Jetze Tepe

Jetze Tepe


– And up next we have Professor Jetze Tepe and he’s a chemistry
professor at Michigan State, and his research is on the synthesis and use of natural
products and their analogs for modulating proteasome activity. And today he is going to speak to us about therapeutic small molecule
proteasome activation. – Thank you Greg, I want to thank the organi
for giving me the opportunity to talk a little bit
about the research that we’re doing in our lab. And as Dave Washburn already said, some of these patent royalties get used by the foundation to
sponsor certain projects. The project I want to tell you about today is one of those projects
that’s been funded in part by the MSU foundation through the strategic partnership grants. The disease we’re focusing on in our lab is multiple myeloma, and multiple myeloma is a cancer
of differentiated B-cells. It’s therefore primarily
found in the bone marrow and where the multiple
myeloma cells really feed on the micro environment
of the bone marrow. As you see here in the slide, if this is a multiple myeloma cell, there are several pathways
that are over expressed or constitutively expressed
in multiple myeloma cells. One of the pathways that
drives growth and proliferation is the NF Kappa B signaling pathway. This pathway is activated
by certain cytokines in which IL 6 is one of
the primary cytokines that activate this pathway. What you see in a bone marrow environment is that the multiple myeloma cells have activated NF Kappa
B which then induces the description of IL 6. So these cytokines in part then go on and activate bone marrow stromal cells which also again activates the NF Kappa B signaling pathway in the stromal cells. Once NF Kappa B is activated here again it induces a wide
range of cytokines, IL 6 being one of the
primary cytokines again, as well as RANKL, which is the receptor activator
for the NF Kappa B ligands, those will activate the osteoclasts, and the osteoclasts
once they get activated they actually start to
decompose, or destruct, the bone density in your skeleton. The osteoclasts, again you
activate this NF Kappa B pathway which induces IL 6 which
then induces NF Kappa B again at your multiple myeloma cells. So what you see here is in this cycle, this vicious cycle in your
bone marrow environment you have these multiple
myeloma cells activated NF Kappa B inducing
growth and proliferation, as well as survival of the
multiple myeloma cell itself, and at the same time the osteoclasts will start to degrade your bone density. So you get this
combination of tumor growth and degradation of bone
density which is obviously a very bad combination. About 30,000 patients are
diagnosed per year in the U.S. The current survival is
a little bit under 50% in five year survival. The standard treatment as of 2003 has been proteasome inhibition
by primarily Bortezomib, there’s two new drugs on the market I’ll talk about in a little bit that are currently taking over the
market from Bortezomib, but it’s really the
standard front line therapy for multiple myeloma. The biggest problem in
this disease is after about five years almost
all patients become resistant to treatment and after that there’s really
no follow up treatment. Bortezomib itself will
inhibit the degradation of this inhibitory protein for NF Kappa B. Bortezomib blocks the proteasome and therefore NF Kappa
B cannot be activated, cannot induce the intrascription
of these cytokines so you really in effect
block that signaling pathway of NF Kappa B and you block the growth of the multiple myeloma and as a side effect, you actually also prevent the degradation of your bone density. If you look in more detail
about the way Bortezomib works, it targets the proteasome
which is shown back here. The proteasome itself is this very large barrel type structure, inside of this barrel
you have catalytic sites shown back here, Beta one,
Beta two, and Beta three. Those are proteolytic sites, so the proteasome will degrade proteins, such as I Kappa B at these catalytic sites inside of the barrel. You have these, the
barrel shown back here, you have these alpha ring, the
top ring of the proteasome, and that has a gate that
controls the gate opening that provides an access
to those proteolytic sites inside of the proteasome itself. Typically this gate is closed and so proteins cannot be
degraded by the proteasome unless these caps, not our multi subunit
complex that will actually dock on top of the proteasome. It has these small peptide
chains, hydrophobic chains, that dock into small pockets
on top of that alpha ring and it will induce this
ring opening to allow access to the gate and
the catalytic sites. These caps will actually
recognize the proteins that need to be degraded in the cell. So proteins will get ubiquitinated and this gets induced by
a variety of different protein modifications that result in the ubiquitinilazation of these proteins. Once the protein is
ubiquitinilated it’s those caps that will recognize the
ubiquitinilated proteins and it will unfold the
protein as shown back here, push it down it’s barrel
and inside the barrel, the catalytic sites will
then degrade these proteins. So Bortezomib structure shown back here, Bortezomib of a small peptide with an electrophylic head group, it’s that head group that
will actually covalently bind to those catalytic sites and therefore block proteasome activity. So when a patient is
