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Using
RNAi To Screen for Drug Targets in Metabolic Diseases
Michael
P. Czech, Ph.D., is a Professor and Chair for the
Program in Molecular Medicine at University of
Massachusetts
Medical
School.
Dr. Michael P. Czech and his colleagues
recently used RNAi as a screen to find putative drug
targets. One study, published in PNAS,
linked MAP4K4, a protein kinase, to the insulin
sensitivity pathway. The other study, published in Journal
of Clinical Investigation,
hit upon a protein called RIP140, a co-repressor that
regulates metabolic genes and pathways in fat cells.
As Czech describes to Pharma DD, below, both these proteins play very interesting roles in biology.
Obesity
and Type 2 Diabetes Are Locked In a Vicious Cycle…
The
context for our work is the fascinating
interrelationship between obesity and type 2 diabetes,
which have truly become pandemics. Insulin resistance
occurs when muscle--and even fat--become resistant to
the action of the hormone insulin, whose role it is to
maintain blood glucose at its normal concentration.
Type 2 diabetes is caused by chronic insulin
resistance, caused by obesity that over time, in some
individuals, leads to failure of the beta cell, which
secretes insulin to overcome insulin resistance. It is
a feed-forward and feedback mechanism all in one.
The
objective of our work was to discover novel genes
encoding proteins operating in the pathway of insulin
signaling and, by inference, insulin resistance. We
wanted to uncover new molecular mechanisms by which
this relationship between obesity and type 2
diabetes/insulin resistance operates.
…Enter
RNAi to Remediate
To
do that, we exploited RNAi-based technology, choosing
RNAi as our tool because it selectively and
systematically silences genes to determine the effect
of knocking them out. We were able to harness and
miniaturize RNAi technology to a high-throughput
screen in one of the master regulator tissues of the
body--the fat cell (or adipocyte). Adipocytes control
insulin sensitivity--even in muscle--by secreting
peptides that act as hormones, as well as fatty acids.
Using RNAi, we knocked down about 1,000 genes, one by
one, in cultured fat cells to see which ones caused a
change in insulin signaling or other metabolic
pathways in fat cells involved in insulin resistance
(and therefore potentially regulating whole-body
insulin systems in the muscle). We got a number of
very exciting hits.
RIP140
Silencing Upregulates a Multitude of Genes
The
first hit was a transcriptional co-repressor called
RIP140. When we knocked it out using RNAi, to our
amazement, we found that large numbers of genes were
coordinately upregulated. To a large extent, those
upregulated genes were ones that regulate fatty acid
oxidation as well as insulin sensitivity and glucose
transport into the fat cell. Knocking out RIP140
created a fat cell with much stronger oxidative
metabolism, increasing the oxidation of both fat and
glucose. We analyzed the genes regulated by RIP140 and
found they include many mitochondrial genes, genes
involved in glycolysis and the TCA cycle, et cetera,
which become upregulated when RIP140 is knocked out.
In summary, this screen uncovered a novel co-repressor
that regulates metabolic genes in fat cells, as well
as pathways that regulate the release of fatty acids
into the blood from the fat cell and control the
uptake of glucose into the fat cell.
We
hypothesized that RIP140 is a major regulator of
oxidative metabolism that might, in the intact body,
regulate an animal’s propensity (a mouse in this
case) to become more or less obese on a high-fat diet.
We collaborated with Malcolm Parker’s group in the
UK
, who had studied this gene previously with respect to
reproductive biology. Parker’s group found that when
RIP140 is knocked out in a mouse, the animal fails to
gain weight even when on a high-fat diet. Their
finding corroborated our own findings from the RNAi
screen we did in the isolated adipocyte.
Can
RIP140 Knock Out Obesity and Type 2 Diabetes?
We
went beyond that in our Journal
of Clinical Investigation article. Again
collaborating with Malcolm Parker’s group, we showed
that the RIP140 knockout mouse, in addition to being
resistant to weight gain, also showed a very nicely
improved glucose tolerance. When glucose was given to
the knockout mouse, it was able to remove glucose from
the blood much more quickly than the wildtype animal
when the animals were fed a high-fat diet. This
finding was very exciting because again, it is
consistent with the idea that RIP140 controls
whole-body glucose uptake in tissues, potentially
muscle, and definitely fat cells. Therefore, RIP140
regulates not only the propensity to become obese but
also glucose tolerance.
Within
this collaboration, we were able to go all the way
from an in vitro
screen and a hit, to in
vivo metabolism. Now we’re off and running on a
sheaf of questions related to how RIP140 works, what
it does to whole-body metabolism, and most
importantly, what it does in human fat cells. We’re
currently running studies that use human fat cells
obtained from patients undergoing gastric bypass
surgery. In these experiments, we’re looking at the
effect of knocking down RIP140 under those conditions.
We
are testing the hypothesis that in humans, RIP140
could be a therapeutic target. Using the conventional
small-molecule approach, we can see if RIP140 itself
or other proteins that work with it bind a
small-molecule drug that would inhibit RIP140’s
co-repressor activity. If such a small molecule could
be found, it might be a very potent antiobesity and/or
antidiabetic drug.
