vivo studies in nude mice
bearing established different malignant human tumors revealed
that i.v. administration of tTF-RGD or tTF-NGR induced partial
or complete thrombotic occlusion of tumor vessels, which was
demonstrated by histological analysis. Furthermore, treatment
studies showed that the targeted tTF fusion proteins but not
untargeted tTF proteins induced significant tumor growth retardation
in human adenocarcinoma of the breast in a nude mice model
without apparent side effects such as thrombosis in liver,
kidney, heart or lung at therapeutic dose levels. Finally,
we illustrate the upscaling process of fusion protein fabrication
in order to produce the amounts needed for clinical studies.
Thus, generation and screening of active fusion proteins,
which induce selective thrombosis in the tumor vasculature,
may be a promising strategy for the development of new drugs
as cancer therapeutics.
[Back to top]
Genomic Signatures for Individualized Treatment of Malignant
Tumors
Manfred Kunz
In recent years, significant progresses have been achieved
in clinical oncology due to a plethora of new chemotherapeutic
drugs and agents for targeted therapy. However, clinical response
and overall survival rates have not improved significantly
for a large number of different tumors. This may at least
in part be due to the enormous genetic heterogeneity among
tumors, even within a tumor entity. Moreover, besides individual
somatic mutations or combinations of these in malignant tumors,
the genetic background of each individual cancer patient appears
to have a major impact on treatment response and overall survival.
Current DNA microarray technology allows the simultaneous
gene expression analysis of all known genes, and large-scale
gene expression studies have provided new molecular classifications
for a series of different tumors. Evidence has also been provided
that gene expression signatures of malignant tumors may predict
treatment response to classical chemotherapeutic or targeted
anticancer drugs. The presented report summarizes the current
knowledge about the role of gene expression signatures as
putative guides for treatment decisions, with the future prospect
of individualized treatment approaches for cancer patients.
[Back to top]
Neurokinin Receptors as Potential Targets in Breast Cancer
Treatment
Bobby Y. Reddy, Katarzyna A. Trzaska, Raghav G. Murthy,
Peter Navarro and Pranela Rameshwar
Despite recent advances in the diagnoses and treatment
of breast cancer, this disease continues to be a major cause
of death. One of the biggest challenges in breast cancer treatment
is bone metastasis. Breast cancer cells (BCCs) are capable
of migrating to the bone marrow and utilizing the marrow microenvironment
to remain quiescent. While exhibiting quiescence in the marrow,
BCCs can evade the effects of conventional cancer treatments
such as chemotherapy. Therefore, scientists must find a new
paradigm to target these quiescent BCCs. The development of
potential targets may require a more comprehensive understanding
of the marrow microenvironment and its regulators. The preprotachykinin-1
(PPT-I) gene encodes for the tachykinin peptides, which interact
with neurokinin (NK) receptors. Studies have correlated this
interaction with BCC integration into the bone marrow and
breast cancer progression. In this review, we discuss the
roles that different factors of the marrow microenvironment
play in breast cancer and targets of NK receptors as potential
treatment options.
[Back to top]
Biocombinatorial Selection of Carbohydrate Binding Agents
of Therapeutic Significance
Sergei A. Svarovsky and Lokesh Joshi
Components of the cell-surface and extracellular matrix
(including cell walls of some microorganisms) present ideal
but often challenging targets for disease detection and targeting.
In the past few years, selection of peptide and oligonucleotide
biomimics against secreted and extracellular targets has resulted
in the isolation of small high affinity binding agents against
target molecules. However, little attention has been paid
to the interaction of these biomimics with glycans (specifically,
cell surface glycans) despite their ubiquitous and abundant
presence on every cell (glycocalyx). Although glycans are
now accepted as critically important class of molecules as
biomarkers, imaging, therapeutic and drug targets, little
advance has been made in exploiting these molecules for clinical
applications. The diversity and complexity of glycan structures
along with time-consuming analytical and synthetic methods
are among the most significant challenges faced by glycoscientists.
Innovative technologies are urgently needed to overcome these
challenges and to develop glycan-based clinical applications.
This review article will provide an overview of existing biocombinatorial
methods that focus on the selection and use of glycan-binding
random oligonucleotide ligands (aptamers) as conceptually
novel therapeutics for a variety of biomedical applications
including anti-microbial and anti-cancer agents. Given the
range and importance of protein-carbohydrate interactions
in biological processes, rapid discovery of agents that mediate
these interactions may provide fruitful venues towards novel
therapeutics.
