|
|
 |
|
PHOTODISC |
|
|
Treating a viral infection is like defending against a guerrilla
strike. Viruses attack like resistance fighters. Instead of setting
up camp and openly multiplying, as bacteria do, they hijack enemy
cells and use the cells' own machinery to launch a new generation
of resistance and new weaponry. They slip into enemy territory
wearing just a simple protein capsid shell enclosing a bit of genetic
instruction. They land on cells, insert the instructions, and take
over.
That's why, when you go to the doctor with an itchy nose or
sore throat, you hope that you are fighting a more conventional
bacterial army. If so, your doctor has a whole arsenal of antibacterials
to choose from.
But if your malady is viral-based--and chances are it is, because
70 to 80% of all respiratory infections are viral--more likely
you will come home from the doctor empty-handed. All existing
over-the-counter cough, cold, and flu medicines may help get you
through the symptoms, but the virus itself is not affected by
those drugs.
The AIDS epidemic and the advent of molecular biology with
its tools of genetic engineering spurred much research and ushered
in a burst of antivirals in the '80s and '90s. Even so, today
there are only six antivirals that target respiratory viruses.
The troublesome group includes influenza, parainfluenza, respiratory
syncytial virus (RSV), coronaviruses (including the severe acute
respiratory syndrome--SARS--coronavirus), picornaviruses (some
of which cause the common cold), adenoviruses, and other still
unknown respiratory-disease-causing viruses.
Treating respiratory infections presents some specific biological
and economic challenges. Nevertheless, a number of strategies
are in the pipeline to fight these infections. And HIV research
has proven that, given the need, therapy for wily viruses can
indeed be achieved.
Finding effective pharmaceutical weaponry to fend off viral
attack has historically been tricky. Development of antibacterials
was much more straightforward. First of all, scientists could
see bacteria long before they ever saw viruses. Using his microscope,
Anton van Leeuwenhoek first observed microorganisms in 1673. Most
viruses are invisible under a light microscope, and it wasn't
until the turn of the 20th century that viruses began to be investigated.
In addition, screening drugs against bacterial growth is relatively
simple. "You just grow bacteria in some medium, throw the compound
in, and then ask the question: Does it affect the growth of bacteria?
You can't do that easily for viruses because viruses by themselves
don't grow," says Norbert W. Bischofberger, executive vice president
of R&D at Gilead Sciences.
To test antiviral drugs, researchers have had to find cell cultures
and animal models in which the virus replicates easily. For some
viruses (such as SARS), researchers still don't have a good animal
model.
Finally, "bacteria are unicellular organisms that differ from
human cells very much in their makeup and in their biochemistry,"
Bischofberger says. Viruses perform most of their functions inside
the human cell, "so it is challenging to find compounds that very
specifically inhibit the virus without affecting the cell. With
bacteria, that has been much easier." For example, bacteria have
a cell wall made of cross-linked peptidoglycan units. Nothing
of the kind exists in human cells, so drugs that inhibit bacterial
cell wall synthesis in one form or another (such as penicillin
and cephalosporins) are effective, safe, and broad-spectrum.
|
 |
|
ENVELOPED FLU The outer membrane of the influenza
virion holds two major transmembrane glycoproteins: hemagglutinin
and neuraminidase.
COURTESY OF GREG TANNOCK |
|
|
Of course, there are some targets unique to viruses. And with the
advent of polymerase chain reaction and genetic engineering, those
targets have become easier to find. Using just a viral genetic
code, researchers can now model the structure of specific viral
proteins. And using structure-based drug design, pharmaceutical
makers can find a small molecule that inhibits an enzyme's activity,
blocks its binding, or otherwise specifically shuts the viral enzyme
down without affecting eukaryotic enzymes.
Yet each virus seems to require its own custom-designed drug.
A small molecule that could inhibit all types of viruses--a pan-antiviral
analogous to a broad-spectrum antibiotic--is not yet on the horizon.
"I rarely use the word impossible," Bischofberger says, "but I
think it's appropriate here. From what we know about viral mechanisms,
I couldn't conceive of what that would look like."
The first oral antiviral was a drug for herpes, acyclovir,
developed by the laboratory of Gertrude B. Elion at Burroughs-Wellcome
in the 1960s. Acyclovir is a nucleoside analog that fits into
the binding pocket of herpes DNA polymerase and terminates the
enzyme's growing nucleic acid chain. AIDS became an issue in the
1980s, and researchers started looking for something--anything--to
give dying AIDS patients. AZT, a cancer drug that became the first
anti-HIV drug, was modeled after acyclovir.
