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Current
Pharmaceutical Biotechnology
ISSN: 1389-2010
Current Pharmaceutical Biotechnology
Volume 9, Number 3, June 2008
Contents
The Therapeutic Potential of Ribonucleases
Guest Editor: Urich Arnold
Editorial Pp. 134
Eosinophil-Derived Neurotoxin / RNase 2: Connecting
the Past, the Present and the Future Pp.
135-140
Helene F. Rosenberg
[Abstract]
The Antipathogen Activities of Eosinophil Cationic
Protein Pp. 141-152
Ester Boix, Marc Torrent, Daniel Sánchez
and Maria Victòria Nogués
[Abstract]
The Therapeutic Potential of Fungal Ribotoxins
Pp. 153-160
Nelson Carreras-Sangrà, Elisa Álvarez-García,
Elías Herrero-Galán, Jaime Tomé, Javier
Lacadena, Jorge Alegre-Cebollada, Mercedes Oñaderra,
José G. Gavilanes and Álvaro Martínez-del-Pozo
[Abstract]
Aspects of the Cytotoxic Action of Ribonucleases
Pp. 161-168
Ulrich Arnold
[Abstract]
Intracellular Routing of Cytotoxic Pancreatic-Type
Ribonucleases Pp. 169-179
Antoni Benito, Maria Vilanova and Marc
Ribó
[Abstract]
Design of Cytotoxic Ribonucleases by Cationization
to Enhance Intracellular Protein Delivery Pp. 180-184
Junichiro Futami and Hidenori Yamada
[Abstract]
Evasion of Ribonuclease Inhibitor as
a Determinant of Ribonuclease Cytotoxicity Pp.
185-199
Thomas J. Rutkoski and Ronald T.
Raines
[Abstract]
A Novel Biological Actions Acquired by Ribonuclease
Through Oligomerization Pp. 200-209
Massimo Libonati, Giovanni Gotte and
Francesca Vottariello
[Abstract]
From ImmunoToxins to ImmunoRNases Pp.
210-214
Claudia De Lorenzo and Giuseppe
D’Alessio
[Abstract]
Onconase and Amphinase, the Antitumor Ribonucleases
from Rana pipiens Oocytes Pp. 215-225
Wojciech Ardelt, Kuslima Shogen and Zbigniew
Darzynkiewicz
[Abstract]
Antibody-Onconase Conjugates: Cytotoxicity and
Intracellular Routing Pp. 226-230
Susanna M. Rybak
[Abstract]
Antibody-Targeted RNase Fusion Proteins (ImmunoRNases)
for Cancer Therapy Pp. 231-234
Jürgen Krauss, Michaela A. E. Arndt, Stefan
Dübel and Susanna M. Rybak
[Abstract]
Abstracts
[Back to top]
Editorial
As the annual number of newly diagnosed cases of cancer is
about 10 million worldwide, combat of cancer is one of the
most outstanding challenges of recent medicine. The considerable
undesired side effects of chemotherapy and/or radiotherapy
caused by their little selectivity call for a specific targeting
of the affected tissue. Exploiting the target specificity
of antibodies, which selectively bind to tumor-associated
antigens presented on the cell surface, reduces the systemic
toxicity of the coupled compounds.
All organisms contain numerous ribonucleolytic enzymes for
RNA digestion, processing, and regulation etc., which
provide a wide palette of potential cytotoxins. Several of
these display a preference for transformed cells per se,
others can be converted into tumor specific drugs by modification
or fusion to respective targeting moieties. Hence, intensive
research is carried out on the use of these enzymes as therapeutics.
This issue of Current Pharmaceutical Biotechnology consists
of a comprehensive compilation of reviews on the therapeutic
potential of RNases, mainly from the RNase A (bovine pancreatic
RNase) superfamily. The cytotoxicity of RNases was discovered
back in the 1950s but cytotoxic effects were observed only
when milligrams of enzyme were injected into solid tumors,
while smaller doses of RNase A had no effect. In parallel,
RNase A evolved as one of the most intensively studied model
proteins in virtually all fields of biochemical research.
