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It
was 1992 and Emory pharmacologist Haian Fu was hot on the trail of an
intriguing family of proteins called 14-3-3. Originally identified in
the 1960s as exclusively connected to the brain, the 14-3-3 family had
been largely ignored ever since. Fu discovered, however, that these proteins
actually play a key role in regulating cell growth throughout the body,
a finding that implicated them in the unchecked growth of tumor cells.
Like a cell phone company activating new handsets, 14-3-3 proteins
bind to other protein targets and initiate a “conversation”
that turns them on or off. Fu discovered that in cancer, this conversation
can promote tumor growth by triggering the activation of proteins. These
cancer-promoting proteins are like armor that allows tumor cells to resist
cancer therapies as well as apoptosis, the natural process of cell death
designed to eliminate damaged cells.
In hopes of halting cancer growth, Fu wanted
to cut off the “conversation” from 14-3-3 to its client proteins.
He began the painstaking, years-long process that would be necessary to
accomplish his goal, using conventional laboratory assays. First he would
need to discover exactly how the protein functioned and where it might
be vulnerable to alteration. Then he would need to conduct semi-random
tests that combined the protein in individual test tubes with a series
of molecular compounds with potential drug-like properties.
Birth of a drug-screening enterprise
Fast forward to 2002. Fu, along with Emory pharmacology chair Raymond
Dingledine, Winship Cancer Institute associate director Fadlo Khuri, and
Emory chemists Dennis Liotta and James Snyder, set their sights on securing
drug discovery technology that previously had been the exclusive purview
of large pharmaceutical companies––robotics equipment that
could automatically handle thousands of liquids and chemical assays in
minute quantities, then compute and analyze the resulting massive amounts
of information. This powerful system can quickly screen hundreds of thousands
of molecular compounds against protein targets.
Researchers at Harvard Medical School had
become the first academic scientists to venture into this new frontier
at the beginning of the 21st century. High-throughput screening equipment
was becoming more affordable, and a number of chemical companies were
offering large libraries of compounds for sale.
In light of Harvard’s success and
Emory’s own experiences in drug development, researchers here decided
it was time to claim a spot for Emory as a national drug-screening leader.
Using existing research grants, combined with funds from the Keck Foundation
and Synaptic Pharmaceuticals, the group began to piece together the necessary
resources, including a library of 100,000 molecular compounds purchased
from ChemDiv.
The Georgia Research Alliance (GRA) contributed
$400,000 for high-throughput robotics screening equipment, Amgen Corporation
offered some planning funds, and the Emory University Chemical-Biology
Discovery Center was officially born.
The new drug discovery center placed Emory
squarely on the front lines of academic drug screening. In 2005, when
the NIH decided to fund a national network of molecular screening centers,
the Emory team was already up and running. Emory was designated one of
nine new centers in the NIH Molecular Libraries Screening Center Network
(MLSCN) and received a three-year grant of $9 million dollars, including
a library of 100,000 additional compounds. Part of the NIH Roadmap’s
“New Pathways to Discovery” initiative, the network aims to
build a better toolbox for medical researchers and to serve as a resource
for academic researchers throughout the country. Scientists submit suggested
proteins to a national MLSCN peer-review process that picks the most promising.
Each center is expected to screen the interactions of about 20 proteins
a year.
Emory scientists now have two chances to
submit a protein for high-throughput screening––either through
Emory’s own drug discovery center or through the national MLSCN.
In some cases, proteins are being screened against the compound libraries
in both centers.
Protein power
High-throughput screening (HTS) technology is one way for scientists to
capitalize on the human genome project and venture into the realm of proteomics,
or the study of proteins that genes produce. Scientists originally believed
that each gene within the DNA sequence corresponded only to one human
protein. Now, however, they recognize that the 25,000 or 30,000 genes
in our DNA may encode five to 10 times that many proteins.
Because proteins play a part in everything that happens inside living
cells, they are the object of intense study by scientists seeking to understand
disease processes. Drug compounds are aimed at either inhibiting or enhancing
the actions of proteins that are related to disease.
For example, Fu identified a tiny fragment
that serves as the “mouth” of his 14-3-3 protein and enables
the cell-to-cell communication that triggers cancer oncogenes. He designed
an assay to connect the protein fragment to a fluorophore, a chemical
marker that lights up when the protein binds to a molecule. Fu was the
first Emory scientist to use the new screening equipment to test his protein
against the entire library of 100,000 molecular compounds in Emory’s
drug discovery center, and his protein also was selected for testing by
the MLSCN network. He has identified several promising compounds that
may be inhibitors to his target protein.
In conventional laboratory experiments,
a researcher pipettes solutions from one test tube to another and writes
down in a notebook what was done and why.
