Symposium 2014: Targeting Mutant KRAS with Small Molecules

Symposium 2014: Targeting Mutant KRAS with Small Molecules


[ Silence ]>>So first speaker of today’s from UCSF,
Kevan Shokat. He’s a chair of Cellular and Molecular Pharmacology. He’s also affiliation
with Department of Chemistry at Berkeley. He’s a member of the National Academy of Sciences,
the Institute of Medicine, the American Academy of Arts and Sciences and I think all of the
typicals, there are two things that are two things that actually show can Kevan Shokat
is in this type of research, he’s a passionate researcher. I think he will never give up
until it’s done. Utilizing chemistry synthesis and protein engineering to investigate signal
transduction pathways into to really understand how do they communicate with each other, but
how also can you utilize them to design new very intelligent drugs. And in recently I
think some started to shed some light how this can be done. But he’s also an entrepreneur
and bringing his discoveries to clinical market which I think is important because at the
end we only use it to treat patient. So the floors to you for your talk on small molecule
inhibitor of KRAS G12C.>>Thank you, [inaudible], thank you very
much. Well, great. Well, welcome. That’s for–thanks for Laura, Frank, Kevin and Martin for putting
together the meeting. And before I start I want to make sure that sort of bring out one
thing Frank said that it’d be great if during the meeting people interrupted with any questions
and we sort of the next day and a half we really dig in to RAS I think in a way that
Frank really envision so don’t hesitate. You don’t have to raise your hand, just start
speaking over me and interrupt me. I would love that. I would love that. And I also want
somebody to keep track of how many times Frank says his favorite word. I think you all know
what that word is so, all right. We’re going to have a counter up here. So we will have
a world cup scores and then Frank’s tabulation. All right. Well, I want to build on many of
the points that Frank made and I think we all aware of how important mutations are in
driving cancer and how important of a signal that is for guiding toward discovery. We know
from much of the success of targeted therapies that once a kinase is activated in cancer
such as Bcr-abl that provides a fantastic direction from the distal chemistry and drug
discovery, creating a drug like Gleevec which shuts down the pathway that is aberrantly
activated in the tumor cells and then by shutting that off because the tumor cell is dependent
upon that signaling pathway, the tumor cells, apoptos and we have a fantastic therapeutic
index. I think we all know how this is a much, much oversimplified picture of how lucky we
are that drugs work in this way and one point that I think we often forget about is that
we don’t necessarily understand the true nature of oncogene addiction and why this cell is
truly dependent upon the activity of this mutant oncogene and cannot survive when that
oncogene is inhibited, and I think Julian Downward will tell us more about oncogene
addiction and we’ll understand how much more we have to understand there. From a chemical
standpoint one thing that this slide sort of glosses over is that the inhibitors that
blocked the mutant proteins, also bind to proto-oncogenes. So other thing this–success
of this kind of strategy relies on is that the inhibition of the non-mutated kinase and
the rest of the body has to be tolerated at a dose that affectively kills the tumor cells
and shrinks the tumor. So because small molecule chemistry in the kinase area has not been
able to be successfully developed just the mutant protein and leave behind them non-mutant
protein we have to rely on sort of oncogene addiction and the ability to tolerate inhibition
of the pathway. So that’s a chemical challenge that still has not been–been met in the kinase
field. The other feature is, is that we know these are proteins that operate in pathways
and if we take a pathway view, then we should be able to just inhibit anything in the pathway
to block the aberrant activation of that pathway. Think of Statins targeting HMG CoA reductase
and cholesterol. That’s not the point of the pathway that’s mutated and on for example
but it works very, very well but signaling cascades are much more intricate and complicated
and as I’ll show you, and as we’ll see in many of the talks here how targeting even
one step below the oncogene but right in the same pathway is very, very much different
than targeting the exact oncogene. So, there are much–many more intricacies of how the
pathway works and how the oncogene collapsed the pathway that we have to take into account
when we think of targeting the drug. But the two points I want to make here is that the
simplest and most direct way would be to make a drug that only inhibited the mutant oncogene
and targeted that mutant oncogene level of the pathway rather than somewhere upstream
or downstream. Other strategies might work but we will see that much more complicated.
