Testimony
Before the House
Committee on Government Reform
Quickening the Pace of Research In Protecting
Against Anthrax and Other Biological Terrorist Agents-A
Look at Toxin Interference
Statement of
Stephen
Leppla, Ph.D.
Senior
Investigator, Oral Infection and Immunity Branch, Division
of Intramural Research,
National
Institute of Dental and Craniofacial Research, National
Institutes of Health
For
Release on Delivery
February
28, 2002
Mr.
Chairman and Members of the Committee:
I appreciate the opportunity
to appear before you today to describe my research regarding
anthrax toxin receptors and the role of the protease furin
in anthrax toxin action. Included in my remarks will be
some discussion about the possible use of furin inhibitors
to block anthrax toxin action, and the potential this
holds for the treatment of anthrax infections.
The National Institute of
Allergy and Infectious Diseases (NIAID) spearheads the
bioterrorism research effort at the National Institutes
of Heath (NIH) and supported the recent studies
by Dr. John Collier, Harvard University, and Dr. John
Young, University of Wisconsin Medical School,
who are present today, which elucidated the mechanisms
by which anthrax toxin destroys cells. The information
gained through these studies will likely hasten the development
of new drugs to treat anthrax.
NIAID
also supports a number of other drug development efforts
for anthrax and other Category A agents of bioterrorism.
Earlier in the month, NIAID sponsored a Blue Ribbon Panel
on Bioterrorism and its Implications for Biomedical Research,
which brought together a distinguished panel of leaders
from the biomedical research community and experts in
Category A agents of bioterrorism to obtain expert advice
and input on NIAID's Counter-Bioterrorism Research Agenda.
This group helped NIAID assess its current research efforts
to counter bioterrorism and identified goals for NIAID
to implement on an immediate and intermediate/long-term
basis. Indeed, one of the immediate goals identified for
anthrax research is to encourage exploration of new targets
for antimicrobial therapies, including strategies to prevent
germination of spores, the synthesis or neutralization
of toxins, and interference with attachment and entry
of toxins into host target cells, which will build upon
the findings of Drs. Young and Collier.
First
I wish to briefly discuss our work on the cellular receptor
for anthrax toxin. Bacterial toxins that attack animal
cells must first bind to the surface of those cells. Several
toxins do this by interacting with a single specific protein
present on the cell surface. By definition, this protein
is the toxin receptor. The receptor is typically a normal
cellular protein that has a recognized role in cell function.
It is only by accident that this protein is used by a
bacterial toxin to enter the cell and damage it. I began
studies intended to identify the anthrax toxin receptor
some years ago while a researcher at the United States
Army Medical Research Institute of Infectious Diseases
(USAMRIID) in Frederick, MD, and continued this work after
transferring to the National Institute of Dental and Craniofacial
Research (NIDCR) in 1989. Our early work showed that all
types of cells have anthrax toxin receptors, that cells
typically have about 10,000 receptor molecules on their
surface, and that the receptors were probably proteins.
We generated cultured cell mutants lacking functional
anthrax toxin receptors. Over a period of years, we worked
intermittently to identify the receptor, using various
biochemical and genetic methods, but our efforts were
unsuccessful. Several years ago, a group at Harvard University
began similar efforts, and they were successful in identifying
the receptor last summer. This work, led by Drs. John
Young and John Collier and supported by the NIAID, was
published in the journal Nature several months
ago. This important work showed that anthrax toxin uses
as its receptor a protein named tumor endothelial marker
8, or TEM8. This cell surface protein had been described
just one year ago in work from the laboratory of Dr. Ken
Kinzler of Johns Hopkins University, work supported by
the National Cancer Institute. The protein was identified
as one that is highly expressed in tumor endothelial cells.
As Drs. Young and Collier
pointed out in their publication, and as I mentioned earlier,
their important discovery opens potential several avenues
toward development of new therapies for anthrax infection.
Specifically, they showed that a portion of the receptor,
produced as a recombinant protein in the common bacterium
Esherichia coli, was able to act as a receptor
decoy and block the action of toxin in cultured cells.
A precedent for receptor decoys being effective therapeutic
agents is provided by the tumor necrosis factor soluble
receptor, marketed as the product Enbrel, which is used
in treating rheumatoid arthritis.
Now let me turn to describing
work performed in my own laboratory on furin. Furin is
a cellular protease which is required for the processing
of many proteins that a cell secretes or delivers to its
cell surface. Furin is a member of a family of similar
enzymes that include those required for generating the
final, active forms of peptide hormones such as insulin.
It is an essential enzyme, as indicated by the fact that
inactivation of the gene in mice causes death during an
early stage of embryonic development.
When I began work on anthrax
toxin, there were already several examples of bacterial
toxins that require proteolytic activation. That is, the
toxins had to be cut at a specific site by a protease
enzyme to be made fully active. During our first efforts
to purify the protective antigen protein of anthrax toxin,
we noted that it was very easily cleaved by proteases
at a single site. By sequence analysis of the fragments,
we determined that the protein was cleaved following a
sequence of four amino acids, arginine-lysine-lysine-arginine.
We then showed that removal of this cleavage site inactivated
the toxin. This was convincing proof that cleavage at
this site is essential for anthrax toxin action. Because
uncleaved toxin was fully active when added to cells,
we suspected that the cells were causing proteolytic activation
of the toxin. In effect, the toxin appeared to be using
a cellular protease to achieve its own activation.
We then set out to identify
the cellular protease required for anthrax toxin activation.
We changed the amino acids within the arginine-lysine-lysine-arginine
sequence, replacing each amino acid with many different
ones. By comparing the toxicity of more than 30 mutated
proteins, we found that any toxin protein having arginine
at both the first and fourth positions was toxic to cells.