[Back to top]
Targeting Toll-Like Receptor Signaling Pathways for Design
of Novel Immune Therapeutics
Elizabeth Hong-Geller, Anu Chaudhary and Sabine Lauer
The Toll-like receptor (TLR) family plays a fundamental
role in host innate immunity by mounting a rapid and potent
inflammatory response to pathogen infection. TLRs recognize
distinct microbial components and activate intracellular signaling
pathways that induce expression of host inflammatory genes.
Extensive research in the past decade to understand TLR-mediated
mechanisms of innate immunity has enabled pharmaceutical companies
to begin to develop novel therapeutics for the purpose of
controlling inflammatory disease. Initially, extracellular
TLR agonists were designed to compete with natural microbial
ligands for binding to TLRs. More recently, basic research
to identify new targets for drug development has begun to
explore modulation of TLR intracellular signaling pathways,
in addition to TLR ligand binding. In this review, we will
discuss recent strategies, including the use of decoy peptides
and mimetics, plant polyphenols, and chemically modified antisense
oligonucleotides, that inhibit different molecular events
in TLR signaling pathways to modulate the inflammatory response.
The molecular mechanisms of these inhibitors range from interference
with protein-protein interactions between signaling proteins,
to inhibition of transcription factor activity, to perturbation
of the plasma membrane, and are derived from host, pathogen,
and plant sources and by rational design. Taken together,
these studies represent promising avenues for the development
of novel tailored immune therapeutics that can relieve the
great toll inflicted by inflammatory disease on human health
and quality of life.
[Back to top]
Targeted Drug Delivery to Bone: Pharmacokinetic and Pharmacological
Properties of Acidic Oligopeptide Tagged Drugs
Tatsuo Takahashi-Nishioka, Koichi Yokogawa, Shunji Tomatsu,
Masaaki Nomura, Shinjiro Kobayashi and Ken-ichi Miyamoto
Site-specific drug delivery to bone is considered to
be achievable by utilizing acidic amino acid homopeptides.
We found that fluorescence-labeled acidic amino acid (L-Asp
or L-Glu) homopeptides containing six or more residues bound
strongly to hydroxyapatite, which is a major component of
bone, and were selectively delivered to and retained in bone
after systemic administration. We explored the applicability
of this result for drug delivery by conjugation of estradiol
and levofloxacin with an L-Asp hexapeptide. We also similarly
tagged an enzyme, tissue-nonspecific alkaline phosphatase,
to see whether this would improve the efficacy of enzyme replacement
therapy. The L-Asp hexapeptide-tagged drugs, including the
enzyme, were selectively delivered to bone in comparison with
the untagged drugs. It was expected that the ester linkage
to the hexapeptide would be susceptible to hydrolysis in situ,
releasing the drug or enzyme from the acidic oligopeptide.
An in vivo experiment confirmed the efficacy of L-Asp
hexapeptide-tagged estradiol and levofloxacin, although there
was some loss of bioactivity of estradiol and levofloxacin
in vitro, suggesting that the acidic hexapeptide
was partly removed by hydrolysis in the body after delivery
to bone. The adverse effect of estradiol on the uterus was
greatly reduced by conjugation to the hexapeptide. These results
support the usefulness of acidic oligopeptides as bone-targeting
carriers for therapeutic agents. We present some pharmacokinetic
and pharmacological properties of the L-Asp hexapeptide-tagged
drugs and enzyme.
[Back to top]
MicroRNAs-Based Therapeutic Strategy for Virally Induced Diseases
Alhousseynou Sall, Zhen Liu, Huifang M. Zhang, Ji Yuan,
Travis Lim, Yue Su and Decheng Yang
MicroRNAs (miRNAs) are endogenous, short, double-stranded
and noncoding RNA molecules that have been identified in a
variety of organisms and certain viruses. This group of new
molecules is transcribed mainly from the introns and/or exons
or intergenic regions and plays important regulatory roles
in development and gene expression. Mature miRNAs are typically
20-24 nucleotides in length and regulate target mRNAs post
transcriptionally by interactions with partially mismatched
sequences in the 3’untraslated regions of these messengers.
These interactions result in the suppression of translation
or degradation of target mRNAs. At the present, although the
biological functions of miRNAs are not completely revealed,
a growing body of evidence implicates that miRNA pathway is
a new mechanism of gene regulation in both normal and diseased
conditions and therefore investigation of miRNA biogenesis
and function may add new tools for gene functional study and
drug development. In this article, we will briefly review
the structure, biogenesis and basic mechanism of action of
miRNAs identified in higher organisms and viruses and then
focus on the recent progress in research for drug development
using the miRNA pathway as a strategy. Particularly, we will
discuss the advance, challenge and future directions on antiviral
drug development using miRNA as a target or a gene silencing
tool for the treatment of viral infections.