So kinases are mutated but also as Frank mentioned we’re here about RAS. How are these two nucleotide
dependent enzymes working? At a very basic level they both co-op and use phosphate as
a switch. In the kinase area, increasing the kinase activity is what mutants–mutations
that drive cancer do because activating the kinase is what transfer the phosphate to substrate
which go on to signal downstream. So this is a situation where in the unactivated case,
kinase has have many ways of keeping themselves off and then the oncogene sort of release
that off state and push towards the on-state. Nucleotide dependent enzymes like the GTPases
however, operate very, very differently, almost in an opposite sense. They are nucleotide
state dependence switches so in the GTP state , they’re held down in that very tight confirmation
that Frank alluded to serving as a switch to produce a surface of protein that is complementary
to effector proteins that are downstream. And in this case then the job of the non-mutated
protein and normal signaling is to activate the GTPase activity, releasing the phosphate
and then relaxing the protein so it’s unable to bind. So in one case, we’re going to find
mutants that drive activity, catalytic activity and in one case mutations that cost a lost
of catalytic activity. OK, now if you understand how a GTPase can act upstream and turn on
a kinase what you can then also readily appreciate as Frank mentioned is just by following two
amino acids, really two proteins in one signaling cascade the RAS/RAF/MEK/ERK pathway, you can
appreciate almost 30–I understand how 30 percent of cancer is produced. So in the KRAS
case, we mutate up the glycine 12 or 13 or 61 position that leaves RAS predominantly
in the GTP state producing the surface that binds and recruits and the RAF kinase which
has a RAS binding, effector domain localizing it to the membrane and activating it and this
is much, much more simple, I will see many, many more examples of how complicated this
in fact, is and Frank already alluded to how there’s many pieces of the puzzle we still
don’t understand. In a mutually exclusive way, even without a mutation, at the RAS level,
RAF can be mutated at the valine 600 position, immediately downstream to activate the pathway
at that level and that explains another large fraction of cancers. So with that understanding
of how RAS activates the pathway and how RAF has been activated independently and we know
how druggable Kinases are–we’ve all seen in the last 15-20 years of drug discovery,
you might think that we should go to the list of approved kinase inhibitors, find the RAF
inhibitor because RAF is below RAS, and pick out vemurafenib or now Dabrafenib and use
that to treat RAS mutant tumors. It works in the pathway, it’s immediately downstream
I think by all of the early logic we have even five years ago, ten years ago. It should
have been a fantastic approach. Well, you need a fantastic drug luckily Flexicon and
earlier many, many MEK inhibitors were developed so we had excellent kinase inhibitors downstream
to target the kinases that are activated by mutant RAS. And not only is a good molecule
in vitro but the early clinical success of vemurafenib in patient that had V600E mutations
was astounding. So, from the Flexicon data we knew that about 81 percent response rate
in phase I, in metastatic melanoma patients that have V600E mutations, PET scans before
and after therapy have fantastic responses. So, it’s a fantastic drug, clinically very
active, inhibits the pathway exactly like we would expect. So everything is looking
very optimistic and my point here is what happens when we take this into a RAS mutant
setting? Well, interestingly, unexpectedly in a very surprising way, Neal Rosen at the
time of the phase I clinical trial learned and told me that in phase I there were patients
that were benefiting their metastatic melanoma tumors were regressing but about 31 percent
of patient in phase I were developing Keratoacanthomas another skin tumor and this were in sites
that didn’t seem to have–have tumors previously. So this is completely paradoxical, how would
that work? Neal’s lab in his postdoc at that point glucose developed a cell line model
which mimic this potentially–this surprising paradoxical activation of the RAS/RAF pathway
by the RAF inhibitor. By–I showed you this side of the gel where cells that have a RAF
mutation treated with PLX-4720 block MEK and block ERK, that as we would expect but the
surprising effect is if the compound is added to a cell that a RAS mutation rather than
a RAF mutation. So only wild type RAF. You see a huge activation of MEK phosphorylation
and ERK phosphorylation. So how would that work? How does an inhibitor of a kinase activate
the downstream pathway? And the Genentech Group and Neal Rosen and our group collaborated
independently to reveal a mechanism that might explain how a kinase inhibitor that targets
wild type RAF in the setting of a RAS mutation could activate the pathway. And the way we
believe this works is that as I told you there are intricate mechanisms for kinases to maintain
