Pathology Grand Rounds March 6, 2025 - Loren D. Walensky, MD, PhD

Transcript

• 00:01You know, it's an absolute
• 00:03privilege and a pleasure for
• 00:05me to introduce Professor Lauren
• 00:06Wilenski
• 00:07for today's Grand Rounds.
• 00:10He graduated from Princeton as
• 00:11the class valedictorian
• 00:13while majoring in chemistry.
• 00:15He completed his MD PhD
• 00:17at Johns Hopkins in seven
• 00:19years.
• 00:20He first came to Children's
• 00:21Hospital Boston as a pediatrics
• 00:23intern, then the Dana Farber
• 00:25Cancer Institute as a pediatric
• 00:26HemOnc fellow, and finally, Harvard
• 00:29is a faculty member where
• 00:30he's currently a full professor,
• 00:32the director
• 00:33of the, Lindy program in
• 00:35cancer,
• 00:36chemical biology, and the director
• 00:38of the Harvard MIT MD
• 00:40PhD program.
• 00:42His research focuses on using
• 00:43chemical biology to target cancer
• 00:45where he helped develop a
• 00:47new class of biologics called
• 00:48stapled peptides
• 00:50and then applied them to
• 00:51numerous pathways, including p fifty
• 00:53three, KRAS, as well as
• 00:55viral and bacterial targets.
• 00:58However, his most significant work
• 00:59has been elucidating the complicated
• 01:01functions of the BCL two
• 01:03family of proteins, both within
• 01:05and outside of apoptosis.
• 01:07And while he's made significant
• 01:08progress in our understanding of
• 01:10MCL one, today he's gonna
• 01:11focus on a little known
• 01:13protein called Bax,
• 01:15which you might recognize from
• 01:17the homologue of its more
• 01:18well known BAC
• 01:20protein.
• 01:22He served as the past
• 01:23chair of the Cancer Molecular
• 01:25Pathobiology
• 01:26Camp study section and has
• 01:28received numerous innovation awards, including
• 01:30an NCI
• 01:31outstanding investigator r thirty five
• 01:33award and one of the
• 01:34top twenty translational researchers
• 01:37of twenty nineteen.
• 01:38This shows in him being
• 01:40the scientific cofounder of five
• 01:41companies with with three lead
• 01:43compounds in clinical testing.
• 01:45However, arguably, the most meaningful
• 01:47awards, at least to me
• 01:48as a former lab member,
• 01:50is of Lauren being a
• 01:51superb mentor shown in his
• 01:53winning the Harvard Medical School
• 01:55Young Mentor Award in two
• 01:56thousand and seven,
• 01:57both the HST and BBS
• 01:59mentoring awards in twenty seventeen,
• 02:02and the MD PhD mentoring
• 02:03award in twenty twenty four.
• 02:06Lauren's ability to create a
• 02:07community and a family that
• 02:09is all inclusive
• 02:10is unsurpassed.
• 02:12As a postdoc in his
• 02:13laboratory with a young family,
• 02:15it was inspiring for me
• 02:16to see him excel with
• 02:18his three children,
• 02:19a wife as busy as
• 02:20himself,
• 02:21very sick clinical patients,
• 02:24multiple lab members with vastly
• 02:26different needs,
• 02:27and all the while advancing
• 02:28the most meticulous and beautiful
• 02:30scientific discoveries.
• 02:32I'm just truly grateful to
• 02:34be introducing my friend and
• 02:35mentor, professor Laura Glenske.
• 02:43Alright. Thanks so much. Thanks
• 02:45for coming.
• 02:46It's
• 02:47hard to top that, but
• 02:48I I,
• 02:50it's such
• 02:51so much fun to be
• 02:52here.
• 02:53I've had such great chats
• 02:55with Sam over the last
• 02:56twenty four hours.
• 02:58Those of you who have
• 02:59labs and have mentees,
• 03:03you should all be so
• 03:04lucky
• 03:05to have your first first
• 03:07group of postdocs have someone
• 03:09like Sam in the cohort.
