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The current project spans various aspects of the design,
synthesis, and analysis of compounds to target the HIV protease,
mutant proteases, and possible related targets, like RNA.
In one sense, the project is focused on drug design and has
all the classic components of such a project—including
structural biologists and modelers, molecular biologists with
expertise in gene expression; synthetic organic chemists who
can make new lead compounds; and biologists who can test them
in cell culture and other model systems. However, the project
is not so straightforward.
"We are actually funded to develop methodology—not
to cure AIDS," says Olson, adding that AIDS is only one disease
out of many in which the evolution of resistance to drugs
threatens to make these diseases harder and more expensive
to treat.
The program project grant is organized around four collaborative
projects supported by two cores. In general terms, the groups
involved in the projects analyze the effect of HIV mutations
on various protease inhibitors computationally and experimentally
in a coordinated and interactive effort.
Olson, along with TSRI Associate Professor David Goodsell
and University of California, San Diego's Department of Cognitive
Science Professor Richard Belew, is taking a computational
"coevolutionary" approach to develop models of mutant forms
of the HIV protease and predict the interactions of these
inhibitors against all the mutant protein.
Torbett's project involves using biological models like
tissue culture. These assays help to derive the computer models,
which can then be used to predict the results of further biological
assays, and these assays can then be tested and used to improve
the computer model in an iteratively improving process. Torbett's
project will also interact closely with the two other projects
in the grant, which are led by Wong and 2001 Nobel Laureate
K. Barry Sharpless, who is the W.M. Keck Professor of Chemistry
at TSRI.
Sharpless, joined by Associate Professor M.G. Finn and Assistant
Professor Valery Fokin are applying their in situ click
chemistry for the rapid development and evolution of inhibitors
to drug-resistant proteases.
Wong is taking a synthetic chemical approach to designing
inhibitors of HIV protease. He is also interested in using
his methodology to target RNA structures associated with the
protease, believing that there may be some targets there that
are not as susceptible to mutation.
All four projects interact with each other and alternatively
suggest experiments and take suggestions from one another.
And they also interact closely with two different scientific
cores funded by the grant.
Protein Expression and Crystallization Cores
One of the two cores of the program project grant is led
by Elder and Assistant Professor Philip Dawson. This Protein
Expression Core, through the efforts of Research Associate
Ying-Chuan Lin of the Elder lab, produces proteases and mutant
forms of the protease, using a combination of synthetic chemistry
and biological expression systems. They also design functional
assays and chemical probes to see how mutations affect the
activity of the enzyme. And they look at the substrate specificity
of the HIV protease, working with substrate and substrate-like
inhibitors.
"In general," says Dawson, "our role is to apply the technologies
that we have been developing to the study of drug resistance."
Dawson and his colleagues use the technique of solid-phase
synthesis to make the peptides. Invented by Robert Bruce Merrifield
in 1963 (for which he was awarded the 1984 Nobel Prize in
Chemistry), solid phase protein synthesis basically entails
building a peptide step-by-step, starting with a single amino
acid that is attached to a polymer resin. Amino acids are
then added one at a time, the resin is washed between each
successive round, and finally the finished peptide is removed
from the resin.
This expertise in chemically synthesizing proteins also
gives Dawson and his laboratory the ability to routinely make
proteins for a number of applied problems. One problem they
work on is the protease encoded by HIV.
Dawson joined the program project grant during the latest
round of funding. His expertise in chemically synthesizing
proteins gives them the ability to routinely make proteins
for a number of applied problems. Actually, says Dawson, "HIV
protease is one of the big success stories of chemical protein
synthesis." The enzyme was synthesized and then crystallized
thanks to the work of Dawson's Ph.D. advisor and former TSRI
Professor Steve Kent (now at the University of Chicago) and
Alexander Wlodawer, who is currently chief of the Macromolecular
Crystallography Laboratory and the Protein Structure Section
at the National Cancer Institute.
Back in the late 1980s, when the push to design potent inhibitors
of HIV protease was in full swing, one major obstacle was
expressing and purifying the protein. Kent successfully synthesized
HIV protease and Wlodawer successfully crystallized it and
solved its three-dimensional structure. This work led to the
structure-assisted design of a number of drugs.
Wlodawer is also involved as one of the co-lead investigators
on the second of the program project grant's two cores. This
core is devoted to elucidating the structure and modeling
of various forms of HIV protease. These structures also provide
important information to Olson, who can use them as the starting
point for designing lead compounds, which can then be handed
off to the chemists, who can use them as guides for synthesizing
new inhibitors. And any compounds that they do develop can
then be tested by Torbett.
TSRI Associate Professor Dave Stout is head of this core,
and he is leading an effort to crystallize the proteins with
RNA in order to look at RNA–protease interactions. Additionally,
this core runs computational protein–inhibitor docking
experiments and other computer aided drug design studies.
It's a very dynamic group of people, says Fokin. "It's always
interesting to see what other people come up with."
Populating Dynamics and Dimer Inhibitors
There are many interesting questions being raised within
the program project grant, including some that take quite
unusual approached to addressing HIV protease resistance.
For instance, Goodsell ties atomic models to population
models.
"You can create a model that estimates all the cells in
the immune system, how they reproduce, and how the virus interacts
with them," says Goodsell. This allows him to ask such questions
as: If a patient takes one particular AIDS drug for a year,
what would the population of mutants in his/her body be like
at the end of that year? Would being able to predict this
generate better-tailored therapies or point the way to the
best possible combination of therapies?
Another unusual approach is asking if parts of the protease
molecule other than the active site can be targeted with drugs.
Specifically, can one target what is known as the "dimer interface"
of the molecule, which is where the two ends of two HIV protease
monomers come together and form a beta-sheet—like interlaced
fingers from two hands that hold the active dimer form of
the molecule together.
The aim is to target this interface and prevent two individual
protease monomers from coming together. Since the protease
enzyme is only active as a dimer, mucking with this dimer
interface should profoundly affect the activity of the enzyme.
"If we target those areas, what does the protease do?" asks
Torbett.
Of course, the problem of mutations to the protease cannot
be avoided here either. Not all of the mutations that arise
in the protease enzyme are in the active site or are functionally
related to the active site. In fact, many of the mutants that
have been raised by the TSRI team in the laboratory and that
have arisen in patients who are taking antiretrovirals are
not in the active site of the protease. Some are in the area
of this dimer interface and altering the stability of the
dimer.
One interesting way that they are looking at this is by
forcing dimerization of the protease monomers by expressing
them together in a linked form.
Next Week: Fighting HIV Resistance at Home and in
the Laboratory
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