themselves in the off-state and as Frank mentioned there are many important steps we still don’t
appreciate but one thing we basically agree on is that wild type RAF in the off state
is in some state of a monomer state and then it becomes activated when RAS is activated
and in that dimeric state, further activates and MEK and ERK. We’ll hear from Debra Morrison
[assumed spelling] more about how much more complicate this can be. But what we realized
was that kinases are not just simple catalysts that chug out protein–chug out their product
all the time that they’re in this two state, the off state and the on state. And what we
reason that that kinase inhibitor although would inhibit the activity, it could also
change the confirmation when it bound to the kinase. And in that new confirmation it might
promote the dimerization, and then at certain intermediate concentrations, intermediate
concentrations that were achieved clinically you might be half occupying one of the protomer
and activating the other unoccupied protomer which would lead to hyper activation. So this,
I think illustrates two points. The first is is that with RAS mutations, there is a
little bit of a trickle of activity sometimes and an inhibitor of a downstream kinase can
further accelerate that so that illustrates the point that going even one step away from
the oncogene can be very detrimental, in this case completely paradoxical, not just–not
work but it can actually amplified the pathway. So that’s that–the sort of bad thing. I’d
say the silver lining however, is that small molecules can do surprising things in complicated
signaling networks because these enzymes are regulated at so many levels. So if somebody
had told you, “Hey, in this disease, I want to turn on the activity of a kinase, I don’t
want to turn it off. Let’s say, OK, that seems tough. But then they told you, the only thing
you can use is an inhibitor of the enzyme then you would say, “OK, that’s really crazy,
I can’t even begin to understand I’ll do that” yet that’s exactly how this works. So I think
the point I like to make is that is with careful structural biology, careful cellular biochemistry,
we can really understand new opportunities for chemical transformation of these proteins
to elicit the kind of therapeutic beneficial affects we want. So even though this is causing
another cancer, and largely I think that’s mitigated now by combination with MEK inhibitors
as you might already imagine, it really tells us that the amazing sort of gymnastics that
small molecule inhibitors can elicit in these pathways might give us new opportunities.
I think we need completely new ways to think about chemistry where it attack some of these
problems. Yeah? [ Inaudible Remark ] I think that people believe it has a–they
have a RAS mutation in them and that they go into in senescence state and then the inhibitor
pushes them over but maybe Martin–>>Yes, about 60, it reported 60 percent of
the Keratoacanthoma have an HRAS Mutation.>>HRAS preexisting, great.>>That is how HPV–>>Either way, they–oh OK. And do those–the
HPV ones become also driven by that RAF inhibitors?>>Yes, they do.>>OK, so they’ll multiple anything that will
trickle to give a little bit of signal, the RAF inhibitor will push. All right. OK. So,
so, a little bit back on RAS and the oncogenes, this is just a timeline of sort of the discovery,
fantastic, early investigation of tumor viruses and the isolation of RAS and SARK is primarily
oncogene. And what I think is amazing from the work back then is that although we have
no reason to expect that those would be the prime oncogenes that we’re driving so much
cancer, they did turn out to be this sort of the prime drivers. And with focusing on
those early oncogenes we’ve been able to understand immediately one binds GTP, one binds ATP,
one is a kinase, one is GTPase, then all the different regulators, structures were solved
and this is amazing that RAS is identified very early then turned out to be the most
frequently mutated oncogene in cancer. So I think this–as Frank said prompted much
early you know, efforts on targeting RAS and what’s also surprising is that the kinase
mutants have been much, much more successfully targeted. We now have over 20 different kinase
inhibitors that target oncogenic kinases that have been approved. And although the farnesyltransferase
inhibitors and the early KRAS focus turned out not to be as beneficial, I think we’ll
hear from Herbert Boldman [assumed spelling] that the idea of mislocalizing RAS by relying
on even new aspect of how RAS farnesylation traffics in the cell is going to be a very
exciting new direction as Frank mentioned that maybe a focus on HRAS by this farnesyltransferase
inhibitors could be useful. There’s a lot of excitement still on the farnesylation,
just not as simple as we thought. So what I’d like to focus on thought, what I talked
about in the first two, first slide is that we really need a kind of chemical approach
that focuses just on the mutant oncogene if we could achieve that we would have the chance
for a huge therapeutic index because we wouldn’t be targeting RAS in the rest of the body.