treated with Bortezomib, you block degradation of all proteins that need to be degraded in the cell. It’s a very effective way
of inducing cell growth, but as you can imagine, it’s also relatively toxic treatment. There is currently two
other drugs on the market, Carfilzomib, it’s much more
tolerated by the patient, and Ixazomib it’s really
the same structure show back here but it’s a pro drug, it’s actually orally available, so it’s likely gonna take
over from Bortezomib. Again, as I said earlier, the biggest challenges right
now is that all patients will become resistant eventually
to Bortezomib treatment. And since all these drugs operate through the exact same mechanism of action, there really is no follow up treatment once a patient becomes
resistant to Bortezomib or any of these proteasome inhibitors. So what we wanted to do was
try to find new molecules that would block that
vicious cycle of NF Kappa B mediated tumor growth as
well as bone destruction. So what we did is we looked at a variety of different natural products, these marine sponge
metabolites that in literature were found to inhibit cytokine production or look like molecules
that inhibit NF Kappa B. This was our starting
point to try to discover new types of proteasome or new types of NF Kappa B inhibitors. So the group itself synthesized
a variety of these molecules and we’re still working on some of these molecules in our lab, but instead of just
synthesizing these molecules, one of the strategies we had
was to make small molecules that kind of resembled these
kinds of natural products. Instead of making a
complex natural product, if I can make a small
scaffold that looks like some of these natural
products, as shown back here, I should have access to a small molecule in a short amount of time and I should be able to
optimize it very quickly for it’s properties. So many of the students over
the years have developed new reactions to access
these small type of scaffolds that in fact resemble
these natural products. Once we’ve made the
molecules we then test them in luciferase assay so for this we used a cell that has a plasma
that expresses luciferase. Luciferase is probably something
everyone has seen before, it’s the same enzyme that’s
present in fireflies, so what happens if we treat
cells with a cytokine, such as IL 1-6 or TNF Alpha, we activate this NF Kappa
B signaling pathway, NF Kappa B becomes activated, translocated into the
nucleus, binds to the DNA, and in this case expressed
this protein called luciferase. Once luciferase is expressed
it will transfer luciferin into oxyluciferin and
it will give off light and we can actually
measure NF Kappa B activity by the light that comes off the cells. If we then treat the
cells with the different types of molecules if we see a decrease in the activity of luciferase activity, we know that somewhere along this pathway we effect this NF Kappa
B signaling pathway. This is the molecule
imidazoline that we’ve been focusing on a lot over the years, and what we see is that
the molecule blocks that signaling pathway
relatively effectively. Here’s the structure shown
back here, it’s TCH-13, if we treat these cells
with various doses of TCH-13 we see an inhibition of NF
Kappa B luciferase activity with an IC-50 of around
one point six micromolars. We subsequently did a whole
bunch of other studies until we got to taking blood
samples from not patients but healthy volunteers who gave use blood, and we then activated
the blood with a cytokine to activate the NF Kappa
B signaling pathway and then we measured
cytokines such as IL-6 or TNF Alpha to see if we actually in a more complicated system blocked the NF Kappa B signaling pathway. And you can see here that we
have very reasonable IC-50s when we exposed them to TCH-13. So at that point we knew we effected the NF Kappa B signaling pathway, we could block cytokine
production through NF Kappa B and we started to look
more in exactly where in this pathway of NF
Kappa B activation did we block or where was our target. As we see here in this picture, cytokines will induce post
translational modifications to I Kappa B and I Kappa B
is the inhibitory protein that sequesters NF Kappa
B in the cytoplasm. Only when you have these post
translational modifications occurring on I Kappa B,
including ubiquinilization, it gets targeted by the
proteasome for degradation. The proteasome will subsequently degrade, or eat up the I Kappa B protein, therefore liberating NF Kappa B which then induces its gene expression. So in one of the assays what we found if you look at just I Kappa B, we can stain that with
antibodies shown back, all the red that you see here
in the cell is I Kappa B, if you then treat it with a
cytokine such as TNF Alpha, within about 10 to 20 minutes you see all the I Kappa B disappearing
meaning it’s being degraded by the proteasome. If you then add in TCH-13 you can block that degradation by that cytokine. So what we found here in the assay, and a variety of other
assays that we described more in this paper, is that these molecules block
the degradation of I Kappa B. And so that led us
further to the proteasome which is responsible
for the degradation of that inhibitory protein. As we started to look for the proteasome as a possible target we found
something very interesting. If you look at the proteasome
itself it has these three catalytic sites. There are probes that bind
specifically covalently to these three catalytic sites. And these probes are shown back here, this is a molecule that has an
