By accounting for the floppy, fickle nature of RNA,
researchers at the Univ. of Michigan and the Univ.
of California, Irvine have developed a new way to search for
drugs that target this important molecule. Their work appears in Nature Chemical Biology.
Once thought to be a passive carrier of genetic information,
RNA now is understood to perform a number of other vital roles in the cell, and
its malfunction can lead to disease. The versatile molecule also is essential
to retroviruses such as HIV, which have no DNA and instead rely on RNA to both
transport and execute genetic instructions for everything the virus needs to
invade and hijack its host. As more and more links to disease are discovered,
the quest for drugs that target RNA is intensifying.
Searching for such drugs is not a simple matter, however.
Most of today’s drug-hunting tools are designed to find small molecules that
bind to protein targets, but RNA is not a protein, and it differs from proteins
in many key features. “So there’s a growing need for high-throughput
technologies that can identify compounds that bind RNA,” said Hashim M.
Al-Hashimi, the Robert L. Kuczkowski Professor of Chemistry and Professor of
Biophysics at U-M.
Al-Hashimi and coworkers adapted an existing computational
technique for virtually screening libraries of small molecules to determine
their RNA-binding abilities. In this approach, the shape of a target molecule
is first determined by x-ray crystallography or NMR spectroscopy; next, researchers
run computer simulations to compute how well various small molecules nestle
into and bind to the target structure. RNA presents a major challenge to this
methodology because it doesn’t have just one configuration; it’s a floppy
molecule, and depending on which small molecule it binds, it can assume vastly
different shapes.
It once was thought that encounters with drug molecules
actually caused RNA’s shape changes, and that it was impossible to predict what
shape an RNA would adopt upon binding to a given small molecule. However, in
earlier research, Al-Hashimi’s team challenged this conventional
“induced-fit” concept by showing that the RNA, on its own, can dance
through the various shapes that it adopts when bound to different drugs. The
team discovered that each drug molecule simply “waits” for the RNA to
morph into its preferred shape and then latches onto it.
The researchers’ previous work involved creating
“nano-movies” of RNA that capture this dance of shape changes. In
this new study, the researchers froze individual “frames” from the
nano-movies, each showing the RNA in a different conformation, and subjected
each of them to virtual screening. To test the method in the “real
world,” they first tried it on compounds already known to bind a
particular RNA molecule from HIV called TAR.
“We showed that by virtually screening multiple
snapshots of TAR, we could predict at a useful level of accuracy how tightly
these different compounds bind to TAR,” Al-Hashimi said. “But if we
used the conventional method and virtually screened a single TAR structure
determined by x-ray crystallography or NMR spectroscopy, we failed to predict
binding of these drugs that we know can bind TAR.”
Next, the researchers tried using the method to discover new
TAR-targeting drugs. They screened about 51,000 compounds from the U-M Life
Sciences Institute’s Center for Chemical Genomics. “From this relatively
small compound library, we ended up identifying six new small molecules that
bind TAR and block its interaction with other essential viral molecules,”
Al-Hashimi said.
What’s more, one of the six compounds, netilmicin, showed a
strong preference for TAR.
“Netilmicin specifically binds TAR but not other related
RNAs,” said former graduate student Andrew Stelzer. “We were very
pleased with these results because one of the biggest challenges in
RNA-targeted drug discovery is to be able to identify compounds that bind a
specific RNA target without binding other RNAs. The ability of netilmicin to
specifically bind TAR provides proof of concept for this new technology,”
said Stelzer.
Further experiments showed that, for the six potential drug
molecules, the method not only successfully predicted that they would bind to
TAR, it also showed—with atomic-level accuracy—where on the RNA molecule each
drug would bind.
Al-Hashimi then turned the six drug candidates over to David
Markovitz, a professor of infectious diseases at the U-M Medical
School, who tested them
in cultured human T cells infected with HIV. The point of this experiment was
to see if the drugs would prevent HIV from making copies of itself, an
essential step in the disease process.
“Netilmicin did in fact inhibit HIV replication,”
Markovitz said. “This result demonstrates that using an NMR spectrometer
and some computers we can discover drugs that target RNA and are active in
human cells.”
In addition to testing compounds in existing molecular
libraries, the virtual screening technique can be used to explore the potential
of new compounds that have not yet been synthesized, Al-Hashimi said.
“This opens up a whole new frontier for exploring RNA as a drug target and
finding new compounds that specifically target it.”
The enabling technology has been exclusively licensed to
Nymirum.
Filed Under: Drug Discovery