We would target just the mutant that is produced in the tumor cell. So that’s one challenge.
The second is I focused on a lot is that because of the nature of the difference between a
GTPase and a kinase and how the enzymatic cycle works, the competition for the nucleotide
GTP is much, much difficult in the GTPase’s case than it is in the kinase case, will much
more difficult? Is it really that big of a problem? Well, if we look at the comparison
of kinases and GTPases, the real key number here is what is the difference in KD between
ATP for kinases and GTP for KRAS and it’s about a million fold. So that means we would
have to be a million fold better in our chemistry than we have been in all of the kinase field
to come up with the molecule what competes for GTP. That’s possible, maybe, very, very,
very, difficult. I think probably the only strategy that has a chance at that is one
that Nathaniel Grays’ lab that Dana Farber is pursuing which relies on a covalent bond
to compete out the nucleotide. That is the one I think window and so it’s great that
people are pursuing that but that still is a very, very high bar but I think that is
the largely the reason for the low. [ Inaudible Remark ] Yeah, that’s a great question.>>Dr. Shokat can you repeat a question?>>Oh sure.>>We couldn’t hear it [inaudible].>>Sure, sure. So, Frank sort of said, well,
why can’t we turn that around on itself? Maybe a drug that looked like GTP would have also
picomolar affinity and then it would compete. The problem is is that, we have not–phosphate
containing drugs are very poor and they’re from a single properties getting across the
cell, prodrugs [phonetic] don’t work great so there is that–that problem that. And the
other problem is nobody has every found a good enough surrogate substitute for phosphate.
There’s something about two negative charges, tetrahedral and it’s very, very hard to get
that to go. Even a single phosphate like in SH2 domains, very, very hard. So to think
about three phosphates is tough. [ Inaudible Remark ]>>–like a hundred thousand peptides, [inaudible]
came out, it was about 60 picomolar. [Inaudible] was the best thing you get when you modify
GTP is something we can achieve.>>Yeah, yeah.>>Could you repeat [inaudible].>>So Herbert said, years ago, they tried
a chemistry around GTP, you started with GTP and added peptide to the base or the phosphate
and you basically came within five folds, six folds of this but could never go better
than that, yeah, yeah.>>Hey, man, if you could just follow up a
little more on this. Even if you imagine the thought experiment of an extremely high affinity,
deep you know, small molecule.>>Yeah.>>Whereas the therapeutic index for all of
the normal proteins in the cell, [inaudible].>>Yeah, yeah.>>–cycle [inaudible] like drug and just
wipe out all kinds of–>>Right, yeah, So Kevin brings up the point
that a GTP drug would just block all the GTPases and you didn’t have much of a therapeutic
index and that goes to this question. I think that’s a little let. It’s a sort of mitigated
maybe a little bit by the kinase experience. I mean kinases we have an even larger problem
that we have ATP mimetic drugs. We have 500 kinases, surprisingly once you find that mimic
you can eke out selectively even if every amino acid that contacts the ATP is the same.
So it’s a good. I mean it’s something to worry about but I sort of feel, it’s better to put.
It’s better not to close off any road. It’s good if somebody have a good idea to replace
three phosphates, start there then probably by decorating, you might be able to get selectivity.
