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Wong is also trying to perfect procedures for attaching the
carbohydrates to a solid phase so that they can be easily
presented for screening against some biological target. He
has already perfected the chemistry needed to do so, and he
can attach designed linkers to his carbohydrate molecules.
The next step is to determine the best way to quickly and
accurately deposit these molecules onto surfaces on a very
small scale, so that a "carbohydrate chip" can be prepared.
Wong is still working out the technology to do this, but in
preliminary demonstrations he and his laboratory group have
assembled and affixed arrays of carbohydrates onto microtiter
plates by pipeting.
"It is possible to assemble millions of molecules on a polystyrene
surface or glass slide," he says. Knowing where the carbohydrate
molecules are on the chip, one can profile a particular carbohydrate-based
receptor–ligand interaction, looking for drug candidates,
for instance.
Targeting RNA Instead of Proteins
Wong is already using the compounds he has created through
his one-pot syntheses to pursue many different biological
targets. One of the key molecules that Wong is looking at
is RNA, and he is trying to come up with carbohydrate structures
that specifically target various RNA sequences.
RNA is a legitimate target. Many viruses use RNA as their
genetic material, so one could attack this RNA. Moreover,
RNA is ubiquitous in biology, so there is not a single pathogen
or human disease state in which it is not an integral part.
And one might consider that RNA's location in the cytoplasm
of cells—out in the open, as it were—makes an even
better target.
Furthermore, many proteins are difficult or impossible to
express and purify in the laboratory, making them simply unavailable
for research. This is particularly true of glycoproteins,
which undergo post-translational modifications that cannot
be duplicated in expression systems.
Other proteins are impossible to target specifically because
their binding sites are too similar to the binding sites of
many other proteins in the body. There are thousands of kinases
that use the same mechanistic means to accomplish a multitude
of biological ends. Giving a person a drug that interferes
with the one would disrupt the function of the whole lot.
"How do you come up with a selective inhibitor against just
one?" asks Wong.
And his answer, simply, is that you forget about targeting
the protein and set your sights on the RNA from whence it
comes. If you can bind to a particular sequence of the RNA
and block its translation into protein, then you are effectively
circumventing the need to block that protein.
Wong turned his attention to RNA a few years ago when he
asked whether it would be possible to come up with molecules
that target RNA with high affinity. Such an RNA-binding motif
is already, conveniently, known in a certain class of antibiotics
currently on the drug market.
The aminoglycosides, which include streptomycin and gentamicin,
bind to the 30S subunit of ribosomal RNA of bacteria like
Staphylococci and Mycobacteria. All aminoglycosides
contain one particular "b-hydroxyamine"
motif that is critical for the interaction with the RNA.
Once Wong had realized that, he set about designing a library
of carbohydrate compounds—"aminoglycoside mimics" he
calls them—based on this b-hydroxyamine
motif that binds RNA.
With the library, he is targeting a number of different
types of RNA, including oncogenic RNA (in collaboration with
TSRI Professor Peter Vogt), HIV and hepatitis C viral RNA,
and bacterial RNA. For each he identifies particular, unique
RNA sequences that he can target and tests these sequences
against his library.
One new antibiotic that Wong developed based on the aminoglycosides
is lethal to over 200 kinds of gram negative (lipopolysaccharide-covered)
and gram positive (peptidoglycan-covered) bacteria.
"It kills almost every bacterial species we test," he says.
The preclinical studies on this antibiotic are now being pursued
by Optimer Pharmaceuticals, Inc., an outside company Wong
co-founded.
Wong has also identified a 5' untranslated region of the
hepatitis C viral genome that is essential for the binding
of the ribosome and the assembly of the virus. This region
is highly conserved and absolutely essential for the viral
lifecycle.
"We want to come up with small molecules that target this
sequence," says Wong. Similarly, he has identified a unique,
conserved, essential sequence in HIV RNA that he uses as another
target for panning.
One Laboratory, Many Targets
Wong is also panning for inhibitors of enzymes involved
in cancer metastasis. When cells move from one tissue to another,
they have to detach from the surface of the old tissue and
make new attachments on the surface of the new tissue. Many
of these interactions involve carbohydrate recognition, and
Wong is designing small molecules to mimic these carbohydrates.
A portion of Wong's laboratory is also working on vaccine
design by identifying antigens that are displayed on the surface
of cancer cells, bacteria, and viruses. Screening his libraries
can reveal which particular parts of the pathogenic surface
are visible to the immune system, and therefore which would
make good vaccine candidates.
One final target that Wong has in his sights is that of
HIV protease, an enzyme that the virus uses to process viral
proteins to make infectious virus particles.
For the last several years, the greatest weapons doctors
have had for treating HIV infections have been antiretroviral
drugs that tightly bind specific viral enzymes necessary for
replication and infection—the protease and reverse transcriptase
inhibitors, for instance. Highly active antiretroviral therapy
(HAART), which combines both classes of drugs together into
one treatment, has proven particularly effective, as demonstrated
by the decline in AIDS mortality in the United States in the
last few years.
However, the last few years have also witnessed the rise
of drug-resistant strains of HIV in patients on HAART. Because
HIV's genome is short and composed of only the essentials
it needs for replication and infectivity, it lacks the luxury
of a proofreading mechanism—which mammals, for instance,
use to ensure fidelity in cell replication and division. The
fidelity of HIV is so low that it makes an average of one
mistake every time it replicates. And because it replicates
so much in an infected host, mutant variants quickly arise.
These variants are often resistant to HAART drugs but are
still able to replicate. The drugs lose and the infection
wins. As a result, more and more inhibitors have been designed
in recent years, alongside a plethora of combinations and
dosage schedules that aim to maximize the effectiveness of
the drug.
"HIV drug resistance is a major problem," says Wong. "If
you design a perfect drug, it may not be a good one from a
therapeutic point of view."
So Wong has taken a different route. He has designed an
inhibitor that is intentionally imperfect, with broad activity
rather than specific. This inhibitor, which targets the viral
protease that HIV uses to assemble infectious virions, is
ten-fold less active than some of the weakest HIV protease
inhibitors on the market, but it is active against almost
all variants of the viral enzyme. Mutations in the protease
are less likely to knock out its effectiveness.
"It is based on our understanding of how the protease evolves
and how the structure changes to escape."
In preliminary studies, the inhibitor is effective against
a broad range of similar proteases from a range of HIV and
related viruses in cell culture assays, and further studies
are being pursued. Interestingly, five years ago, one might
not have guessed that a less potent inhibitor could be a good
thing, or that sometimes the road less traveled would be a
better route to drug discovery.
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