Still, cytotoxicity has remained one of the most attractive
characteristics of RNases because these enzymes could be used,
alone or conjugated to ligands or antibodies, as therapeutic
agents for cancer treatment. The subject is timely, as an
important series of new discoveries and developments in this
area has emerged over the past years. With Onconase®
(Alfacell Corp., Somerset, NJ, U.S.A.), an RNase from the
Northern Leopard frog Rana pipiens, an RNase has
reached phase III clinical trials for treatment of non-small
cell lung cancer and confirmatory phase IIIb for treatment
of malignant mesothelioma. Furthermore, a recently generated
fully human fusion protein composed of an anti-ErbB2 single-chain
antibody fragment and the human pancreatic RNase (ERB-hRNase
immunoRNase) has portended the development of RNase-based
drugs from human-sourced building blocks. Thus, the promising
medicinal applicability of RNases emphasizes the lasting topicality
of these rather old enzymes.
With techniques to follow molecules on the cellular and subcellular
level, the understanding of the mechanisms and the interplay
by which RNases exert cytotoxicity begins to become lucid.
On their way from administration to the final intracellular
target, the proteins face numerous obstacles and there are
tremendous differences in how efficient the particular RNase
can cope with the respective prerequisites. The reviews of
this issue cover the different stages and underline the sophisticated
requirements an RNase-based drug must meet.
ACKNOWLEDGEMENTS
The editor gratefully acknowledges the contributions
of all authors of the reviews for this special issue of Current
Pharmaceutical Biotechnology. In the following, the corresponding
authors are listed in alphabetical order:
Wojciech Ardelt, Ester Boix, Giuseppe D’Alessio, Junichiro
Futami, Jürgen Krauss, Massimo Libonati, Álvaro
Martínez-del-Pozo, Ronald T. Raines, Marc Ribó,
Helene F. Rosenberg, and Susanna M. Rybak
Guest Editor
Ulrich Arnold
Martin-Luther University Halle-Wittenberg
Institute of Biochemistry and Biotechnology
Kurt-Mothes Str. 3
06120 Halle
GERMANY
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Eosinophil-Derived Neurotoxin / RNase 2: Connecting the Past,
the Present and the Future
Helene F. Rosenberg
The eosinophil-derived neurotoxin (EDN, also known as
eosinophil protein-X) is best-known as one of the four major
proteins found in the large specific granules of human eosinophilic
leukocytes. Although it was named for its discovery and initial
characterization as a neurotoxin, it is also expressed constitutively
in human liver tissue and its expression can be induced in
macrophages by proinflammatory stimuli. EDN and its divergent
orthologs in rodents have ribonuclease activity, and are members
of the extensive RNase A superfamily, although the relationship
between the characterized physiologic functions and enzymatic
activity remains poorly understood. Recent explorations into
potential physiologic functions for EDN have provided us with
some insights into its role in antiviral host defense, as
a chemoattractant for human dendritic cells, and most recently,
as an endogenous ligand for toll-like receptor (TLR)2.
[Back to top]
The Antipathogen Activities of Eosinophil Cationic
Protein
Ester Boix, Marc Torrent, Daniel Sánchez
and Maria Victòria Nogués
The eosinophil cationic protein (ECP) is a secretory
ribonuclease, which is found in the eosinophilic leukocyte
and involved in the innate immune system. Its cytotoxic activity
is effective against a wide range of pathogens, suggesting
a relatively non-specific mechanism of action. We review here
the specific antipathogen activities that have been characterized
for ECP. Although eosinophils and ECP are primarily associated
with the host defense against nonphagocytosable pathogens,
such as helminthic parasites, ECP has also an antibacterial
activity, which is not shared by the other, closelyrelated
eosinophil ribonuclease, the eosinophil derived neurotoxin
(EDN). Although there is no evidence for direct involvement
in vivo of eosinophils in the host response to bacterial
infection, ECP is active against both Gram-negative and Gram-positive
bacterial strains and its mechanism depends on its action
both at the bacterial cell wall and cytoplasmic membrane levels.
Other antipathogen activities, including antihelminthic activity,
are also discussed. Modulation of the protein activity by
posttranslational modifications and the currently identified
polymorphisms are reviewed. Antimicrobial RNases, as innate
immune proteins with anti-infective and immunomodulatory properties,
present substantial therapeutic potential in the drug development
industry, both in the search of alternative antibiotics and
for the treatment of inflammatory disorders.