So get potency first then selectivity.>>In phosphates is what you mean.>>Two, two should be enough, sorry, yeah,
let’s make it simpler. Take one away. Yeah, because a three would keep on. So exactly
we want it off. We want it off very, good, very good. So, now we’ll get into the RAS
cycle a little bit. I’m sure we’ll hear about this much more, more detail but what appreciate
and I told you about so far is that RAS is in the GDP state which is off and it is sort
of in the relaxed state and switch 1 and switch 2 are in the open confirmation when it binds
GTP, the switch 1, switch 2 are tied down. Now, other RAS effectors that helped convert,
and are convert from the GDP to the GTP state are enzymes called guanine exchange factors
that bind to open up the nucleotide pockets so that GDP can fall out and then by virtue
by the ten fold higher concentration of GTP then GDP in the cell, GTP will become loaded
and the cycle will go in the forward direction. Once it’s in the GTP state they’ll be the
intrinsic GTP’s activity as well as the GDP is activating protein that introduces an arginine
to complement the transition state to greatly accelerate the conversion back. Then the two,
classes or examples of effectors that I’ve showed you before, the RAF interaction here,
with the GTP state as well as PI3 kinase. So many, many people have looked at drugging
every aspect of this. Drug companies have put the entire cycle together, run the entire
deck of compounds through and it’s been very, very difficult to come up with molecules that
can interrupt this cycle but we’ll see opportunities from Steve later in this portion how some
of these complexes provide other pockets that can manipulate the activity of the protein
which are very exciting directions. So how do the mutations cause the transformation?
If we put the localized–the G12, G13, glutamine 61, on the surface of the inactive form of
the protein and the active form you see that in the inactive state the residues are far
apart but they cluster very close to one another in the act of state suggesting that these
residues are important in actually disrupting this active state of the protein. How does
the 61 position cause transformation. So, in this close up you can see that we have
a crystal structure of GDP in it bound with aluminum fluoride mimicking the gamma phosphate
and in gray and light blue is RAS and then the gap in the lighter blue color is the gap
putting in the arginine finger. So glutamine-61 is aiding the catalytic step by activating
water for attack on the mimic here of the gamma phosphate so mutating this catalytic
residue is going to destroy catalytic activity dramatically, and Carla Mattis [assumed spelling]
will–can tell us much more details about this. If we flip the protein around and highlight
where the glycine 12 and glycine 13 are, you see that they’d lined the side chain of the
arginine from the arginine finger so named by Frank and Harry Bourne [assumed spelling]
here at UCSF is complementing catalysis and what’s important to note is that the side
chains are absence in the glycine and so any side chain, any mutation to any residue largely
will clash with the arginine, misaligning the arginine from the gap, greatly decreasing
the gap, facilitated hydrolysis of GTP. So that’s the basic mechanism that neutralized
the transformation, the biochemical inactivation of the protein. So what–what we decided to
look at is that since any amino acid can sit at the twelve or thirteen position, and disrupt
this catalytic mechanism, what kind of features are accessible chemically by the different
residues that are put there? So here are some of the pie charts sort of like what Frank
showed before about the dominance of aspartate, the most frequent glycine 12 mutation, the
next most is valine but the cysteine is a significant fraction of all KRAS mutations
and we if looked in lung cancer it’s actually the most frequent residue that is replaced
for glycine 12. And in this lung cancer situation we know that a lot of that preference for
cysteine or valine is driven by smoking-induced mutations because in current smokers, the
cysteine and valine are the predominant mutations and the never smokers that’s in the aspartate
so there’s some amount of carcinogen induced specific allele specificity but as Frank mentioned,
there’s a lot, lot to understand about why, why glycine gets mutated to what it does get
mutated in various cancers. For our purposes we saw the cysteine as a chemical handle that
is basically to a biological chemist, cysteine is like, you know, honey to a bear. I mean
that is like the one thing that will save you. It’s like water in a desert. I don’t
know if you know, if you know what the analogies are but you need that. So that cysteine was–going
to be the focus of our effort. As we solve the crystal structure of the glycine 12 to
cysteine and the GTP state, we expected to show the cysteine was exposed to the surface
of the protein. Here’s a switch 1 it was ordered in the inactive state, switch 2 was disordered
and in fact some of the residues cannot be defined as our many of the cases of GDP structures
of the GTPases. And then–so how do we find a drug that uniquely relies on attachment
to the cysteine? Very, very luckily for me, my colleague here Jim Wills [assumed spelling]
for many years developed a very, very elegant chemical strategy for focusing on molecules
that attached to cysteine and identified pockets neighboring that are adjacent to the cysteine.
