General Anthrax Q&A
Summary: Intel, Microsoft, United Devices, the University
of Oxford and the NFCR are teaming up to launch a new project on
the Internet focused on Anthrax. The same infrastructure, application
and processes will be used to analyze the Anthrax toxin protein
as are being used to analyze cancer proteins. The project will launch
January 22, 2002 and is expected to run anywhere from 3 weeks to
1.5 months.
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- Q. What is the Anthrax Research Project?
- A. The Anthrax Research Project is the collaboration of technology
and science leaders to help find a treatment for the Anthrax toxin.
The project will screen 3.5 billion molecular compounds against
one Anthrax toxin protein to hopefully identify a molecule that
will block the toxicity of the protein.
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- Q. How would this treatment differ from current treatments already
on the market?
- A. While antibiotics can wipe out the bacteria in many cases,
unfortunately anthrax is frequently diagnosed only after a victim's
bacterial count has escalated. At this point, antibiotics can
do nothing to clear the toxin. Furthermore, antibiotics, especially
if taken inappropriately, can actually increase the danger from
Anthrax by allowing resistant strains to develop. Clearly, with
an increased threat, there has never been a more appropriate time
to find a more effective therapy.
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- Q. What does a treatment for Anthrax mean?
- A. A suitable drug could block the entry of the lethal toxin
into a cell.
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- Q. How long will it take to find a potential treatment?
- A. Turning a drug candidate into a useable therapy is a major
undertaking costing millions of dollars and taking a few years.
Q. How is this different/similar to the cancer research program?
- A. Actually, it is very similar. We are using the same technology
to analyze the same molecular compounds as the cancer research
project. The only real difference is that we will be working with
an Anthrax toxin protein rather than cancer proteins.
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- Q. Anthrax hasn't impacted very many people. Why focus resources
on Anthrax?
- A. Although Anthrax has yet to cause significant loss of life,
it has affected a great deal of people, emotionally as well as
psychologically. Additionally, we now have the technology to potentially
stop the threat of Anthrax as a weapon. This will prove to be
a great starting point to address further possible biological
threats in a similar fashion.
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- Q. Why hasn't this already been done?
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A. The structure of the target protein has only recently become
available and the unique Oxford software has identified a target
site on the protein.
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- Q. Who is sponsoring the project?
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A. Microsoft and Intel are providing the capital, United Devices
is providing the edge distributed computing technology and Oxford
is supplying the science.
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- Q. How do individuals get involved?
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A. Any concerned individual around the world can easily participate
by going to www.intel.com/cure and downloading the screensaver.
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Figure. Anthrax toxin is assembled on the surface of cells
and once this is complete enters and kills the cell. The anthrax
toxin is produced by the bacteria Bacillus anthracis and
is the cause of the symptoms of anthrax poisoning. The bacteria
produces three proteins which when combined together act to kill
cells. The three proteins are a single receptor moiety - called
protective antigen (PA), an enzyme - called lethal factor (LF) and
another enzyme called edema factor (EF). These three proteins are
not toxic and when released by the bacteria they diffuse around
the host's blood stream until the following steps occur:
- The PA binds to receptors on cell walls
- The PA is cleaved into two parts by a protease enzyme, one is
released and the other part remains bound
- The bound PA momers now self-associate with others on the surface
to form seven moiety units - a heptamer
- The heptamer now acts as a prepore, which can allow toxins to
enter the cell
- The factors LF or EF bind to the heptameric prepore
- The whole toxin is internalized into the cell by endocytosis
- The low pH inside the cell causes the PA heptamer to insert
through the cell wall
- The factors are then released into the cell. EF and LF acts
as enzymes disrupting the cell biochemistry, causing cell death
and ultimately host death
References
The biochemistry of the action the anthrax toxin on cells and an
approach to design toxin inhibitors was very recently published
c.f.
"Designing a polyvalent inhibitor of anthrax toxin" Michael
Mourez, Ravi S. Kane, Jeremy Mogridge, Steve Metallo, Pascal Deschatelets,
Bret R. Sellman, George M. Whitesides & R. John Collier Nature
Biotechnology - October 2001 Volume 19 Number 10 pp 958 - 961
Abstract Screening peptide libraries is a proven strategy
for identifying inhibitors of protein–ligand interactions.