[Back to top]
The Therapeutic Potential of Fungal Ribotoxins
Nelson Carreras-Sangrà, Elisa Álvarez-García,
Elías Herrero-Galán, Jaime Tomé, Javier
Lacadena, Jorge Alegre-Cebollada, Mercedes Oñaderra,
José G. Gavilanes and Álvaro Martínez-del-Pozo
Ribotoxins constitute a family of toxic extracellular
fungal RNases that exert a highly specific activity on a conserved
region of the larger molecule of rRNA, known as the sarcin–ricin
loop. This cleavage of a single phosphodiester bond inactivates
the ribosome and leads to protein synthesis inhibition and
cell death. In addition to this ribonucleolytic activity,
ribotoxins can cross lipid membranes in the absence of any
known protein receptor. This ability is due to their capacity
to interact with acid phospholipid-containing membranes. Both
activities together explain their cytotoxic character, being
rather specific when assayed against some transformed cell
lines. The determination of high-resolution structures of
some ribotoxins, the characterization of a large number of
mutants, and the use of lipid model vesicles and transformed
cell lines have been the tools used for the study of their
mechanism of action at the molecular level. The present knowledge
suggests that wild-type ribotoxins or some modified variants
might be used in human therapies. Production of hypoallergenic
mutants and immunotoxins designed against specific tumors
stand out as feasible alternatives to treat some human pathology
in the mid-term future.
[Back to top]
Aspects of the Cytotoxic Action of Ribonucleases
Ulrich Arnold
By virtue of their RNA degrading catalytic activity,
ribonucleases are potentially cytotoxic. For the application
of these enzymes as therapeutics, however, they have to overcome
several obstacles whose interplay is not yet fully understood.
Ribonucleases with a basic pI are not only able to interact
with the (negatively charged) cellular membrane but they are
also distinctively selective for tumor cells. After the (endocytotic)
uptake into the cell and release into the cytosol from the
endosomes where they have to resist the attack by proteases,
they face the cytosolic ribonuclease inhibitor. Only if they
are able to evade the tight binding to the inhibitor (or if
enough ribonuclease molecules enter the cell to neutralize
the inhibitor protein) they are able to attack their target
RNA, for which a sufficient ribonucleolytic activity is indispensable.
Each of these steps can turn into an insurmountable hurdle
spoiling the cytotoxic potential of these enzymes.
In the present review I will summarize the status quo of the
knowledge on the mechanisms and their interdependence as well
as to develop strategies to overcome possible limitations.
[Back to top]
Intracellular Routing of Cytotoxic Pancreatic-Type
Ribonucleases
Antoni Benito, Maria Vilanova and Marc
Ribó
In addition to their ribonucleolytic activity, several
ribonucleases (RNases) play important roles in other specific
biological activities, such as dendritic cell activation,
certain pollen-induced allergies, blood vessel formation and
defense against parasitic or microbial infections. Among these
diverse actions, cytotoxic activity, which relies in most
cases on ribonucleolytic activity, has attracted a considerable
attention because of the potential for using RNases as therapeutic
agents for the treatment of different malignancies. In addition
to use naturally existing RNases, major efforts have been
made in the development of engineered variants, which display
more potent cytotoxic activity and greater selectivity for
malignant cells. This review focuses on the molecular and
cellular aspects of the internalization, intracellular trafficking
and final sorting of cytotoxic RNases. Knowledge about the
strategies used by these promising toxins provides us with
essential information about the mechanisms that can be used
to gain access to different subcellular compartments and intracellular
sorting.
[Back to top]
Design of Cytotoxic Ribonucleases by Cationization
to Enhance Intracellular Protein Delivery
Junichiro Futami and Hidenori Yamada
The cytotoxic properties of naturally occurring or engineered
RNases correlate well with their efficiency of cellular internalization
and digestion level of cellular RNA. Cationized RNases are
considered to adsorb to the anionic cellular surface by Coulombic
interactions, and then become efficiently internalized into
cells by an endocytosis-like pathway. The design of cytotoxic
RNases by chemical modification of surface carboxylic residues
is one of the powerful strategies for enhancing cellular internalization
and is accompanied with a decreased sensitivity for the cytoplasmic
RNase inhibitor. Although chemically modified cationized RNases
showed decreased ribonucleolytic activity, improved endocytosis
and decreased affinity to the endogenous RNase inhibitor conclusively
contribute to their ability to digest cellular RNA. Fur-thermore,
the cytotoxicity of cationized RNases can be drastically enhanced
by co-endocytosis with an endosomedestabilizing peptide. Since
efficient cellular internalization of proteins into living
cells is an important technology for biotechnology, studies
concerning the design of cytotoxic RNases provided general
perceptions for protein-based drug design.