Newer antiviral drugs, primarily anti-HIV agents, target other
steps in the viral life cycle. Some attempt to block the initial
binding of virus to receptors on the cell surface, and some block
the fusion of viral envelope with the cell membrane. A number
of drugs target the proteins encoded by the viral genetic material
that are made once the virus has started directing cellular activity.
For example, viral polymerases create viral nucleic acids, and
viral proteases cut apart the newly created polypeptides.
Respiratory viral targets aren't yet as well known as HIV targets,
although diseases caused by influenza virus and RSV cause significant
death and disease. "If you look at commercial interest," Bischofberger
says, "there are first the herpes viruses, and that was addressed
by acyclovir, at least partly. And then there were the retroviruses,
including HIV. And the last holdout is the respiratory viruses."
There are reasons for that situation.
Biologically, respiratory viruses are challenging to treat.
First, the treatment window is narrow. After initial infection,
respiratory viruses replicate rapidly. Quickly, "the density of
virus in the nasal cavity grows exponentially, on the order of
12 to 24 hours," says Siegfried Reich, head of viral and ophthalmic
diseases at Pfizer,
La Jolla, Calif. And the body responds with a strong immune defense.
"It's almost like an overreaction," Reich says. Most of what we
identify with a respiratory infection--the sneezing, the congestion,
the runny nose--is "really an extreme immune response to an invader."
The viral load often decreases after a few days, while a cascade
of trailing sniffles and leftover cough may last for weeks. Therefore,
a treatment, to be effective, must get at the virus early--within
24 to 48 hours of onset of symptoms.
Second, respiratory viruses can be vastly different from each
other, yet the symptoms that they cause are almost indistinguishable.
"Take, for instance, the commonly known influenza, parainfluenza,
respiratory syncytial virus, and coronavirus," Bischofberger says.
"The disease symptoms are the same: cough, fever, muscle ache.
You know how you feel when you have the flu. It's just called
the flu." Yet in fact, it could have been caused by any number
of viruses. The only thing, really, that keeps respiratory viruses
in the same category is this similar clinical manifestation. Respiratory
viruses, according to Reich, just "happen to replicate very nicely"
in the respiratory tract (the lungs, epithelial tissue, and nasal
cavity).
The trouble is that a drug designed to inhibit the parainfluenza
polymerase probably would do nothing against a coronavirus polymerase
and vice versa. Without knowing which virus is the troublemaker,
treatment is hit-or-miss.
INFLUENZA.
Influenza tends to be easier to pick out of the crowd
than the others. "You can tell when the flu epidemic is on the
march," says Greg
Tannock, professor of virology at RMIT University in Melbourne,
Australia. It is a seasonal disease. During flu season and in
a community experiencing an outbreak, the percentage of respiratory
illnesses attributable to influenza is well over 50%.
That pattern certainly makes it easier to perform a clinical
trial, and perhaps that is why influenza now has the best antiviral
arsenal of all the respiratory diseases. Four drugs are available
that specifically target influenza.
Two older drugs, amantadine (Symmetrel) and its derivative
rimantadine (Flumadine), fight just the influenza A virus. (Influenza
comes in many subtypes, but A and B are the primary categories
that cause human disease.) These symmetric tricyclic amine compounds
interfere with proton transport of the viral transmembrane protein
called M2.
Two newer and broader drugs are also available: the neuraminidase
inhibitors, oseltamivir (Tamiflu) and zanamivir (Relenza).
"Flu is an enormous problem," says Gilead's Bischofberger,
who helped develop Tamiflu. According to the Centers
for Disease Control & Prevention, 10 to 20% of the population
is infected every year. And then a pandemic rolls around about
every 25 years, Bischofberger says. Gilead scientists knew they
wanted to develop an influenza drug that would be effective against
as many strains of influenza as possible because the virus mutates
yearly. "Influenza replicates in three main species: pigs, birds,
and humans. And viruses from the three species can rearrange,"
Bischofberger says. In addition, the scientists knew they needed
to target a portion of the virus that was essential to its function.
Otherwise, a resistant version could easily arise.
The scientists chose the neuraminidase enzyme. When influenza
viruses land on human cells, one of the outer viral glycoproteins,
hemagglutinin, binds to the sialic acids found on the outside
of all human cells. This works well the first go-round, but when
new viruses bud off the human cell, they're enclosed in human
membrane, and sialic acid is now part of their own coat as well.