The new robotics equipment automatically
pipettes liquids containing both a target protein and one of the 100,000
compounds into a tall stack of rectangular, 3x5-inch plastic “plates”
containing 384 tiny holes or “wells.” A computerized plate
reader analyzes the miniature chemical reactions that occur when the compounds
are combined with their targets in the wells. An assay this small must
elicit a simple on or off reaction—either the compound has the desired
effect or it doesn’t. A program lists each of the compounds that
reacted with the protein target. These compounds can then be cherry picked
from the library for further study. It takes about one to two weeks to
screen the protein interactions with a library of 100,000 compounds using
HTS equipment.
Using
the cherry-picked compounds from the initial screenings, scientists like
Fu then go back to their laboratories to perform a more “old-fashioned”
biological experiment that sorts out the false-positive hit compounds.
The end result can be up to 20 promising compounds. They then relay these
compounds to the chemistry department, where a bioinformatics and modeling
group directed by Jim Snyder combines its knowledge of chemical compounds
with specialized computer software to analyze the detailed molecular structures
of all the hit compounds. A model of molecular activity called the QSAR,
for quantitative structure activity relationship, helps predict which
structural modifications of the compounds will be more active against
a target. This analysis is the first step in the “hit-to-lead”
process that puts a compound on the drug-candidate pathway.
Medicinal chemists use this information
as a guide to remodel the structures of the validated hits by laboratory
synthesis to improve the biological profiles. Fu’s lead compounds,
for example, are now being optimized by a chemistry team led by Dennis
Liotta. The gold standard is a compound that exerts a strong and selective
effect on a target protein at a very low concentration. This means it
is less likely to be toxic than a less selective, less potent compound
would be. Desirable drug-like properties also include high solubility
in water. “A drug with zero solubility would be like ingesting a
small rock,” says Liotta.
Now the modeling group and the synthesis
group are developing their own small library of compounds. These might
include modified compounds from the screening-modeling-synthesis process.
Or they might be structurally similar compounds identified by the researchers
that are available for purchase but not yet part of Emory’s library.
“This kind of work would be almost
impossible without our screening center,” says Fu. “There
is a huge difference between academia and industry in screening target
proteins. We design the screening assays in our own laboratories, whereas
many pharmaceutical companies often purchase ready-made assays. Academic
scientists know these targets inside out because we’ve been studying
them for years. Emory’s screening centers, combined with the ability
of our expert chemists to modify compounds, can serve as the glue to enhance
the interaction between basic and clinical science and to promote the
translation of basic science to the bedside.”
“We are not competing with pharmaceutical
companies, although some of our findings may well be useful to them on
down the road,” Dingledine says. “We intentionally are not
looking for the next blockbuster antidepressant or antireflux drug. That’s
not our ball game. We are trying to identify targets that are not being
pursued by major pharmaceutical companies yet might have real research
value or clinical impact. Academic medical laboratories like ours have
a Zen-like focus on understanding the properties of proteins.”
Why Emory got the grant
Emory’s
history in successful AIDS drug discovery and its track record of collaboration
between the medical school and the college’s chemistry department
gave it a leg up in the selection process for the new NIH screening centers.
“When we went up against some of the
biggest-name universities in the country, we were very competitive,”
says Liotta. “This was because we had ongoing collaborations between
chemistry and the School of Medicine that we could point to as real examples
of our expertise and our desire to move in that direction. It wasn’t
a forced interaction––it is what we’ve done for years.
“Drug discovery is an intimate partnership
between biologists and chemists,” Liotta continues. “In most
places, there is little or no interaction between those two groups, which
means that each group is by definition limited in terms of the kinds of
questions they can ask and the kinds of answers they can get. Emory’s
screening center allows for a smooth interface between these two critical
groups that enables us to ask and answer the bigger questions. We hope
this will make Emory more competitive for external funding and may lead
to alternative revenue sources such as drug royalties. Overall, it’s
an opportunity for us to pool our complementary skill sets and do important
work. We hope this also will enable us to recruit the best people, because
this extra opportunity is something they don’t have at other research
institutions.”
Former Emory faculty member Jeffrey Conn,
now professor and director of Translational Neuropharmacology at Vanderbilt,
heads another of the newly designated NIH Molecular Libraries Screening
Centers. “Emory is in an excellent position to play a major leadership
role in what may be the early stages of a major shift in biomedical research,”
said Conn. “Because of its culture of strong collaborations between
clinical and basic scientists, I see a translational mindset of scientists
across Emory. The Department of Pharmacology has an uncommon combination
of strength in classical pharmacology, and the chemistry department is
world-class—in fact one of the rare chemistry departments with an
established track record in discovery of marketed drugs.”