[Back to top]
Evasion of Ribonuclease Inhibitor as
a Determinant of Ribonuclease Cytotoxicity
Thomas J. Rutkoski and Ronald T.
Raines
Onconase® (ONC) is an amphibian member
of the bovine pancreatic ribonuclease (RNase A) superfamily
that exhibits innate antitumoral activity. ONC has been granted
both orphan-drug and fast-track status by the U.S. Food and
Drug Administration for the treatment of malignant mesothelioma,
and is poised to become the first chemotherapeutic agent based
on a ribonuclease. Investigations into the mechanism of ribonuclease-based
cytotoxicity have elucidated several important determinants
for cytotoxicity, including efficient deliverance of ribonucleolytic
activity to the cytosol and preservation of conformation stability.
Nevertheless, the most striking similarity between ONC and
bovine seminal ribonuclease, another naturally cytotoxic ribonuclease,
is their insensitivity to inhibition by the potent cytosolic
ribonuclease inhibitor protein (RI). RI typically binds to
its ribonuclease ligands with femtomolar affinity—an
extraordinary feat considering the modest sequence identity
among the bound ribonucleases. Mammalian ribonucleases such
as RNase A or its human homologue, RNase 1, have the potential
to be more attractive chemotherapeutic agents than ONC owing
to their higher catalytic activity, low potential for immunogenicity,
favorable tissue distribution, and high therapeutic index,
but are limited by their sensitivity to RI. These non-toxic
mammalian ribonucleases can be transformed into potent cytotoxins
by engendering them with RI-evasion using protein engineering
strategies such as site-directed mutagenesis, multimerization,
fusion to a targeting moiety, and chemical modification. In
several instances, these engineered ribonucleases exhibit
greater cytotoxicity in vitro than does ONC. Herein,
we review the biochemical characteristics of RI• ribonuclease
complexes and progress towards the development of mammalian
ribonuclease-based chemotherapeutics through the elicitation
of RI-evasion.
[Back to top]
A Novel Biological Actions Acquired by Ribonuclease
Through Oligomerization
Massimo Libonati, Giovanni Gotte and
Francesca Vottariello
After a short introduction with some examples of cytotoxic
ribonucleases, the importance of natural or artificial dimerization
(oligomerization) as a way for a ribonuclease to acquire novel
functional properties has been pointed out. In particular,
the role of the three dimensional domain swapping mechanism
in bovine pancreatic ribonuclease A oligomerization, as well
as its impact for the acquisition of novel biological functions
(among which a remarkable antitumor action) by the enzyme
protein in oligomeric form have been discussed. Finally, the
structural and functional features that could explain why
oligomeric ribonuclease A becomes able to display a cytotoxic
activity, and the possible use and limits of the three dimensional
domain-swapped oligomers of ribonuclease A as anticancer therapeutic
agents have been described and discussed.
[Back to top]
From ImmunoToxins to ImmunoRNases
Claudia De Lorenzo and Giuseppe
D’Alessio
Immunotoxins are chimeric molecules that specifically
target tumor cells, as they are made up of toxins linked to
an antibody directed to a specific, cell-surface tumor-associated-antigen
(TAA). When the immune moiety is internalized by the tumor
cell, it will carry the conjugated toxin into the cell, so
that the cell will be selectively killed in a way postulated
more than a hundred years ago by Paul Ehrlich, the first author
to use the term magic bullet. To date, toxicity and
immunogenicity have complicated the clinical use of most immunotoxins.