Compounds identified in these screens often bind to their targets
with low affinities. When the target protein is present at a high
density on the surface of cells or other biological surfaces,
it is sometimes possible to increase the biological activity of
a weakly binding ligand by presenting multiple copies of it on
the same molecule. We isolated a peptide from a phage display
library that binds weakly to the heptameric cell-binding subunit
of anthrax toxin and prevents the interaction between cell-binding
and enzymatic moieties. A molecule consisting of multiple copies
of this nonnatural peptide, covalently linked to a flexible backbone,
prevented assembly of the toxin complex in vitro and blocked toxin
action in an animal model. This result demonstrates that protein–protein
interactions can be inhibited by a synthetic, polymeric, polyvalent
inhibitor in vivo.
The structure of the heptamer we are using to screen molecules
against was reported in 1997. We kindly thank Carlo Petosa for sending
to us the crystal structure data of the heptamer.
"Crystal structure of the anthrax toxin protective antigen"
Petosa, Carlo; Collier, R. John; Klimpel, Kurt R.; Leppla, Stephen
H.; Liddington, Robert C. Biochemistry Dep., Univ. Leicester, Leciester,
UK. Nature (London) (1997), 385(6619), 833-838.
Abstract Protective antigen (PA) is the central component
of the three-part protein toxin secreted by bacillus anthracis,
the organism responsible for anthrax. After proteolytic activation
on the host cell surface, PA forms a membrane-inserting heptamer
that translocates the toxic enzymes, edema factor and lethal factor,
into the cytosol. PA, which has a relative mol. mass of 83,000
(Mr 83K), can also translocate heterologous proteins, and is being
evaluated for use as a general protein delivery system. Here we
report the crystal structure of monomeric PA at 2.1 .ANG. resoln.
and the water-sol. heptamer at 4.5 .ANG. resoln. The monomer is
organized mainly into antiparallel b-sheets and has four domains:
an amino-terminal domain (domain 1) contg. two calcium ions and
the cleavage site for activating proteases; a heptamerization
domain (domain 2) contg. a large flexible loop implicated in membrane
insertion; a small domain of unknown function (domain 3); and
a carboxy-terminal receptor-binding domain (domain 4). Removal
of a 20K amino-terminal fragment from domain 1 allows the assembly
of the heptamer, a ring-shaped structure with a neg. charged lumen,
and exposes a large hydrophobic surface for binding the toxic
enzymes. We propose a model of pH-dependent membrane insertion
involving the formation of a porin-like, membrane-spanning beta-barrel.
The structure of the anthrax toxin Lethal Factor has been recently
reported in 2001.
"Crystal structure of the anthrax lethal factor" Pannifer,
Andrew D.; Wong, Thiang Ylan; Schwarzenbacher, Robert; Renatus,
Martin; Petosa, Carlo; Blenkowska, Jadwiga; Lacy, D. Borden; Collier,
R. John; Park, Sukjoon; Leppla, Stephen H.; Hanna, Philip; Liddington,
Robert C. Biochemistry Department, University of Leicester, Leicester,
UK. Nature (London, United Kingdom) (2001), 414(6860), 229-233.
Abstract Lethal factor (LF) is a protein (relative mol.
mass 90,000) that is crit. in the pathogenesis of anthrax. It
is a highly specific protease that cleaves members of the mitogen-activated
protein kinase kinase (MAPKK) family near to their amino termini,
leading to the inhibition of one or more signaling pathways. Here
we describe the crystal structure of LF and its complex with the
N terminus of MAPKK-2. LF comprises four domains: domain I binds
the membrane-translocating component of anthrax toxin, the protective
antigen (PA); domains II, III and IV together create a long deep
groove that holds the 16-residue N-terminal tail of MAPKK-2 before
cleavage. Domain II resembles the ADP-ribosylating toxin from
Bacillus cereus, but the active site has been mutated and recruited
to augment substrate recognition. Domain III is inserted into
domain II, and seems to have arisen from a repeated duplication
of a structural element of domain II. Domain IV is distantly related
to the zinc metalloprotease family, and contains the catalytic
center; it also resembles domain I. The structure thus reveals
a protein that has evolved through a process of gene duplication,
mutation and fusion, into an enzyme with high and unusual specificity.
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