• 03:11It's just
• 03:12the trust that folks have
• 03:13in you at the beginning
• 03:14of their,
• 03:15of your careers is like
• 03:16a very special
• 03:18thing. And so those always
• 03:20remain among the most special
• 03:21folks in your life because
• 03:22they kind of trusted you
• 03:23when you probably had no
• 03:25right to be trusted in
• 03:26terms of mentoring their careers.
• 03:30So thanks for that.
• 03:33So what I was gonna
• 03:34do today is kind of
• 03:35a little bit of a
• 03:36nostalgia for me. I decided,
• 03:38you know, it's like twenty
• 03:39years since I started working
• 03:40on this protein,
• 03:42and I thought it would
• 03:43be a good opportunity to
• 03:44look back to kind of
• 03:46talk about all the things
• 03:47that we've learned. And and
• 03:48one of the beauties of
• 03:49academic medicine is that you
• 03:51could pick something that you
• 03:52care about and really stay
• 03:53focused on for many, many
• 03:54years. In a world where
• 03:56attention spans are always way
• 03:58too short, we have the
• 03:59luxury of being able to
• 04:00focus on things that we
• 04:01believe are very, very important
• 04:02and stick to them because
• 04:04we have that conviction.
• 04:06And I would say during
• 04:07these times, that is more
• 04:09important than ever.
• 04:11Stick to the things that
• 04:12you care about. Focus on
• 04:13them. Don't be distracted,
• 04:15and and and really kind
• 04:16of embrace and double down
• 04:18on your scientific and medical
• 04:19missions.
• 04:21So to start off,
• 04:24I got interested in this
• 04:26field,
• 04:27because when I was looking
• 04:28for a postdoctoral fellowship,
• 04:30Stan Korsmeyer
• 04:32basically started out with a
• 04:33verbal version of this slide.
• 04:35And he used to say,
• 04:36like, apoptosis is the best
• 04:38thing you could possibly study.
• 04:39Why? Because, like,
• 04:41every
• 04:42disease, and and overall health
• 04:44depends on a balance between,
• 04:45you know, new and dying
• 04:46cells. And he kind of
• 04:48convinced us that, like, okay.
• 04:50You could figure out
• 04:52this balance, and if you
• 04:53can modulate it in one
• 04:54direction or the other, you
• 04:55could tackle all the diseases
• 04:56where there's too much cell
• 04:57survival,
• 04:58like cancer and autoimmunity, inflammation.
• 05:00And then on the flip
• 05:01side, you know, you could
• 05:02tackle all the diseases where
• 05:04there's premature cell loss, like
• 05:05neurodegeneration and infertility and stroke,
• 05:07heart attack. And that was,
• 05:09like,
• 05:10very exciting concept that if
• 05:11you could understand this balance,
• 05:13you could potentially impact a
• 05:14whole host of diseases. So
• 05:16I was sold,
• 05:18and I started as a
• 05:19postdoc
• 05:20in an area where I
• 05:21knew absolutely
• 05:23nothing.
• 05:23I had never studied these
• 05:25proteins. I knew nothing about
• 05:26b c l two,
• 05:28and and in a way,
• 05:30I always try to encourage
• 05:31all the newbies in my
• 05:32lab that that having no
• 05:34background in an area of
• 05:35science is actually your biggest
• 05:37asset.
• 05:38Because, you know, I have
• 05:39to try to convince and
• 05:40remind myself over and over,
• 05:42like, the older you get
• 05:43in a field, the more
• 05:44you think you know things
• 05:45about it. And and that's
• 05:47actually a liability because then
• 05:49you start to turn off
• 05:50to things that may be
• 05:51completely new and unexpected that
• 05:52you hadn't appreciated before.
• 05:55So what I learned, was
• 05:56that there were two classes
• 05:57of survival proteins and death
• 05:59proteins, and the survival proteins
• 06:00in orange were like BCL
• 06:02two
• 06:03and, you know, its brothers
• 06:04and sisters, BCLXL and MCL
• 06:06one, which we'll talk about.
• 06:07And then there were death
• 06:08proteins, And at that time,
• 06:10it was BACs and BAC,
• 06:11and that one inhibited the
• 06:12other.
