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Georgia Tech researchers
examine petri plates hosting yeast colonies containing re-engineered
nuclear receptors.
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"We are hijacking these nuclear receptors for a new set of
purposes," explained Donald Doyle, assistant professor in the School of
Chemistry and Biochemistry at the Georgia Institute of Technology. "We want to
change the nuclear receptors themselves so they don't recognize what they
normally recognize, and instead recognize the small molecules we want to detect.
That would allow us to develop a new type of sensing mechanism."
A paper published in the September 27 – October 1, 2004 issue
of the journal Proceedings of the National Academy of Sciences describes how
Doyle's research team – which also included Lauren Schwimmer, Priyanka Rohatgi,
Bahareh Azizi and Katherine Seley – modified one type of nuclear receptor to
bind a drug compound to which it previously did not respond. Based on this
success, the researchers hope to demonstrate broader application with other
small molecules.
The work was sponsored by the Research Corporation, the
Seaver Foundation and the National Science Foundation.
Nuclear receptors are ligand-activated transcription factors
contained in cells. When activated by specific small molecules, the nuclear
receptors initiate a complex process that results in gene expression. Because
these receptors play a vital role in controlling cellular response to these
small molecules, scientists have been attempting to understand them and solve
their molecular structures – with a goal of creating pharmaceuticals able to
turn them off or on. The research often involves placing nuclear receptors into
yeast cells, which because they do not have nuclear receptors of their own,
allow scientists to isolate the activities of specific receptors.
For their study, Doyle's team chose the nuclear receptor
retinoid x receptor (RXR) whose molecular structure has been well documented by
other researchers. Using a technique known as structure-based codon
randomization, the Georgia Tech researchers used their knowledge of RXR's
structure to modify the 20 different amino acids that make up the receptor
pocket. The goal was to create a library containing 32,768 different variations
in the hope of creating – and then finding – a few re-engineered receptors that
would have the ability to bind to a molecule known as LG335. (LG335 is
structurally similar to Targretin®, a pharmaceutical developed to bind RXR.
However, LG335 does not effectively bind unmodified RXR.)
Though the goal was 32,768 variants, the method actually
produced approximately 380,000 samples – which would have been impossible to
test using conventional screening methods. However, Doyle and his research team
used a new protein engineering technique known as chemical complementation that
allowed the variants to be tested in parallel – a task akin to finding genetic
needles in thousands of haystacks simultaneously.
Using the technique, the researchers placed each variant,
which had been inserted into yeast cells, onto petri plates containing LG335. A
small number of yeast cells (less than 0.1 percent of the total) grew into
visible colonies, suggesting they might contain nuclear receptors for the LG335.
To verify that the colonies were growing in response to LG335
and not some other compound contained in the yeast, samples were then smeared
onto another petri plate that did not contain LG335. Any colonies that grew
there were discarded. From their initial 380,000 candidates, the researchers
ended up with about a dozen nuclear receptors whose recognition pockets had been
re-engineered to respond to the LG335.
"Using this technique, we don't have to evaluate each member
of our library," Doyle said. "The yeast actually do the work for us. If only one
in a million respond to the compound on the plate, the yeast will form a colony
around that one. This allows us to do highly parallel and rapid screening to
find a few functional receptors in a large collection of nonfunctional receptors."
The receptors found by the researchers varied in their
responsiveness, with some significantly more sensitive than others. Some
performed the function of on-off switches, while others were more like dimmer
switches, responding in proportion to the amount of LG335 bound to them.
Doyle's team also studied their re-engineered receptors in
mammalian cells – and found they behaved much the same as in yeast.
The re-engineered receptors could be used in gene therapy
against cancer, or as research tools to investigate gene function. But more
importantly, the new receptors serve as proof of principle for the protein
engineering technique used to produce them. Doyle envisions using that technique
to produce other nuclear receptors that could be the basis for sensing arrays in
which a variety of receptors, each sensitive to a different compound, could be
used to quickly analyze an unknown agent. A sensor array might also be used in a
hospital emergency room to rapidly test for chemical agents in the blood of an
unconscious patient.
"If we could take the receptors, express them and put them
into a device where there is a color change or another signal produced, we could
potentially detect small molecules in a robust way that could complement or
replace other detection technologies," he explained.
The same technology could also be used to produce enzymes,
and to regulate metabolism in cells, Doyle said.
Having demonstrated the ability to re-engineer one nuclear
receptor to respond to a small molecule to which it previously did not bind, the
research team next wants to demonstrate that the technique could apply to other
small molecules.
"Now we have to see how far we can push this and how many
small molecules we can accommodate with this technique," Doyle said. "We are
trying to generalize this approach to genetic selection. There is a lot of
diversity we can work with in terms of different binding pockets and shapes, so
this is only the first step." |