More recently, based on the immunotoxin principle, immunoRNases
have been proposed, in which the toxin moiety of immunotoxins
is replaced by a non-toxic RNase. An immunoRNase (IR) is in
fact an immuno-pro-toxin, as it can travel in the bloodstream
without any damages to cells devoid of the targeted TAA, while
magically selecting the cells targeted by the immune
moiety. Once internalized, the RNase moiety will exert its
RNA degrading activity, which will readily lead to the death
of the targeted cell. By choosing a human RNase, and a human
antibody fragment as immune moiety, an IR would be not only
non-toxic, but also non-immunogenic. As for the possible inhibitory
action of the cytosolic RNase inhibitor, exerted on all non-toxic
vertebrate RNases, it can be opposed by flooding the cytosol
with high levels of IR, which will neutralize the RNase inhibitor,
or by using RNases resistant to the inhibitor.
[Back to top]
Onconase and Amphinase, the Antitumor Ribonucleases
from Rana pipiens Oocytes
Wojciech Ardelt, Kuslima Shogen and Zbigniew
Darzynkiewicz
Rana pipiens oocytes contain two homologues of pancreatic
ribonuclease A that are cytostatic and cytotoxic to human
cancer cells. Extensively studied Onconase is in advanced
Phase IIIb clinical trials against malignant mesothelioma,
while Amphinase is a novel enzyme in pre-clinical development.
Onconase is the smallest (104 amino acid residues) member
of the ribonuclease A superfamily while Amphinase (114 residues)
is the largest among amphibian ribonucleases. Both enzymes
share the characteristic frog ribonucleases C-terminal disulfide
bond but another signature of this group, the N-terminal pyroglutamate,
an integral part of Onconase active site is not conserved
in Amphinase.
Although Onconase and Amphinase are weak catalysts their enzymatic
activities are required for cytostatic and cytotoxic activity.
While it was postulated that tRNA is the primary substrate
of Onconase in vivo there is also extensive indirect
evidence that suggests other RNA species, in particular micro
RNAs, may actually be the critical target of these ribonucleases.
The cytostatic effects of Onconase and Amphinase are manifested
as cell arrest in the G1
cell cycle phase. Apoptosis then follows involving activation
of endonucleases(s), caspases, serine proteases and transglutaminase.
Onconase was shown to be strongly synergistic when combined
with numerous other antitumor modalities. Onconase and Amphinase
are highly cationic molecules and their preferential toxicity
towards cancer cells (having distinctly higher negative charge
compared to normal cells) may depend on increased binding
efficiency to the cell surface by electrostatic interactions.
Here we will discuss the structures of Onconase and Amphinase
and the molecular basis for their enzymatic and anticancer
functions.
[Back to top]
Antibody-Onconase Conjugates: Cytotoxicity and
Intracellular Routing
Susanna M. Rybak
Onconase, a member of the pancreatic ribonuclease A superfamily,
is currently in Phase III clinical trials for treatment of
unresectable malignant mesothelioma. The anticancer effect
of onconase may relate to its intracellular target, a non-coding
RNA. Some non- coding RNAs are aberrantly expressed in cancer
cells. This discovery is creating new interest in drugs that
target RNA. Conjugating onconase to agents that recognize
tumor associated molecules further increases its potency and
specificity. Analysis of onconase activity when directed to
two different internalizing and one non-internalizing receptor
reveals that the ideal targeting agents would rapidly enter
lysosomal compartments before onconase escaped to the cytosol.
Antibody-onconase conjugates are effective in preclinical
models, cause little non-specific toxicities in mice and have
favorable formulation properties. Understanding the reason
for their potency coupled with understanding novel RNA-based
mechanisms of tumor cell death will lead to improved variations
of targeted onconase.
[Back to top]
Antibody-Targeted RNase Fusion Proteins (ImmunoRNases)
for Cancer Therapy
Jürgen Krauss, Michaela A. E. Arndt, Stefan
Dübel and Susanna M. Rybak
Ribonucleases (RNases) of the superfamily A exhibit potent
antineoplastic activity yet do not mediate appreciable immunogenicity
or non-specific toxicity in both animal models and cancer
patients. Ranpirnase (Onconase®), the first
ribonuclease being evaluated as a therapeutic in humans, has
progressed to phase III clinical trials in patients with unresectable
mesothelioma. Conjugation of RNases to internalizing tumor-targeting
monoclonal antibodies was shown to enhance specific cell killing
by several orders of magnitude both in vitro and
in animal models. In this review we describe the development
and current status of genetically engineered 2nd
generation immunoRNases as promising novel anti-cancer therapeutics.
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