electrophylic site back here so these catalytic sites, the threonine in that
site will actually attack that position and you
covalently bind that probe to the catalytic site. On the other end of this
probe you have biotin so you can stain the
protein for an antibody for that and you can actually
visualize the catalytic site as shown back here. So if you take the proteasome itself and you run it down a gel, and you expose it to
this probe you can see the beta two, the beta one, and
the beta five catalytic site now covalently bound to that probe. However what we found when
we treated the proteasome first with our molecule, we still saw that this
probe very effectively still bound to that catalytic site. However if you treat
something like Bortezomib, we see that we covalently
block that site from binding. So what we saw here was
that our molecule did effect the proteasome and did prevent
a degradation of I Kappa B, but really did not
interact at all with these catalytic sites for the proteasome. And that was something
very new at the time when we first discovered that. So we didn’t know what the mechanism was, we knew that it effected the proteasome, we just did not see any
of the traditional ways of inhibiting the proteasome through and interaction with the catalytic sites. This mechanism translated very
well in both in cell cultures as well as in vivo studies. Here we see a range of leukemia
and multiple myeloma cells. We effectively killed them and we had some good cell activity for the bone marrow stromal cells or the normal cells that we
tested against these compounds. But I think more importantly, if you look at resistant cell lines, so this is a THP-1 cell
line with different levels of resistance to Bortezomib, the current leading drug, you see that you go from
eight nanomolar inhibitor to a micromolar inhibitor there. So those cell lines are very
resistant to Bortezomib. If you treat those cells
lines with our molecule, even though we don’t have the
potency what Bortezomib has, you see that we’re completely overcoming that resistance to Bortezomib. Which again would imply that
the mechanism of inhibition is very different than what we see with traditional proteasome inhibitors. We then subsequently treated
mice that had multiple myeloma tumors with a large dose of TCH-13. This was done by IP injection, here you have the tumor growing over time and both Bortezomib and our
molecule very effectively blocked the tumor from growing. So at that point we started
to look at a wide range of different imidazoline, see if we could optimize the activity, see if we could make them
a little bit more potent. And to do so we developed a
variety of different reactions, I won’t go into the details of
the scope of these reactions, but we just needed to
develop new reactions to access different types
of imidazoline scaffolds. We are currently still
working on new reactions to make these types of imidazolines that again can access
different functionalities that we cannot access
with the current methods. These molecules are then
tested now that we know the proteasome is a target
in an in vitro assay where we take the proteasome, we have a small peptide
chain that is functionalized with a fluorescent probe, if the proteasome is active it will digest or cleave this small peptide and the release of the probe will indicate that we have an active proteasome. If you inhibit it you
will obviously inhibit the release of the probe and
not see the fluorescence. What we found with this molecule, TCH-165, is that instead of
inhibiting the proteasome, we actually saw that we
activated a proteasome. And that is something
that was very very new. There really have been
no proteasome activators reported as of recently, and what we see here is
when we take the proteasome, treat it with our molecule, we actually get a very strong increase in the proteolytic activity
of the proteasome. So we can alisterically
activate the proteasome. Double the concentration
of the proteasome activity of each of the three catalytic
sites are shown back here. We get about eight to ten fold increase in proteasome activity. This only worked for the 20 S proteasome and we don’t see any effect
on the 26 S proteasome, and that is the one
that has been assembled with these caps. So wanted to look more into
the mechanism of action of these molecules and
the person really that spearheaded the study is Eva, who is in the audience
today, I’m not sure where, she’s right there, so she did a lot of this work
I’m gonna be showing you next. We worked also with another group in University of Texas health science center, Maria Gaczynska, whose using
atomic force microscopy to actually look at the proteasome. In atomic force microscopy
you can use a small needle that actually taps over the surface of a biological molecule and
you can actually outline the surface of whatever
you’re investigating. Because of the size of the proteasome, it’s a very large complex
you can actually do this on the proteasome itself. And so what we see here
is an actual outline of the alpha ring, of the
cap shown back here, of the proteasome. If you then treat the proteasome
with this molecule TCH-165 you can start seeing that
this gate actually opens up. So if you add in a
higher dose of molecule, a higher dose of TCH-165, you see more particles
with the open gate itself of the proteasome. Cory Jones is another