Carson Loomis, program director of the NIH
Molecular Screening Libraries Division of Extramural Research at the National
Human Genome Research Institute, says Dingledine’s leadership in
assay-development and high-throughput screening helped Emory capture the
molecular libraries designation. “The award was based on the quality
of his scientific staff, his development plan, and the proven record of
scientific success at Emory,” he says.
Cooperation versus competition
The
MLSCN has a really nice feature that’s different from any other
NIH award with which I have been involved,” says chemistry’s
Jim Snyder. “There are ten centers around the country, one of which
is at the NIH, and this network is meant to be collaborative. We all get
on the phone once a month and share information. We try to lower the cost
of software and pool ideas about how we can more efficiently do the tasks
of molecular screening.”
Emory will continue screening proteins identified
by its own faculty members in the Emory Chemical-Biology Discovery Center
at the same time it screens the NIH-identified proteins. A great deal
of the equipment and personnel in the two centers will overlap. Intellectual
property issues have required complex negotiations between the NIH and
Emory’s Office of Technology Transfer. The NIH wants all results
of the MLSCN to be transferred quickly into the publicly accessible PubChem
database. After much debate, however, it agreed to allow the screening
centers and investigators a short time to file patent applications on
their discoveries. Compounds not immediately patented will go into PubChem.
Many Emory scientists feel strongly that protecting intellectual property
is critical for the future of therapeutic development and for the ultimate
good of patients.
“If the targets are important and
if we discover novel modulators of those targets, then it’s our
responsibility to file patent applications on them,” says Liotta.
“If we don’t, and they get into the public domain, no one
will ever develop them commercially. It would be terrible if we found
something that was potentially an important therapy but no one would spend
the money to develop it because we didn’t patent it. That’s
when the public loses. It’s not about money; it’s about getting
these discoveries into the marketplace for the benefit of patients.”
“This screening center is going to
have a huge impact on tech transfer for a number of reasons,” says
Todd Sherer, director of technology transfer at Emory. “Venture
capitalists and large pharmaceutical companies are not as interested in
commercializing early-stage technologies and target proteins as they were
five years ago because of changes in patent laws. That means we have to
find ways to move target technology further down the innovation pathway
and create better investment opportunities.
“This screening center will help us
cross the ‘valley of death’ that stands between many laboratory
research discoveries and getting drugs into the marketplace. This ability
to create more compelling intellectual property technology is a crucial
part of our ability to be successful in the future.”
A winning combination
Two
of the first three proteins selected by the NIH MLSCN for screening at
Emory included Fu’s 14-3-3 protein and a protein called BAP-1, identified
in 1998 by Emory biochemists Keith Wilkinson and Frank Rauscher. BAP-1
interacts with the BRCA1 protein, which is a product of a breast cancer
suppressor gene that when mutated promotes the growth of breast cancer.
Wilkinson and Rauscher hope they may be able to slow the growth of breast
tumor cells by finding a compound to inhibit this protein. After first
screening the BAP-1 protein in Emory’s library of compounds, they
now are screening it through the MLSCN library.
“Before these centers existed, we
would have been reduced to trying to talk a drug company into doing this
kind of screening,” says Wilkinson. “That is difficult if
you can’t point to the disease and to the nature of a drug that
might develop from such a screening. Pharmaceutical companies are not
in the business of generating reagents for curious biologists.”
Scientists from throughout the School of
Medicine are beginning to use the Emory Chemical-Biology Center to advance
their research. Neurologist Jonathan Glass is looking for compounds that
might inhibit a protein that causes toxic nerve reactions to the anti-cancer
drug taxol. Microbiologists Richard Plemper and Richard Compans, along
with Jim Snyder and Dennis Liotta, are trying to develop an antiviral
drug for measles. In the first round of high-throughput screening based
on a cell fusion assay, they identified more active and promising compounds
than they had identified in the past two years working with conventional
chemical synthesis. Biochemist David Lambeth is studying a target protein
related to the inflammation involved in cardiovascular disease and cancer.
Stem cell expert Marie Csete and Dingledine are planning to look for compounds
that would reduce the ability of cultured stem cells to differentiate
in the laboratory or direct them to differentiate into specific types
of cells.
“Emory recognized the opportunity for this
kind of high-throughput molecular screening earlier than most academic
institutions and was fortunate to receive the support to move forward,”
Dingledine says. “Others are now recognizing the advantages of combining
chemistry and biology and pharmacology. The national network will move
us in exciting new directions, with expertise and interests that by design
are complementary rather than competing. No individual center could do
all this on its own, but together we can accomplish a great deal.”
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