And so what this relies on is a clever chemical trick which is an inner conversion between
several different disulfides. So we start with the protein with a cysteine that is either
is a natural one or is oncogenetically induced or is introduced into the protein. And in
the presence of beta-mercaptoethanol there’s an inner conversion between the cysteine and
the disulfide of the beta-mercaptoethanol. And then through synthesis of a collection
of molecules that have different functional groups, little different fragment, pieces
of drugs, small molecular weight that have a sulfide that are in disulfide with beta-mercaptoethanol,
there’s an exchange. Now if one of the features in this molecule are complementary to a shape
that is adjacent to the cysteine on the protein then these disulfides will become relatively
resistant to reduction and therefore they will build up in concentration and beta-mercaptoethanol
will be out-competed by a specific drug. What’s great about this technology is that the early
leads in drug discovery in the fragment case which are small molecular weight molecules
of 100 or 200 molecular weight don’t have enough intrinsic affinity for you to pick
them up very easily by noncovalent mechanism. Steve Fesik’s group developed elegant biophysical
methods using SAR by NMR to identify pockets using noncovalent chemistry but that is–I
mean he enabled it when he was at Abbott and that is a Herculean task. Now it’s been unable
to [inaudible] in many places but this technique is a very nice method because you know immediately
that the molecule in the collection that bound is what you think it is because you measure
the intact protein mass of the entire complex. So using that approach we collaborated with
Jim’s lab and identified several hits from his collection that bound to the KRAS in the
G12C form of the protein but the wild type form which has several other cysteines did
not bind at all. So these are two features, one in dark grey, one in light grey. If we
introduce the cysteine into HRAS we can also get binding even though we didn’t screen it
against HRAS. So that says this pocket is somewhat conserved. If we take G12C RAS and
put it into GTP state and we ask if these molecules can form the disulfide only minimally
if at all. So that was actually very, very depressing because we have worked so hard
to get a molecule that bound to the cysteine, that bound very selectively to just the cysteine
we wanted but when we put the protein in the active state where we think the state of the
protein is in the tumor we see really, really no binding. No worries, let’s just keep going
hoping that some little bit of unexpected luck will come our way and also we said immediately,
we screened against the G12C in this GDP state, we screen the collection so we said immediately
let’s go back and screen in the GTP state. We did that screen, we came up with zero.
So that’s when we really said we have no chance–we couldn’t abandon these molecules, we have
to keep going forward. So we then solve the crystal structure of this molecule bound to
RAS and at this point it could have bound in the nucleotide pocket. This covalent binder
it could compete out with picomolar GTP or it could bind somewhere else and very, very
beautifully it bound in a pocket adjacent to the nucleotide so we don’t have to compete
with the picomolar affinity and it looks like it has a pocket that usually was unappreciated
before because of the flexibility of the switch 2 in the GDP state. So although we knew that
GDP–the switch 2 has to fold in to this pocket, our drug is binding effectively what is the
switch 2 pocket in the GTP state but not drug–without the drug there this pocket is really not fully
formed. So with that as a guide we started to optimize the chemistry and think how this
molecule would disrupt RAS function. And this is a little animation of that inhibitor bound
structure, switch 2, switch 1 and the conformational changes that would happen in the absence of
the drug. In the absence of the drug switch 2 would fold into that pocket producing the
surface for RAS binding and activation. So our first inclination was that we had a switch
2 pocket binder and we would disrupt any interaction with RAS that require switch 2. So that seemed
fine to us but we’re still thinking about the GDP state problem, you know. Is this going
to be a killer for us and so we actually had a very nice guide, it’s always nice to see
that nature discovers something before you did because then maybe you have a chance of
having yours to work and there is a natural product that binds to the GTPase Gq, very,
very potent inhibitor, very, very selective. When its crystal structure was solved it was
shown to bind to the GDP state of this GTPase and bind under switch 1 so that’s very, very
analogous to our GDP binding switch 2 pocket binder. So that reassured us. The other feature
is that right before we published our work, the Genentech group and Steve Fesik’s group
identified using SAR by NMR another pocket in RAS that was behind switch 1 and switch
2 and what we liked about our pocket is that it was closer to the catalytic machinery and
so even though we have this GDP problem we at least appreciated that our pocket was closer
to the catalytic machinery of the protein. So we push forward on that basis as well.