Newly formed viruses would clump together if it weren't for a
second viral membrane glycoprotein, the neuraminidase enzyme.
This enzyme clips off sialic acid from newly formed viral particles,
freeing them from clumping and allowing them to bind to new human
cells.
|
 |
|
SARS POCKET Crystal structure of SARS 3CL
protease reveals its active site.
COURTESY OF PFIZER, LA JOLLA |
|
|
The Gilead team used structure-based drug design to develop
oseltamivir, which blocks the active site of neuraminidase.
Successful neuraminidase inhibitors have inspired the development
of new ones, and a number of companies have candidates in the
pipeline, Tannock says.
Because the effectiveness of oseltamivir depends on receiving
the drug as soon as possible after infection, Gilead has explored
the administration of the drug even before infection, as a preventive
agent, and oseltamivir is approved for prophylactic use. "I think
the most likely use would be what we call 'post-exposure prophylaxis,'
" Bischofberger says. When one member of the household gets sick,
all others start taking Tamiflu. "In my mind, that would be the
best use of our drug."
The ultimate in preventive therapy against viruses is, of course,
vaccination. One or two shots before initial exposure can protect
an individual for years or even for life because the immune system
builds up antibodies against the weakened or dead virus in the
vaccine. But some respiratory viruses, including influenza and
rhinoviruses, mutate so rapidly that a vaccine's effectiveness
expires quickly. Because influenza is such a threat, a new influenza
vaccine is re-created every year, and that vaccine is a strong
protection for the old, the young, and the immunocompromised.
Unfortunately, sometimes, like this year, the vaccine makers incorrectly
predict which strain will dominate. In addition, Tannock says,
"it's hard to develop an effective immune response in the respiratory
tract." The immunity often doesn't last long.
RSV.
Physicians would love to have a vaccine for the infection
that is the major cause of lower respiratory tract disease in
infants and children: respiratory syncytial virus. RSV is the
number-one reason why babies under one year old are hospitalized
in the U.S., according to Alan Cohen, a pediatrician and senior
director of medical affairs at MedImmune.
A formalin-killed vaccine was actually created in the 1960s against
RSV. But when tested, it backfired. Children who received the
vaccine, when subsequently exposed to live virus, developed even
more severe symptoms than children who received no vaccine.
Researchers now think that the vaccine actually helped prime
the part of the host immune response that contributes to an RSV
infection (the Th2 subclass of helper T cells). "It basically
revved up the immune response so that when children were exposed
to the real virus, it unleashed a more dangerous response," Cohen
says. Creating a vaccine that avoids priming this part of the
immune response while supporting the immunity-building response
has been tricky. The National Institutes of Health "has been working
on a program for about 30 years," Tannock says. "I think a drug
is the only answer for RSV."
Two antivirals are approved by the Food
& Drug Administration for use against RSV: ribavirin and
palivizumab.
Ribavirin is a purine nucleoside analog that was first synthesized
in 1970 and was shown to have some general antiviral effects in
1972. It is effective in combination therapy against the hepatitis
C virus and was actually one of the first drugs tried when SARS
began to emerge, although it was quickly found to be ineffective
against SARS.
Ribavirin's mechanism of action is not well understood. In
vitro, it does not act as a direct antiviral. Some suspect that
it boosts the human immune response or encourages a mutating virus
to mutate too much. The treatment is cumbersome and expensive,
according to Janet England, associate professor of pediatric infectious
disease at the University of Washington Children's Hospital and
Regional Medical Center in Seattle, Washington. "Ribavirin is
available, and it is used, but not very much," she says.
One other preventive treatment option currently exists for
RSV: MedImmune makes a human monoclonal antibody: palivizumab
(or Synagis). Approved by FDA in 1998, Synagis is a genetically
engineered antibody specific to a highly conserved glycoprotein
on the surface of the respiratory syncytial virus: the F protein
(F because it helps with fusion). "It was the first monoclonal
antibody approved for use in humans to protect against any kind
of infection," Cohen says.
Antibody therapy is somewhere in between a vaccine and a drug.
Instead of priming the immune system to create its own antibodies
against a certain disease, antibody therapy provides prefabricated
antibodies. Babies receive the same kind of short-term immune
help from a mother's antibodies in breast milk.
Synagis must be taken before exposure, so it is injected into
muscle on a monthly basis during the RSV season, and it is primarily
prescribed for premature infants and other high-risk babies. Expensive
monthly shots aren't for every child, but because it is taken
beforehand, antibody therapy does bypass the problem of getting
the drug in the first 48 hours of symptom onset, Cohen notes.