• 06:13And that most cells do
• 06:14fine because for the most
• 06:16part, the the system is
• 06:17rigged to keep your cells
• 06:18alive, keep tissues healthy.
• 06:20And then there's this third
• 06:21class of proteins that
• 06:24are very diverse and that
• 06:26have very heterogeneous
• 06:28structures and functions, but their
• 06:29job is like the antenna
• 06:30proteins
• 06:31that are situated all over
• 06:33the cell to sense different
• 06:35types of stress and then
• 06:36deliver that message to the
• 06:37kind of the big players,
• 06:39you know, at the mitochondria.
• 06:41And so these are the
• 06:41b h three only proteins,
• 06:43and I'll show you why
• 06:43they got their name in
• 06:44a minute.
• 06:45But the idea is is
• 06:46that when they get triggered,
• 06:48one of their ways to
• 06:49turn on death is to
• 06:51inhibit the inhibitor.
• 06:53And then the other way,
• 06:54which was much more controversial,
• 06:56was this idea that maybe
• 06:57they could also directly activate
• 06:59the activator.
• 07:00Okay. And most of the
• 07:01drug development, you know, in
• 07:03this area for the last
• 07:04twenty plus years has been
• 07:06the inhibit the inhibitor pathway.
• 07:09So the family
• 07:10grew and grew and grew
• 07:12based upon this homology map,
• 07:13and they're divided into multi
• 07:15domain proteins and then single
• 07:17domain proteins. So the multi
• 07:18domain homology is derived from
• 07:20what's called these BH or
• 07:22BCL two homology domains, one,
• 07:24two, three, and four. There's
• 07:25multidomain death proteins like BACs
• 07:27and BAC. But these eclectic
• 07:30antenna proteins are called BH
• 07:32three only because they only
• 07:34share the commonality of this
• 07:35one domain, and that domain,
• 07:37as I'll talk a lot
• 07:38about today, is a single
• 07:39alpha helix.
• 07:41And the idea was was
• 07:42that that alpha helix is
• 07:44the vehicle for communication
• 07:46between b h three only
• 07:47proteins and the downstream players.
• 07:50So to kind of put
• 07:51all this in motion,
• 07:53BAX lives in the cytoplasm
• 07:55for the most part as
• 07:56a latent inactive protein. And
• 07:58the way I kind of
• 07:59describe it is it's kind
• 07:59of like a hand grenade,
• 08:01and the pin is, like,
• 08:02firmly in place. And it's
• 08:04just kinda sitting there. And
• 08:05it's sitting there in cancer
• 08:06too,
• 08:07which is kind of the
• 08:08basis for my excitement about
• 08:10it. And then in response
• 08:11to stress, it undergoes this
• 08:13very extensive conformational change. It
• 08:16goes to the mitochondria.
• 08:17It self associates, and then
• 08:18it disrupts the outer mitochondrial
• 08:20membrane, and then second messenger
• 08:22signals come out and, you
• 08:23know, give you the irreversible
• 08:25cell death pathway.
• 08:27And so this is a
• 08:27very dynamic process. Right? You
• 08:29take something that starts in
• 08:30one compartment,
• 08:31completely changes its shape, shuttles
• 08:33to another compartment, then changes
• 08:35its behavior again, self associate.
• 08:37So there's a lot going
• 08:38on here. And and this
• 08:40slide is kind of, like,
• 08:41pretty much a systematic
• 08:43road map for what I've
• 08:44been dissecting for twenty years.
• 08:47And so
• 08:49when this system is blocked
• 08:51by cancer, for example,
• 08:53it's based upon this idea
• 08:54that a survival protein can
• 08:56go and latch on to
• 08:57that single alpha helix there
• 08:59and kind of arrest the
• 09:00whole process. And that single
• 09:01alpha helix that pops out,
• 09:03that's one of the b
• 09:04h three helices that I'll
• 09:05be talking a lot about.