The identity of the amino acids in the two middle positions
occupied by lysine in the original sequence could be changed
to other amino acids with little effect. At the time this
work was being done, other researchers had finally identified
the long-sought human proteases which process biosynthetic
precursors of hormones such as insulin. It had been thought
that these proteases recognize only paired basic amino
acid residues such as the sequence arginine-arginine.
However, the new evidence suggested that one of these
proteases, named furin, cleaves proteins having arginines
at the first and fourth positions. Because this sequence
exactly matched the one we had identified, we speculated
that furin was the cellular protease that was needed to
activate anthrax toxin protective antigen. We then contacted
Dr. Gary Thomas, of the Vollum Institute, University of
Oregon. Dr. Thomas was already a recognized expert in
study of these cellular proteases, and he agreed to collaborate
in further studies. He quickly determined that purified
furin rapidly cleaves the anthrax toxin protective antigen
protein.
Subsequently, we generated
mutated cultured cells lacking functional furin, and showed
that these were highly resistant to anthrax toxin. Similar
mutant cells had been produced some years earlier by Thomas
Moehring, University of Vermont, but the genetic defect
in the cells was not known. We went on to show that the
furin-deficient cells are also resistant to several other
bacterial proteins that require protease activation, and
Dr. Moehring had already shown that such cells are resistant
to certain viruses. In was later shown through work in
other laboratories that furin is involved in the activation
of many viral envelope proteins, including those of influenza
virus and HIV. My lab has not been actively working on
furin in the last several years, although we continue
to aid others investigators in this field by supplying
the furin-deficient cultured cells when requested. My
original collaborator in the furin studies, Dr. Gary Thomas,
has continued to work actively and productively in this
field, and he can provide details about the current state
of research on furin.
I now would like to offer
some comments comparing possible therapeutic opportunities
for anthrax infections. Researchers working on anthrax
have identified at least eight distinct stages at which
one theoretically
could interfere with anthrax toxin action. Studies in
cell culture models have demonstrated in principle that
each of these stages can be blocked. Drs. Collier, Young,
and Dr. Arthur Friedlander, USAMRIID, have provided much
of the data proving that these separate stages each represent
a target for therapeutic interventions.
In trying to find targets
for therapeutic intervention in infectious diseases, most
researchers focus on identifying target molecules that
are unique to the pathogen. For example, one attractive
target is the anthrax toxin lethal factor. Bacillus
anthracis bacteria lacking lethal factor are greatly
weakened in their ability to cause anthrax. Because of
the success with which AIDS is treated by inhibitors of
the HIV viral protease, many researchers believe that
there is a great opportunity for treatment of anthrax
with inhibitors of the lethal factor protease. Pharmaceutical
companies and academic researchers have extensive experience
in developing inhibitors of proteases, and some of that
expertise is being redirected toward developing lethal
factor inhibitors. NIAID has for several years been supporting
at least two research groups studying lethal factor structure
and inhibitor development. An important advance in this
area occurred several months ago with the publication
of the crystal structure of the lethal factor protease,
work done in the laboratory of Robert Liddington, Burnham
Institute, La Jolla, CA. Dr. Collier and I were collaborators
in that work. The availability of the complete crystal
structure of lethal factor has encouraged many researchers
to begin new efforts or intensify existing efforts to
develop lethal factor inhibitors. My lab is providing
purified lethal factor protein to several of these groups
so as to facilitate their work. I have considerable hope
that a carefully selected lethal factor inhibitor will
prove to be an effective therapeutic for anthrax.
The other protease involved
in anthrax toxin action is the one discussed above, the
cellular protease furin. Because of the important role
furin has in normal physiological processes, NIH has supported
many studies involving furin and the family of proteases
which are closely related to it. For example, NIH has
supported the work of Dr. Thomas over a number of years,
during which time he developed the potent inhibitor which
he calls the "Portland" inhibitor. Potent furin inhibitors
have also been developed by two other NIH-funded researchers,
Drs. Iris Lindberg, of Louisana State University, and
Robert Fuller, of the University of Michigan. The inhibitors
developed by these three NIH-funded researchers employ
three different approaches to inhibitor design, and together
identify a number of opportunities for development of
even more potent furin inhibitors. It should be mentioned
that NIH intramural researchers have also made important
contributions in furin research. Drs. David FitzGerald
and Ira Pastan of the National Cancer Institute proved
that furin has an essential role in the action of Pseudomonas
exotoxin. Dr. Juan Bonifacino of the National Institute
of Child Health and Human Development has provided important
knowledge about the movement of furin between various
compartments within a cell. Several other NIH-funded studies
include analysis of the properties and functions of furin
as a part of larger studies of various disease processes.
This portfolio of investigator-initiated extramural and
intramural research is producing a strong knowledge base
on which to base therapies for those diseases in which
furin plays a role.
I mentioned earlier that
drug developers prefer to target molecules that are unique
to a pathogen. For this reason, furin has received less
attention as a target for drug development. The expectation
has been that inhibition of this enzyme, which plays an
essential role in many normal processes, might cause significant
physiological damage to normal tissue. Consistent with
that prediction is the fact mentioned above, that genetic
inactivation of furin causes death of mouse embryos. Nevertheless,
I do believe that inhibition of furin should be examined
as one possible therapy for anthrax. Given the renewed
interest in anthrax, I anticipate that the furin inhibitors
mentioned above, as well as others, will be evaluated
for anthrax toxin inhibition in appropriate cell culture
models in the near future.
That concludes my testimony.
I would be happy to respond to any questions that you
or Members of the Committee may have.
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revised: March 7, 2002