On the research front, a few anti-RSV compounds have shown
some activity both in vitro and in cotton rats.
Parainfluenza virus is also of particular concern for children,
and a newly discovered virus, the metapneumovirus, which was first
described in humans in 2001, may be another agent to which children
are especially susceptible. Neither virus has any approved antiviral.
Metapneumovirus has actually been around a long time. But because
many low-lying respiratory viruses go undiagnosed, it was not
recognized until recently.
SARS. Unlike
metapneumovirus, other viruses are enough of a problem
from their first appearance in humans that they demand research
attention. The SARS viral genome, for example, was entirely sequenced
within three or four months of the SARS outbreak in China last
year.
Many academic laboratories and some companies are now working
on leads for an anti-SARS drug. To be truthful, an antiviral for
SARS will likely not be a big moneymaker, Pfizer's Reich says.
SARS may, however, become a significant public health concern,
and Pfizer is one of the main pursuers of a SARS antiviral.
Pfizer got somewhat of a jump-start when Rolf Hilgenfeld, director
of the Institute of Biochemistry at the University
of Lübeck, in Germany, described the first crystal structure
of a coronavirus protein: the main protease, also called the 3C-like
protease. Pfizer had been actively working to develop an inhibitor
for picornaviruses (a possible treatment for the common cold),
and one of their molecules looked like it might fit very nicely
inside the active site of the SARS protease.
It turns out that this molecule, ruprintrivir (C&EN,
May 19, 2003, page 13), is not an effective drug against either
picornaviruses or SARS coronavirus. However, Pfizer was able to
leverage a great deal of its work on picornaviral infections toward
creating a new SARS program. At the moment, they are pursuing
drug candidates that inhibit the SARS protease.
Hilgenfeld and collaborators in Beijing and Shanghai continue
to characterize more SARS proteins that may be good targets for
antiviral therapy--for example, the RNA-dependent RNA polymerase
and SARS helicase (which helps unwind the viral genetic material).
They are also elucidating the structure of proteins whose function
is still unclear.
"The approach that we follow is really a structural genomics
approach," Hilgenfeld says. "The problem is, this virus has a
very big genome for an RNA virus: 30,000 bases. It is the largest
RNA genome known. It is quite dynamic. During evolution, these
coronaviruses sometimes lose genes and proteins or then they make
new ones, and no one knows what they are good for." SARS makes
between 22 and 28 proteins, Hilgenfeld says, "but we only know
the function of a handful of them."
Other laboratories are exploring different anti-SARS coronavirus
strategies. One strategy is to use heptad repeats as fusion inhibitors,
mimicking one of the most recent tacks in anti-HIV therapy. The
protein that gives coronaviruses their name is called the spike
protein. It sticks out from the surface like a halo--or a corona
during a total eclipse of the sun. This protein is responsible
for binding and fusing with human cells, and it has a recurring
seven-repeat sequence in a triple-helix coil. "The idea is to
synthesize peptides which correspond to these heptad repeats,"
Hilgenfeld says. "It has been shown, at least for HIV--and I am
convinced it will work for SARS--that these heptad-repeat-derived
peptides compete with the virus for binding to the receptor."
Interferons also are candidates for an anti-SARS drug. These
glycoproteins, produced naturally by the human body, are not directly
antiviral. Yet a shot of recombinant human interferon does help
the immune system fight a number of maladies, including viral
infection. All three types-- ,
, and --are
already FDA approved to fight other illnesses, including hepatitis
C, cancer, and multiple sclerosis.
Interferons "are like the Paul Revere of the cell," says Dale
Barnard, a professor of microbiology at Utah State University.
Once a virus has taken over a cell and starts to make double-stranded
RNA or a DNA/RNA hybrid, the defeated human cell begins to make
interferons. Interferons then leave the cell and bind to neighboring
uninfected cells, triggering the production of antiviral proteins.
"Interferon is the warning messenger," Barnard says.
Barnard is affiliated with Utah
State's Institute for Antiviral Research, which is led by
Robert W. Sidwell. The institute is one of two facilities in the
U.S. that has funding from the National
Institute of Allergy & Infectious Diseases to screen anti-SARS
compounds in cell culture. Utah and the Southern
Research Institute (SRI) in Birmingham, Ala., receive compounds
from across the U.S. for initial screening in African green monkey
kidney cells.