• 09:07And so if you were
• 09:08thinking about drug making, you
• 09:10would kind of wanna simulate,
• 09:11you know, this
• 09:13to disarm this inhibition in
• 09:15cancer. And you could imagine
• 09:16that, like, the mitochondria has
• 09:18these orange survival proteins literally
• 09:20sitting on it and as,
• 09:22like, a force field, like,
• 09:23to prevent the incomings, right,
• 09:24and protect the mitochondria. And
• 09:26that's a huge part of
• 09:27cancer. Right? Because cancer need
• 09:29you look at leukemia cells
• 09:30and do a mitochondrial stain,
• 09:32it is the scary thing
• 09:33to look at. Right? These
• 09:34cells, these little, like, lymphocytes
• 09:35that are now lymphoblasts are
• 09:37so small. They have all
• 09:38DNA, and then that thin
• 09:39rim of cytoplasm is choked
• 09:41with mitochondria after mitochondria. And
• 09:43they they need that protected
• 09:45so that they can keep
• 09:46cranking out energy.
• 09:48And so these antenna proteins,
• 09:49again, can inhibit the inhibitor,
• 09:51or they could potentially activate
• 09:53the activator.
• 09:55And so this field really
• 09:56kicked in,
• 09:57once the structure
• 09:59of the first survival protein
• 10:00was
• 10:02collaboration between Craig Thompson and
• 10:04Steve Fesic. And and Steve
• 10:06Fesic was at Abbott Labs
• 10:07at the time.
• 10:09And so here's the structure.
• 10:11And what was discovered in
• 10:12the subsequent paper was that
• 10:14there's a surface groove
• 10:16shown in green, and that
• 10:17is the acceptor for these
• 10:19b h three helices. And
• 10:20this is kind of the
• 10:21basis for the protein protein
• 10:22interaction. This b h three
• 10:24helix fits into the groove,
• 10:26and that's the interaction. That's
• 10:27the wrestling match between life
• 10:28and death.
• 10:30And that kind of gave
• 10:31the road map for drug
• 10:32development in this field starting
• 10:33in the late nineties.
• 10:35And so Abbott went on
• 10:37to
• 10:37basically simulate
• 10:39a helix that bound to
• 10:41b c l two, b
• 10:42c l x l, and,
• 10:43you know,
• 10:44turned it into a drug
• 10:45eventually. It took probably, like,
• 10:47fifteen to twenty years to
• 10:48get there.
• 10:49And then now we have
• 10:50the selected BCL two inhibitor,
• 10:52venetoclax. And how we went
• 10:54from and how Abbott went
• 10:55from, you know, left to
• 10:56right there was just an
• 10:58amazing story that we won't
• 10:59talk about today, but just
• 11:01amazing fortitude, unbelievable
• 11:03persistence,
• 11:04people that were incredibly able
• 11:06to convince folks to keep
• 11:08on keeping on even when
• 11:09something seemed like it was
• 11:10never going to work, and
• 11:11even what happened in the
• 11:12early clinical trials and have
• 11:14that made folks have to
• 11:15go right back to the
• 11:16drawing board and start anew.
• 11:18It's a miracle that this
• 11:20drug actually made it to
• 11:21prime time.
• 11:22And now when you see
• 11:23as a clinician the amount
• 11:25of good that it's doing,
• 11:26it's really a remarkable story
• 11:27that I hope someone will
• 11:28write a book about someday.
• 11:31Now the challenge is is
• 11:32that there's other anti apoptotic
• 11:34proteins that this drug does
• 11:35not bind to because, again,
• 11:36it was engineered to be
• 11:37a selective BCL two inhibitor
• 11:39to avoid
• 11:40toxicities and avoid being too
• 11:41kind of panactive. And so
• 11:43MCL one and BFL one,
• 11:46are very commonly overexpressed,
• 11:48you know, to cause resistance
• 11:49to venetoclax.
• 11:51And then, in recent years,
• 11:53as more and more patients
• 11:54get the drug, it's become
• 11:55clear that b c l
• 11:56two very cleverly,
• 11:58can,
• 11:59basically mutate. So it doesn't
• 12:00bind the drug, but it
• 12:01still binds the b h
• 12:02three helix, which is quite
• 12:04a magnificent,
• 12:05structural biology trick that this
• 12:07protein figured out. Right? Because
• 12:09theoretically, the molecule on the
• 12:10the natural effect, they're all
• 12:12going at the same pocket,
• 12:13but this protein figured out
• 12:15how to mutate to not
• 12:16bind the drug but still
• 12:17bind the the natural target.