graduate student in the lab whose doing a lot of modeling and he really tried to find out what could possibly be the mechanism of
this gate opening of TCH-165. What I’m showing here is
the result that he received when he docked TCH-165
to the proteasome itself. And what he found was that these molecules dock in those small hydrophobic pockets in which these caps
will actually dock into. So as shown back here, typically in the body the 20 S proteasome is in equilibrium with
the 26 S proteasome, these caps will then dock
these small peptide chains into the grooves on top of this alpha ring and therefore open up the gap. What he seen is that TCH-165 actually docks in those hydrophobic pockets. We can actually test this experimentally, we can make these small peptide chains and then do competition experiments and so when we look at the RBT chain, this small peptide terminal chain, we can actually see that in
the presence of this chain we will actually block the TCH-165 from activating the proteasome. So at this point we
think that this molecule blocks on the alpha ring and prevents, or blocks in one of those openings and opens up the gate of
the proteasome itself. Eva then tried to look at the
assembly of the proteasome, because if the small
molecule actually blocked on that alpha ring the question was does it actually effect
the caps from docking onto the proteasome itself. And that’s exactly what we’re seeing here, so this is a western blot
where we look at the 26 S proteasome, it’s shown back here, and this is a double cap and a single cap means you only have one of these caps on either the top or the
bottom of the proteasome. Here is the cap itself,
here is the 20 S subunit and these are additional
controls showing that we have the same amount of proteasome
in each of our lanes. And so what we see if we
increase the dose of TCH-165 in cells that we actually
get a disassembly of the proteasome where we
end up with only the 19 S caps and the 20 S proteasomes by itself. So in terms of proteasome
activation we see that this molecule can
actually activate the 20 S core of the proteasome and it will
also drive this equilibrium to generating more of
the free 20 S proteasome. Now why is this important? That’s really I think the
most important question. All proteasome inhibitors
thus far just reacted with the catalytic site. If we could change this
equilibrium from this form to the small form we
actually have a completely different cellular mechanism. What we see in cells themselves, this equilibrium is actually dictated by the cellular environment. Meaning that if a cell is
exposed to, for example, oxidative damage, this
fully assembled complex will disassemble to form more of
the 20 S non assembled complex. And the reason for that is
that this unassembled complex does not have these 19 S caps, it will therefore not be able to recognize ubiquinilated proteins,
it cannot unfold proteins, but what this catalytic unit can do is degrade proteins that
are already unfolded. And so these are what we call intrinsically disordered proteins. When a cell of course oxidative stress, proteins get oxidized,
proteins get damaged, and they will start to unfold, and therefore the proteasome
will quickly disassemble to form more of the 20 S
proteasome to start degrading those unfolded proteins. Once unfolded proteins
start to accumulate in cells they start to aggregate and causing neuro
toxicity or auto toxicity. So typically speaking,
these disordered proteins are only present in very
short amount of times if you can detect them at all, because when a protein is not unfolded it’s typically immediately
degraded by the 20 S proteasome. Couple of examples of these unfolded or disordered proteins
are shown back here, as I already said
oxidatively damaged proteins have a tendency to be unfolded. Alpha nucleinated is not a protein that’s an unfolded or disordered protein. TAO, SOD-1, beta amyloids, and I assume a lot of
these are very well known targets for neuro degenerative diseases. Meaning that if these proteins accumulate they start causing neuro degeneration. Other proteins more
involved in cancer growth are ornithine decarboxylase,
c-Fos, and c myc, those are all proteins that are also intrinsically disordered. What that means is when you have a cell and it starts to express
for example a large amount of c myc to start to transform the cells from a normal cell to a cancer cell, it starts to drive tumor growth and it also has a major role
in relapse of patients as well. Same with c-Fos, c-Fos
also a very strong effect, once you start over
expressing c-Fos you get enhanced inflammatory
responses and growth as well. So anytime these pathways
over express themselves we either have the cell
will start to induce cancer or you will start to have
neuro degenerative diseases. We tried to test this to
see if these molecules will actually start to target those over expressed disordered proteins. And what Eva did in the
group is that she fused a structured protein with
a disordered protein. If we now express this in a cell, if you activate the 20 S proteasome, you should selectively start to target your disordered proteins and you should not effect
your structured proteins. And therefore you would have
fragments of different size if you start activating
the 20 S proteasome. And that’s exactly what she saw, when you take cells and you
treat them with TCH-165, you start having this large
