The Resistance Part III:
Chemical Innovations and New Approaches
By Jason Socrates
Bardi
"Perhaps
the eye of a scrutinizing observer might have discovered
a barely perceptible fissure, which, extending from the
roof of the building in front, made its way down the wall
in a zigzag direction"
—Edgar
Allen Poe, The Fall of the House of Usher, 1839.
Human immunodeficiency virus (HIV) plays a simple game of
overwhelming numbers.
The game is analogous to what would take place in an honest
casino that has been infiltrated by thousands of crooked gamblers
who are trying to break the bank. The gamblers constantly
try different strategies of cheating, until eventually the
house loses.
Imagine that the casino tries to control these losses by
hiring private detectives who walk around the casino floor
and grab any crooked gamblers they see, incapacitating them
and preventing them from cheating. But the crooked gamblers
soon learn how to outwit the house detectives by disguising
their faces so that they are no longer recognized.
The same thing happens with HIV.
The virus replicates prolifically in the body, and has such
a high frequency of mutation that it can rapidly evolve resistance
to the drugs that are used to treat it. A scientific consortium
led by Professor Arthur Olson of The Scripps Research Institute
(TSRI) was created to counter this HIV drug resistance.
See:
The
Resistance Part I:
From Petri Dishes to Population Dynamics
The Resistance
Part 2:
At Home and in the Laboratory
Funded by a program project grant, the consortium combines
the efforts of dozens of investigators, postdoctoral fellows,
graduate students, and other researchers at TSRI and several
other institutions. It seeks to establish a drug design cycle
aimed at developing, testing, and refining novel approaches
to making specific inhibitors that will disable resistant
mutants of HIV protease, and it combines molecular and cellular
biology, computational and structural approaches, and chemistry.
Two of the projects focus on the design and synthesis of
compounds that inhibit HIV protease and drug-resistant strains
of HIV protease. One of these two projects is led by TSRI
Chemistry Professor Chi-Huey Wong.
A New Strategy
Against the wall in Wong's office is a shelf lined with
volumes of chemistry and biology texts. Outside and down the
hall is a reading room that has stacks of some of the leading
journals in his field. Also in this reading room are several
networked computers.
And yet when Wong and his colleagues began thinking of ways
to combat HIV drug resistance several years ago, the answers
were not to be found in any of these sources.
"We needed to have a new strategy," recalls Wong.
Wong is doing synthetic chemistry work for the project and
coming up with new ways of targeting the virus, including
using technology of his own design to identify unique spots
in the viral mRNA that are highly conserved, even in the mutant
strains. He has found that one good target is the RNA frameshift
region of the virus.
Frameshifting, in biology, is something that takes place
during the translation of the nucleotides into protein amino
acids. It happens when a ribosome that is translating the
RNA into protein shifts slightly, moving up or down the RNA
sequence by a nucleotide or so. But this seemingly slight
change can have a radical change on the amino acid sequence
of the protein that is being translated—it may result
in a completely different protein.
For instance, a sequence that originally is read: (UGG)
(GCA) (UGU) (UGA) (CGU)... might be frameshifted into a sequence
that would now be read: (...U) (GGG) (CAU) (GUU) (GAC)...
In HIV, the frameshift region is where the gag and pol regions
of the viral RNA meet, and is highly conserved because frameshifting
is necessary for the expression of the POL polyprotein, which
contains the all-important viral enzymes, the reverse transcriptase,
the integrase, and the protease. The virus cannot tolerate
mutations to this region of RNA because mutations would block
the frameshift and knock out the all-important viral enzymes.
Wong reasoned when he began this work that by designing
an inhibitor that targets this non-mutating RNA, he could
potentially have a compound that would be broadly effective
against a wide variety of mutant HIV strains. In order to
achieve this, he turned to the aminoglycosides.
One-Pot Synthesis
Aminoglycosides are compounds that interact with interact
with RNA, and there are already drugs of this class on the
market—like the antibiotic streptomycin, which targets
specific pieces of RNA.
These drugs all target the RNA of one portion of the bacterial
ribosome, known as the "30S subunit." The aminoglycosides
bind to this RNA and prevent the ribosome from accurately
translating protein. Wong reasoned that he might be able to
use these compounds as scaffolds upon which to design a chemical
that would specifically target the HIV mRNA frameshift region.
The basic technique that Wong uses is one he designed a
few years ago for the synthesis of oligosaccharides in general,
called programmable one-pot synthesis. This technique basically
involves placing a large number of specific chemical building
blocks into a reaction vessel and then making sequential chemical
reactions in the soup. The final products depend entirely
on the particular reaction scheme followed.
The one-pot technique, as Wong calls it, allows him to quickly
assemble many types of carbohydrate structures—in just
minutes or hours, depending on how complicated the chemistry
is. Wong even developed software that he uses to streamline
the process. "The computer will tell you which building blocks
to use and you simply add them in sequence," he explains.
Depending on the type of building blocks used and the size
of the final structures, the pool of compounds made in this
way can be quite large. For instance, placing 300 building
blocks in a pot and randomly combining them into tetramers
will yield over eight billion compounds.
Once the oligosaccharides are made, these carbohydrate "libraries"
can be used for any number of biological studies. One of Wong's
main uses for the libraries is screening against various types
of biological targets to look for interactions.
For instance, he created an array of several hundred aminoglycosides
and screened for molecules that target HIV RNA by taking a
20- to 40-base piece of the viral RNA attached to a "biotin"
molecule, which allows the bound aminoglycosides to be detected.
While this technology is promising, warns Wong, any potential
drug that could be produced by this method still has to overcome
the obstacle of cell permeability.
"If any [aminoglycoside compound] is going to be useful,
it has to get into a cell," he says. While aminoglycosides
are useful antibiotics already, they cannot get into human
cells as easily as they can get into bacterial cells.
The eventual solution, says Wong, might be to develop a
"prodrug" form of the compound, which would be converted to
an active form after it enters a cell or to employ some "shuttle"
molecules that can transport the drugs into the cells.
For now, he is at the beginning of this research. The structure
of the program project grant is helpful because it allows
him to use the structural and biochemical data that the other
investigators on the project produce to help him come up with
the molecular design of the molecules he is going to synthesize.
"Then you can go back to the biology and see if the chemistry
works," says Wong. "An environment like Scripps is very good
for a chemist. Through collaboration, you can solve a lot
of interesting problems."
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