So then the question is converting the disulfide to a carbon-based electrophile that can work
in cells so we converted and kept the same reversible binding piece and then converted
to electrophiles at either vinyl sulfonamides which are kind of too reactive for a real
drug or acrylamides that occur now relatively commonly in kinase inhibitors that hit non-conserved
cysteines. So we start with reactive groups and we incubate in a sort of very standardized
way, 10 micromolar, 24-hour incubation, this molecule gives 18 percent modification but
through subtle changes to that blue ring, we can increase right up to 80 percent and
then we can even go to less reactive electrophiles and then further basically combine, mix and
match all of the pieces to get the optimal compounds. So this was about an effort from
that disulfides to the optimized compounds I’ll show you of about 200 molecules that
we synthesized. Yeah.>>So in the absence of the electrophilic
warhead, do these molecules bind to a RAS?>>Not that we can appreciatively measure.
They must bind without the electrophile but it’s probably in the, you know, tens of millimolar
range too difficult for us or too big to do a sort–treat as a fragment and bind. I think
Steve one time I talked mentioned that things, using things bind in this pocket in SAR by
NMR screens.>>That’s actually in my talk. I’ll–>>You will, OK.>>I’ll mention it.>>OK. Sorry–yeah?>>Yeah, with covalent inhibitors you have
this within traditional [inaudible] covalent inhibitors. You usually optimized both KI
and KNS>>Yeah.>>In this situation your enzyme is essentially
dead so you can’t–you–and we can’t do the exact same experiments but yeah.>>Right.>>[inaudible] are you optimizing reactivity
or is there actually an affinity components that you’re improving by making this one [inaudible].>>Yeah, yeah.>>Can you repeat the question?>>Yeah, yeah. So she–this question is that
you know in traditional drug discovery you would maybe start with a reversible binding
molecule, get the molecule in there and then change the warhead and keep tuning that in
order to get faster and faster inactivation. We have a harder time in this case because
it’s–we’re trying to optimize essentially both at the same time. So what we’ve done
is we’ve basically abandoned–we sort of kept with one electrophile so we kept with the
acrylamide and then we’ve systematically investigated the reversible binding mode in the context
of the irreversible reaction. So we don’t really have a good measure of the pure reversible
binding yet. I think probably people at Well Spring are getting closer to that but we don’t
have that yet. It’s a great question.>>May I interrupt for you a minute?>>Oh sorry.>>So we have a handheld mic out because the
people who are streaming and people who are in [inaudible] if you have a question please
interrupt anytime during the talk or raise your hands so we can give you the handheld
mic so that the people who are streaming can hear it.>>Great.>>But we also has one in the back.>>Great.>>You’ve got five to ten minutes.>>What’s that?>>You’ve got five to ten minutes.>>I have five to ten minutes. No problem,
I got that. That sounds like one minute. I could even do that. So we had a molecule,
binds to RAS, how does it disrupt the function? I told you disrupted switch 2 confirmation
so immediately asked does it–should disrupt the GEF, that ability to do the exchange reaction
because it relies on contact with switch 2. So in a mant exchange reaction we have GDP-mant.