fused complex over time being degraded to smaller fragments. If you then treat it first with Bortezomib you would block that degradation. So this is one of the
slides that show that we are selectively activating
the 20 S proteasome and selectively degrading
only disordered proteins and not structured proteins. And we see here we have a
nice dose response as well. If you then look at other
proteins such as c-Fos, c-Fos is a protein that’s
involved in cell proliferation, so protein that’s
involved in the formation of AP-1 induced cytokine production. It’s also a disordered protein, so when you now treat it with our molecule you start to get a complete degradation of that protein in cell culture. If you block the proteasome again, you would then block that
induction of degradation so therefore you don’t
see it’s mechanism at all. We have no effect GAPDH
is a structured protein and we see have no effect
on structured proteins. So what it comes down to is this molecule will selectively induce
one part of the proteasome and will start to target proteins
that are already unfolded. This translates very well in
cell culture so if you look for example in a normal cell
line we have no toxicity, if you look at a multiple
myeloma cell line we get IC-50 of about one
point six micromolars. This is a glioblastoma cell line and we have about two micromolars IC-50. The nice thing about this molecule it’s actually orally
available so when we take it into a mouse model we see here this is a multiple myeloma mouse model, we see that the tumors are
growing in the vehicle control and again we block tumor growth after oral with a 100 micromolars per kilogram. The exposure is very good at
one point four micromolars, again that’s the c max, it kind of matches the IC-50
we obtained in the cell line. Recently we also had
the opportunity to work with a veterinary school
so in the labs Yuzbasiyan Gurkan what she did is she’s focusing on Bernese Mountain Dogs. Now the reason that we’re
focusing on Bernese Mountain Dogs is that these dogs, there’s a Bernese Mountain Dog right here, at the age of about seven
years they start to get a disease, a c-Fos driven disease, which again is a disordered protein, and there’s really no treatment
option for these dogs. So after about seven years if
the dogs have been diagnosed they actually have approximately
80 to 90 days left to live. There’s no treatment options,
it’s a very aggressive cancer. She tested our compound in a
variety of histiocytic sarcoma cell lines and found that
of the many compounds she has tested, many
chemo-therapeutic she’s tested, this was one of the only
ones that would actually able to kill these
histiocytic sarcoma cells. So we then pushed this forward and tried to match the
efficacy that we had, or at least the PK that
we had in the mice, we treated the dogs with
50 milligrams per kilogram of TCH-165 to match
approximately the same c max as well as the curve is about the same at the 50 milligrams per kilogram in beagles. After we did that we looked specifically at the toxicity of this approach, what we saw here is that
by clinical observations or by body weight, or we
did a complete blood panel on the treated dogs, we saw that the blood count as well as the clinical chemistry
showed no changes at all compared to the pre treated animals. So at this stage we have given the dose, what we think based on my studies, an effective dose, but we
see no toxicity as of yet. Currently we’re scaling the
molecule up to a larger scale. Unfortunately these are very large dogs and we need a lot of material for and I wish they were
chihuahuas but they’re not. So we’re scaling this up and we’re initiating a clinical
trial at the MSU vet school in this particular dog which hopefully will start at the end of October. With that I would like to
thank a lot of the students who obviously have been
working on this over the years. A couple I want to point
out, Terry Landstill, she really did all the
original work on THC-13 when she worked in the group. Adam Mosey is shown back here, he made TCH-165 for the very first time. Eva did all the biological
studies I’ve shown back here. Corey did all the modeling studies and then we have several
other people still working on the synthesis of more
drug like molecules, try to still increase the
potency of these scaffolds. I want to thank the NIH
and also again the SBC, the strategic partnership grant that had funded some of this work over the years, and I’d like to thank
you for your attention. (applause) – [Greg] Thanks Jetze, do we have one quick question for Jetze? And if not we can head off
to the, oh there’s one. – [Audience Member] What
do you think will be the mechanism for the
resistance for Brotezomib. – So that’s a great question. In the cell lines I’ve
showed you right here, it’s because of mutations
in the catalytic sites. In patients it’s not sure
what the resistant yet. It’s a very heterogeneous tumor. So it could be, it’s a wide range, it’s dependent on the micro environment, it starts to regulate some
of the catalytic sites like the B 5 subunit is over regulated or over producing cells in
some multiple myeloma patients. But there is not one specific
resistance mechanism, there’s multiple different ones. – [Greg] Okay, let’s thank Jetze again. (applause) So now we’ll move onto a break and it will be a full 15 minute break, we’re about 10 minutes behind right now but we’ll take the full 15 minutes. So please come back at 10
minutes after 4 o’clock.

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