One
caveat is that RIP140 doesn’t appear to be a classic
protein that would bind small molecules. Therefore,
the conventional small-molecule approach might not
work. It’s certainly worth a try, but because RIP140
may be more “difficult” than a typical protein
drug target, a second approach would be to use RNAi
itself as the therapeutic. According to this approach,
RNAi would be used initially in mice, and ultimately
in humans, to silence RIP140 for a beneficial effect,
just like we did in the original screen. We
hypothesize that knocking down RIP140 in some part of
the mouse adipose tissue would cause a whole-body
effect like we saw in the entire knockout. The data
suggest it’s an interesting hypothesis because when
RIP140 is knocked out in adipose tissue in
vitro, there is a huge increase in oxygen
consumption, essentially converting the adipose tissue
from fat depositing to fat burning. We’re hoping
that will also be the case in
vivo, in which case it would be possible to create
a part of the adipose tissue that is highly oxidative
and burns fatty acid, thereby increasing energy
expenditure and hopefully increasing the propensity
toward leanness despite a high-fat diet. We’re
collaborating with other investigators to develop new,
stabilizing chemistries for RNAi that can also deliver
RNAi to specific tissues (adipose tissue, in our
case), furthering our goal to ultimately be able to
use it in humans. If successful, this would convert an
in vitro set
of findings to something with enormous clinical and
commercial impact.
MAP4K4 Acts on PPARg
To Enhance Adipocyte Function
Using
RNAi as a screen, again in cultured adipocytes, our
other hit was the protein kinase MAP4K4, which appears
to be involved in the stress-response pathway. It was
previously thought this pathway is also involved in
inhibiting insulin sensitivity. But prior to our
findings, no one had ever definitively connected
MAP4K4 to the pathway of insulin sensitivity. We
subsequently knocked out, one by one, every other MAP
kinase that is known to be expressed in adipose
tissue, and we found that none other had the dramatic
effect that MAP4K4 has.
We
discovered this hit has fascinating biological
properties. When MAP4K4 is silenced, it acts on
transcriptional factors such as PPAR gamma, which is a
known proadipocyte that enhances the adipocyte’s
ability to deposit and burn fat. PPAR gamma is one of
the major drug targets for diabetes; TZD drugs (e.g.
rosiglitazone is a marketed TZD for treatment of
diabetes) act on PPAR gamma. When MAP4K4 is knocked
out, the adipogenic PPAR gamma works better, resulting
in fat cells that store and burn fat very efficiently.
We are excited about testing that hypothesis by
knocking out MAP4K4 specifically in fat cells in the
mouse. Because a full-body knockout of MAP4K4 is
lethal in a mouse, we have to do a conditional
knockout in the adipose tissue only in order to test
our questions: Is MAP4K4 a drug target? Does knocking
it down in vivo have a beneficial effect? Ultimately, it would be a great
thrill to see this hypothesis--were it to be
correct--extended into human studies.
RNAi
Is a Broadly Applicable and Powerful Tool…
There
are ongoing screens using RNAi in various diseases,
and work employing RNAi has been published in many
different fields. The application of RNAi technology
is broad. Not only did the discovery of RNAi reveal a
novel, exciting biological process in all cells, it
provided us with a tool that can be widely used in all
types of disease. It has fantastic benefit and
potential for impact in screening and ultimately for
use at the therapeutic level.
RNAi’s
greatest advantage is that it can be used very
selectively and systematically to silence genes. It is
arguably the major biological discovery of the last 30
years. It has transformed the way people do biology.
It is applicable to many different disease processes,
many different cell types, and it is selective in
knocking down only the desired gene, making it
possible to isolate and dissect hypotheses and attack
them very specifically.
…But
Has Its Limitations Too
Working
with RNAi does have it challenges, both in
vitro and in
vivo.
In vitro, the challenges are related to off-target effects, which
occur when the RNAi works by a nonspecific mechanism.
That can happen, but control experiments can be done
to test for that. These control experiments prove very
valuable in data interpretation. While RNAi is a
spectacular tool, it must be done within the context
of careful controls and judicious data interpretation.
On
the in vivo side, there are major
hurdles to be overcome. One problem is related to
RNAi’s stability--serum and even tissues rapidly
degrade naked RNA. Therefore, modifications may have
to be made to render the RNAi stable for days, weeks,
and eventually, hopefully, even longer. There is also
an RNAi delivery problem—following injection into a
particular tissue type, it might not knock down the
same gene in every tissue, which could have adverse
effects. Those are the challenges we have in using
RNAi, but fortunately labs around the world are
working to address them.
Quite
a bit has been published on RNAi chemistry. Terrific
work has been done toward extending the length of time
that RNAi can work in
vivo, by making modifications to it. There are new
data from many labs that have either modified the RNAi
or conjugated it with other types of molecules that
can target tissues in a specific way. Here at
University of Massachusetts Medical School, there is
work being done on the delivery challenge, which uses
nanoparticles coupled with the RNAi to achieve
delivery. So there has been great progress, and
publications that document this are beginning to
appear from labs worldwide.
Dr.
Czech will be a speaker at CHI's Molecular
Medicine Tri-Conference, which will be held
February 27-March 2, 2007 in San Francisco.
URL:
http://www.pharmadd.com/exclusivecontent/RNAi.asp
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