• 12:19It's amazing.
• 12:21And so I kind of
• 12:22consider this whole area
• 12:24of cancer biology and drug
• 12:26development on the anti apoptotic
• 12:27side as the whack a
• 12:28mole situation where drug companies
• 12:31really want selective inhibitors so
• 12:32that they don't have a
• 12:33lot of toxicities.
• 12:35But when you have selective
• 12:35inhibitors and then you have
• 12:37all of these brothers and
• 12:38sisters that have homologous functions,
• 12:39they're, of course, gonna pop
• 12:41up and take over, and
• 12:41that's exactly what's happened. And
• 12:43so, you know, the industry
• 12:44and, you know, academia and
• 12:46pharma alike have been going
• 12:47after, you know, different MCL
• 12:49one and BFL one and
• 12:50and to try to target
• 12:51them. And as some of
• 12:52you, I'm sure, have heard
• 12:52that these MCL one inhibitors
• 12:54have had toxicities, mostly cardiac
• 12:56in the clinic, and that's
• 12:58there's a biological reason for
• 12:59that because MCL one is
• 13:00very important in,
• 13:02what we've discovered in regulating
• 13:03fatty acid oxidation. And,
• 13:06I'm not gonna say more
• 13:07about antipoptopics
• 13:08side because I decided today
• 13:09to talk about BACs, but
• 13:10there's just still some amazing
• 13:12biology,
• 13:13being discovered on the anti
• 13:15apoptotic side about what these
• 13:16proteins do outside of doing
• 13:18latching on to b h
• 13:19three helices and shutting down
• 13:21apoptosis.
• 13:24But the other question
• 13:25aside from inhibiting the inhibitors
• 13:27was this idea that, you
• 13:28know, Stan Korsmeyer had and
• 13:30wrote about it in the
• 13:31earliest papers about discovering
• 13:33bid interacting with backs. What
• 13:35does that mean? You know,
• 13:37what could the activation look
• 13:38like? And the idea was
• 13:40that this interaction, instead of
• 13:42being a static interaction between
• 13:44b h three and groove
• 13:45that, you you know, sits
• 13:46there, you could immunoprecipitate
• 13:47it, you could solve a
• 13:48crystal structure of it, that
• 13:50this was somehow different, that
• 13:51this was gonna be a
• 13:52triggering interaction. It was gonna
• 13:54be dynamic,
• 13:55and they called it in
• 13:56the earliest paper a hit
• 13:58and run,
• 13:59mechanism.
• 14:01Now
• 14:01a lot of this idea
• 14:03was debunked
• 14:05because
• 14:07you couldn't immunoprecipitate
• 14:08b h three proteins with
• 14:10BAX. You couldn't
• 14:11solve the structure or even
• 14:13measure an interaction
• 14:15between a b h three
• 14:16helix and BAX. And so
• 14:19when I kind of started
• 14:20in at this,
• 14:21and was working on bid
• 14:22BAX, you know, b h
• 14:24three BAX interactions,
• 14:25you know, imagine writing your
• 14:27first r o one grant,
• 14:29okay, about how b h
• 14:31threes might be able to
• 14:32directly bind and activate BAX.
• 14:34But then this paper comes
• 14:35out in science and says
• 14:37aptosis is activated, you know,
• 14:39when you inhibit the inhibitors,
• 14:40and that's it, comma, not
• 14:42BAX or BAX.
• 14:43So that you know, that's
• 14:44pretty tough, you know, when
• 14:46your grant is, when you're
• 14:47asking someone to give you
• 14:48a million dollars, you know,
• 14:49to study direct BAX activation,
• 14:51and you have a science
• 14:53paper that says no. Great
• 14:54great hypothesis, but that doesn't
• 14:56occur. Now at that time,
• 14:58I had already known, that,
• 15:01like, they showed that these
• 15:02peptides did not bind to
• 15:04VAX very well. You couldn't
• 15:06really detect it. So we
• 15:06have the same results, and
• 15:07that's the flat red line
• 15:09here. You take a BID
• 15:10b h three peptide, a
• 15:11b h three only peptide.
