The Resistance Part I:
From Petri Dishes to Population Dynamics
By Jason Socrates
Bardi
"The
air was free from gnats, the earth from weeds or fungi;
everywhere were fruits and sweet and delightful flowers;
brilliant butterflies flew hither and thither. The ideal
of preventive medicine was attained. Diseases had been stamped
out."
—H.G.
Wells, The Time Machine, 1898.
When the history of science and medicine in the 20th century
is eventually written, human immunodeficiency virus (HIV)
will likely get an entire solemn chapter. Emerging in the
early 1980s, ironically just after the major public health
victory against smallpox, HIV had spread to all corners of
the globe by the end of the century.
Today, the disease shows no signs of abating. There is no
vaccine to prevent the spread of HIV and no cure for AIDS,
the disease the virus engenders.
Science and medicine, however, have had their share of successes
controlling HIV. The rise of antiretroviral drugs in 1980s
and 1990s proved that AIDS could be a treatable disease. These
drugs, which target virus-specific enzymes like HIV protease
and reverse transcriptase, showed great efficacy at keeping
the virus in check, which often dramatically improved the
prognosis of those infected. They also provided a relatively
inexpensive way of reducing mother-to-child transmission of
the virus. These are significant breakthroughs, and there
are many people alive today who owe their lives to antiretrovirals.
The virus has fought back, however, with the emergence of
strains resistant to these drugs.
See:
HIV Pathogenesis and Drug Resistance
The Coming Resistance
Now a large research consortium that brings together investigators,
students, postdocs, and other scientists at The Scripps Research
Institute (TSRI) with their colleagues at several other institutions
is beginning to use resistant HIV protease to develop methodologies
for drug evolution.
Led by Molecular Biology Professor Arthur Olson, the group
seeks to establish a drug design "cycle" aimed at developing,
testing, and refining novel approaches to making specific
inhibitors of HIV protease that would be capable of limiting
or eliminating drug resistance.
The group is trying to understand resistance by looking
at what happens as the virus changes in response to protease
inhibitors. They are looking to identify the sequential protease
transition mutants and to understand their most basic to advanced
biology, including sequence, biochemical reactivity, and structure.
The researchers are also looking at resistance as a phenomenon—how
it can be predicted to how it can be countered.
"How do these changes work at the atomic level?" asks Associate
Professor Bruce Torbett, one of the investigators in this
consortium, which is funded by a program project grant from
the National Instuitutes of Health called Drug Design Cycle
Targeting HIV-Protease Drug Resistance.
Rather than aiming solely to make the next useful AIDS drug—which
is naturally one goal of all the investigators—the team
is seeking to bring all of its knowledge and technology to
bear on developing a methodology that will allow them to understand
something much more complex.
"We want to be able to predict what really happens when
[a patient] takes a drug," says Torbett. "We're trying to
figure out how mutable the protease is biologically, structurally,
chemically, and we're asking what are the possible mutations,
when those mutations happen, and what they do in terms of
fitness of the virus."
In particular, the researchers are focusing on active site
mutations. There are 10 amino acids in the binding site of
the protease that have contact with the protein chain that
the protease cleaves (in actuality 20, since the protease
is a dimer composed of two identical 99 amino acid chains).
"The way most people design drugs is to fill up the [binding]
site and get the most binding energy," says Olson. "But certain
mutations can easily knock [these drugs] out."
For instance, a mutation that substitutes a phenylalanine
for almost any other amino acid among the 10 in the active
site of the HIV protease enzyme will add a significant amount
of bulk. This added bulk will effectively hinder any drug
that was designed to fill the binding site.
The goal for the next several years, says Olson, is to ask
questions to get a sense of the protease's ability to mutate.
What are the rules the virus follows in acquiring mutations?
Can one develop a model that will predict how the virus
will respond to various drug regimens? Are there compounds
that could effectively box the protease in?
The Cat's in that Corner
The program project grant has a long history at TSRI and
is currently beginning its third round of funding. The first
round, in the early 1990s, was primarily concerned with comparing
HIV to its cousin, the feline immunodeficiency virus (FIV).
FIV was discovered in California in 1986 by Niels Pedersen,
who is currently director of the Center for Companion Animal
Health at the University of California, Davis, and Janet Yamamoto,
who is now a professor in the University of Florida's College
of Veterinary Medicine.
As the story goes, there was a kindly woman who took in
strays—many strays—housing them in her large kennels.
She noticed an odd thing. Several cats under her care became
sick and eventually died, seemingly as a result of sleeping
in the same pen as one particular feral cat. So she contacted
Pedersen, who took samples and eventually isolated a virion,
which under the electron microscope looked like an RNA virus
belonging to the lentivirus family.
Shortly thereafter, TSRI Professor John Elder, who had been
working on retroviruses for 10 years by 1986, began to collaborate
with the Pedersen laboratory to work on the virus taken from
that isolate and from another isolate from a cat belonging
to former TSRI investigators Fred Hefron and Maggie So.
"They had a cat that came down with FIV, and we isolated
the virus from it," says Elder.
When they started, not much was known about the structure
of the FIV protease. In the first round of funding, Elder
and his colleagues isolated, cloned, and purified proteases
for their crystallographer collaborators to solve. Their hope
was that their discoveries about FIV would shed light on the
problem of HIV.
The highlight of the second round of funding was the development
of the TL3 inhibitor, which could effectively inhibit both
HIV and FIV.
FIV and HIV, it turned out, are closely related, which makes
FIV a good model for studying an HIV-type infection. HIV and
FIV proteases have a 32 percent amino acid identity and essentially
the same three-dimensional structure.
"If I showed you two pictures of FIV protease and HIV protease,
you couldn't tell them apart," says Elder.
Yet, strangely, the two proteases respond differently to
the same inhibitors. Common drugs that inhibit HIV protease
do not work on the FIV protease at all.
This observation led Elder, in collaboration with TSRI investigators
Chi-Huey Wong, who is the Ernest W. Hahn Professor and Chair
in Chemistry, Torbett, Olson, and several others, to ask what
subtle differences between the structures of FIV protease
and HIV protease could cause such a great distinction.
It was Research Associate Taekyu Lee of Wong's group who
noticed a region of the FIV protease that was smaller than
the corresponding region in the HIV protease.
"We looked at the sequences and structures of all the known
mutants," says Wong. "We found that there was a common trend
in the mutants [whereby] one site becomes smaller and smaller."
This reduction is in what is known as the P3 binding site
of the protease—where the enzyme makes contact with one
end of the inhibitor and where it normally would interact
with residues of the protein chain it naturally cleaves three
amino acids down from where the protease breaks the chain.
Most commercial HIV protease inhibitors, says Wong, are designed
to fill a large P3 site, but the virus often acquires resistance
to these drugs when it mutates the amino acids in the P3 site
swapping small amino acids for bulky ones. The team recognized
this, and the knowledge helped them design a new inhibitor
to fit this smaller space.
This prompted Wong to replace the inhibitor residue that
fits in that site with a smaller amino acid. When they did
this, the potency of this inhibitor increased 1,000-fold for
FIV.
"This was the best [inhibitor] we had ever seen against
FIV," says Elder, adding that it was also efficacious against
the wild-type HIV and nine out of thirteen protease-resistant
HIV isolates tested. The molecular changes that allowed many
variants of HIV to escape drug therapies were the same as
those that made FIV distinct from HIV.
"That observation told us that we could use FIV protease
as a drug resistant model for HIV protease," says Wong.
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