We add either SOS or EDTA as the chemical exchange mediator and we ask for loss of fluorescence
by competition with non-labeled GDP and so the G12C mutant without any added SOS or EDTA
has a very slow exchange reaction. If we add SOS, we have very accelerated exchange. If
we add EDTA and yank out the magnesium it’s instantaneous. Now we put our drug on and
we see that we blocked completely the SOS-mediated exchange though we have no effect with EDTA
because that is independent of switch 2 just pulling out the magnesium. So we learned that
we could block the GEF-mediated exchange. I think that’s one area I’m very interested
in understanding is whether the mutant RASes really rely on the GEF very much. I think
that’s one of the basic kind of questions Frank highlighted. There are many, many unanswered
questions. I think that’s a very important one to think about. The other thing we started
to realize when we looked at the crystal structures was that if we look at the textbook, a picture
that Frank showed of the–wait, still on further? [ Inaudible Remark ] There we go. Release the phosphate and the
switch 1 and switch 2 opens up. We realize that our compounds sitting under switch 2
is actually further disrupting the confirmation of switch 2 residues. And if we looked closer
and asked well, what would happen when GTP would try to come in to the drug-bound state?
We could see that glycine 60 in switch 2, threonine 35 and tyrosine 32 which have the
hydroxyls that key into the gamma phosphate and make that confirmation change would be
disrupted by the presence of the drug. So they’ll start to move in but if the drug is
here you’ll see that the glycine 60 in particular would not be able to complement the phosphate
position in GTP. So this made the prediction that our molecule could disrupt GTP affinity
in preference over GDP. And with similar exchange reaction we can titrate in GDP in wild type
G12C or two different drug-bound forms and the GDP affinity is unaffected by the presence
of the drug. But if we compare now and titrate in GTP we see wild type and mutant have high
affinity. This is sort of limited by the concentration of protein we have so it’s not the true affinity
that’s why it doesn’t look like picomolar. But then with the drug-bound we see a rightward
shift and it is lower affinity for GTP. This is fantastic because it kind of unraveled
one of the puzzles that we had at the beginning is that how can we compete with the nucleotide
17 picomolar? Well, what this drug really does is it doesn’t compete for the entire
nucleotide, it just disrupts the gamma phospate which is the Achilles’ heel of RAS and if
that’s true then the molecule should have some ability to kill G12C cells in preference
over other alleles and so we took a number of lung cancer cells in colons that had G12C
mutations in KRAS versus serine, valine or aspartate. And although it’s not perfect with
this early stage molecule you can see very nice dose-dependent killing and the G12C cells
are uniformly more sensitive than the non-G12C cells. And for example, this cell has one
copy of G12C, this copy has six copies of G12C so there’s some–we think there’s some
reasonable hypothesis about why cells that have G12C are differentially sensitive although
one of them like 23 is not particularly sensitive. So we still have a lot of understand about
specificity but importantly I think we always want to see a very, very good correlation
between biochemical ability to bind to the protein and the cellular effects and so what’s
very important is that these reactive molecules could have very nonspecific effects so we
want to compare specific electrophiles like this compound which binds to G12C at 100 percent
and compare it to a molecule that has the same warhead but doesn’t bind at all by virtue
of poor reversible binding and then there’s an intermediate molecule here, number 10.
And the rank order in cells should be 12, 10, 17 and that’s 12, 10, 17 and completely
inactive. So that is all very, very gratifying in [inaudible] that we believe are on target.
So now it’s a game of optimizing the chemistry here for more and more potency and the like.
So to summarize what we’ve identified as a molecule that keys in to just the oncogene,
blocks the GEF-mediated exchange and since it can’t load GTP we think that we’re using
the cellular GDP as our inhibitor in keeping the protein in this state and that will then
preclude it from binding to factors. So the summary of the challenges that I set out were
inhibiting just the oncogene. I think inhibiting just the G12C versus the wild type solves
that problem and we thought we would have to compete with the nucleotide but really
all we needed to compete with was the gamma phosphate. And so with that, the people who
did this work were too fantastic, people in the lab, postdoc Ult Peters [assumed spelling]
and graduate student John Ostrom [assumed spelling] who’ve both gone on now and Martin
Sos who we put on the project only because his name was one of the RAS effectors and
then a new student Danny Gentile [assumed spelling], so a fantastic group and really
couldn’t have done this without Jim Wells and a lot of help from this postdoc Jeff Sidowsky
[assumed spelling]. We started a company with Frank to pursue this in the clinic and we’re
pushing that forward now so thank you very much. [ Applause ]

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