"As far as what we have been sent," Barnard says, "the interferons
have been the most potent and the most reliable. Plus, many are
already FDA approved" and could be quickly put to use in SARS
patients. Barnard qualifies, however, that none of them has been
tested in animal models. In fact, a good animal model for SARS
that reflects how the virus causes disease in humans does not
yet exist, although SRI and others are actively working to create
one.
COMMON COLD.
The common cold, caused by a number of strains of picornaviruses
(rhino- and enteroviruses) has, so far, eluded the reach of any
antiviral drug, in part because any drug that FDA approves for
a non-life-threatening disease must be "very, very safe," says
Jack Secrist, vice president for drug discovery at SRI.
Pleconaril,
a drug candidate made by ViroPharma,
is one compound that recently made it to Phase III clinical trials
for treatment of the common cold. This molecule fits into a hydrophobic
pocket on the capsid of rhinoviruses and enteroviruses and blocks
attachment and uncoating of the virus.
In 2002, the FDA advisory committee for antiviral drugs reviewed
a tablet version of pleconaril. "It did appear that it had some
efficacy--about a half a day or a day's reduction in cold symptoms,
but as it turned out, it had a pretty pronounced drug interaction
with birth control pills, making the birth control perhaps ineffective,"
says Jeff Murray, deputy director of the division of antiviral
drug products at FDA's Center
for Drug Evaluation & Research. "And for a self-limiting
illness like a cold, the drugs need to be squeaky clean. For a
drug that would perhaps result in unwanted pregnancies, the risk/benefit
was considered not to be favorable." ViroPharma has reformulated
pleconaril and entered into an agreement with Schering-Plough
to market an intranasal version of the drug, which would limit
its interaction with other drugs.
ECONOMIC CONSIDERATIONS.
The challenges of developing drugs for respiratory
illnesses are not only scientific but also economic and political.
"I think antiviral drugs have a role," Tannock says. "But they
won't be cheap." Developing an antiviral drug that specifically
inhibits one protein in one type of virus and performing clinical
trials on that drug are expensive. Treatment with Tamiflu costs
$50 for a child and $75 for an adult.
In Europe and other places with socialized health care, the
prospects for antiviral drugs are particularly dim, Tannock says.
Governments do not have a lot of money for antivirals--especially
for respiratory infections, which are hard to distinguish from
each other and tend to be self-limiting illnesses.
|
 |
|
BUDDING VIRUS SARS coronavirus, magnified
in inset, replicates readily in African green monkey kidney
cells.
CDC PHOTO |
|
|
When Gilead approached some European governments about offering
Tamiflu in their countries, the idea wasn't so warmly welcomed.
"This was never explicitly said," Bischofberger says, "but we were
met a little bit with the sentiment, 'Oh this is just going to
cost us a lot of money. We'll just deal with it with chicken soup--it's
cheaper than having a prescription medication used broadly for
the whole population.' That's not an issue in the U.S. It's all
a market economy. But in Europe, some governments were not jumping
up and down. They thought it would cost them a lot of additional
money. If [the drug] is broadly used in a flu season for prophylaxis,
[the cost] adds up. And it is money that these health care systems
don't have." Bischofberger adds that the needs of public health
sometimes don't mesh with the commercial realities of pharmaceutical
companies. "If you look at public health, it would probably be
worthwhile to have a coronavirus agent," he says. But because
coronaviruses cause only a minority of all respiratory infections,
you would probably need a diagnostic test to distinguish coronavirus
from all other respiratory viruses. "And if you need a diagnostic,
you have to think about the costs and the administration," he
continues. The test "should be quick. It should be at the doctor's
office. It really complicates things. A pharmaceutical company
developing a coronavirus agent may not be able to recoup the costs
associated with bringing a drug to the market."
Antiviral drug development is costly and takes a lot of perseverance,
agrees Pfizer's Reich, but for a chemist, this combination presents
an exciting opportunity. "There aren't a whole slew of viral targets
out there. And then when you get to doing structure-based design
on them, they can be quite challenging." The HIV protease inhibitors,
for example, have multiple asymmetric centers, and lots of effort
went into conquering those viral targets.
"In the early days," Reich says, "viruses seemed horrendous.
They mutate. In the case of the respiratory viruses, they have
this immune response. But major milestones have been reached in
getting antiviral compounds to the marketplace in spite of all
the challenges. Antiviral drug research is a great testing ground
for chemists--if you're up for it."
|
|