[Back to top]
Current In Vitro Kinase Assay Technologies: The Quest
for a Universal Format
Yong Jia, Christopher M. Quinn, Silvia Kwak and Robert
V. Talanian
The rapidly growing interest in kinases as drug targets
has prompted the development of many kinase assay technologies.
These technologies can be grouped into three categories: radiometric
assays, phospho-antibody-dependent fluorescence/luminescence
assays, and phospho-antibody-independent fluorescence/luminescence
assays. This article will review some of the major kinase
assay technologies on the market, with particular emphasis
on the newest systems. We will describe the physical principles,
the practical advantages and drawbacks, and the potential
applications of these technologies in kinase drug discovery.
Most of these technologies are suitable for HTS, but only
a few can be utilized for kinetic and mechanistic studies.
Significant progress towards development of generic assays,
free of radioisotopes and custom reagents such as phospho-specific
antibodies, has been made in recent years. However, due to
various limitations of each format, none of these generic
assay technologies can yet claim to be truly universal. Several
factors, including the intended applications, cost, timeline,
expertise, familiarity, and comfort level, should be considered
prior to pursuing a particular kinase assay technology.
[Back to top]
Emerging the Role of the Structure of Brain Membrane Targets
Recognizing Glutamate
Ágnes Simon, Ákos Bencsura, Anna Palló,
László Héja and Julianna Kardos
Ligand-bound and free structures of brain membrane targets
for L-glutamate (Glu) suggest the view, that quaternary rear-rangements
are associated with ligand binding. Rearrangement of the machinery
of the signaling apparatus, such as molecular switches, recognition
sites and the target structures for ligand binding of Glu-operated
ion channel and heptahelical G-protein-coupled family receptors
have been quantified and compared with the use of the root
mean square (RMS) values. In addition to conformational rearrangement
of the Glu receptor structures in complex with a series of
ligands, conformations of Glu in various target structures
became available. High resolution data revealed that the extended
Glu conformation is conserved in the binding crevice of all
ionotropic Glu receptors (iGluRs). Furthermore, the extended
conformations of Glu that characterize iGluRs and mGluRs are
distinguishable by distance and torsion angle parameters,
such as δC1-C2
and Cα-Cβ-Cγ-C2.
By contrast, a bent Glu conformation is recognized in Glu
transporters.
[Back to top]
Targeting Apoptosis to Treat Multiple Sclerosis
Andrea L.O. Hebb, Craig S. Moore, Virender Bhan and George
S. Robertson
Accumulating evidence implicates a failure of myelin-reactive
immune cells to undergo apoptosis in the pathological events
contributing to multiple sclerosis (MS). We have recently
demonstrated that members of the inhibitor of apoptosis (IAP)
family of anti-apoptotic genes are elevated in peripheral
blood immune cells (monocytes, T cells) of patients with aggressive
forms of MS (secondary progressive) or those with relapsing-remitting
MS suffering a disease replase. These findings suggest that
the IAPs may be novel diagnostic markers for distinguishing
subtypes of MS. Moreover, antisense-mediated knockdown of
the IAP family member known as X-linked IAP (XIAP) reverses
paralysis in an animal model of MS suggesting that treatments
targeting XIAP, and perhaps other IAPs, may have utility in
the treatment of MS.
[Back to top]
Controlling Receptor Downregulation by Ubiquitination and
Deubiquitination
Masayuki Komada
Growth factor-activated receptor tyrosine kinases (RTKs)
undergo rapid endocytosis and degradation in lysosomes. This
process, known as receptor downregulation, is essential to
prevent the overgrowth of cells by terminating signal transduction
from activated RTKs. Thus, defects in RTK downergulation lead
to cell proliferative disorders such as cancer. Upon endocytosis,
RTKs are delivered to endosomes, from where they are further
transported to lysosomes. Ubiquitin serves as a sorting signal
that is tagged on activated RTKs and directs their trafficking
from endosomes to lysosomes. On the endosomal membrane, ubiquitinated
RTKs are sorted by coordinated actions of the class E vacuolar
protein sorting (Vps) proteins some of which form complexes
that directly recognize the ubiquitin moieties of RTKs. UBPY
and AMSH in mammals, as well as Doa4 in yeast, are deubiquitinating
enzymes (DUBs) that associate with class E Vps proteins on
endosomes. Here I review the recently unveiled roles and regulatory
mechanisms of these DUBs in the endosomal sorting of ubiquitinated
cargo proteins. These findings suggest that RTK downregulation
is controlled not only by ubiquitination but also by deubiquitination
of RTKs as well as other endosomal proteins. Therefore, elucidating
the entire functions and regulation of the endosomal DUBs
potentially provides novel molecular targets for the treatment
of cancer accompanied by overexpression or constitutive activation
of RTKs.
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