• 15:12It's unstructured.
• 15:13You put it on to
• 15:14BAX, and you don't see
• 15:15binding. But if you do
• 15:16this to an anti apoptotic
• 15:18protein, you get beautiful binding.
• 15:19No problem. Right? So there's
• 15:21your yes and no, right,
• 15:22that underlies that paper.
• 15:25But I had been working
• 15:27on
• 15:29this problem for my postdoc,
• 15:30which was,
• 15:31you know, wouldn't it be
• 15:33great
• 15:34if bioactive
• 15:36motifs and proteins like alpha
• 15:38helices could actually be drugs?
• 15:40Because most drugs are modeled
• 15:41after bioactive motifs, and then
• 15:43you spend two billion dollars
• 15:45to come up with a
• 15:45small molecule that mimics that.
• 15:47But what if you could
• 15:48bypass that whole thing and
• 15:49then just take the peptide
• 15:50that nature already gave you
• 15:52and, you know, solve the
• 15:53problems of peptide therapeutics, which
• 15:55they're unstable, they get proteolyzed,
• 15:56but, you know, get rid
• 15:57of the unfolding problem, and
• 15:59then maybe you can make
• 16:00drugs much faster. That was
• 16:01the idea. And so when
• 16:03you look at, you know,
• 16:04helical motifs like a b
• 16:05h three in the context
• 16:07of a protein, yeah, you
• 16:08synthesize it, you know, in
• 16:09your lab and you do
• 16:10analyses and this is just
• 16:12a simulation.
• 16:13But, like, that thing's never
• 16:14going back to an alpha
• 16:15helix, okay, when you make
• 16:16it.
• 16:18And so the idea was,
• 16:20what if you installed
• 16:21non natural amino acids that
• 16:23you could kind of form
• 16:24a cross link with
• 16:25and try to reestablish
• 16:28structure
• 16:29to this peptide sequence that
• 16:30actually is meant to be
• 16:31an alpha helix?
• 16:33And so I I grew
• 16:34up as a, you know,
• 16:36undergrad that got bitten by
• 16:37the organic chemistry bug. So
• 16:39I started out with synthetic
• 16:40organic chemistry and always wanted
• 16:42to go back to it
• 16:42during my post doc even
• 16:43though my PhD was in
• 16:44molecular biology and and did
• 16:46a lot of biochemistry and
• 16:47animal work.
• 16:48And so I actually knew
• 16:50how to make these things.
• 16:51Like, these non natural amino
• 16:52acids were ten step chiral
• 16:53synthesis.
• 16:55Total nightmare, like, three and
• 16:56a half months of your
• 16:57life to be able to
• 16:58generate these non natural amino
• 16:59acids just to install them
• 17:00by hand, you know, into
• 17:02manual peptide synthesis back in
• 17:03the day.
• 17:05You know, while your beeper
• 17:06is going off and your
• 17:07patients are paging you as
• 17:08a first year fellow, and
• 17:09then you go back to
• 17:10the hood, you're like, now
• 17:11what step was I on
• 17:11of this manual peptide synthesis?
• 17:13And then you didn't remember,
• 17:14so you had to throw
• 17:15it all out and start
• 17:16over.
• 17:18But it looked like this
• 17:19could do the job. And
• 17:21so, you know, these are
• 17:22some of the first data
• 17:23that I got as a
• 17:24postdoc. So here's a b
• 17:25h three peptide of BID.
• 17:27Circular dichroism gives you the
• 17:29sense of the shape and
• 17:30solution. This contour is a
• 17:32piece of spaghetti.
• 17:33And then you put the
• 17:34staple in and all of
• 17:35a sudden you could see
• 17:36the shape change, double hump
• 17:37pattern consistent with a with
• 17:39a pure alpha helix.
• 17:40You take these two things
• 17:41and you throw trypsin at
• 17:43them from, you know, sigma,
• 17:44like, right out of the
• 17:45vial, and the unstapled one
• 17:47is just destroyed. And the
• 17:49stable one magically seems to
• 17:50withstand this, and that's because
• 17:52amide bonds that are twisted
• 17:53are not great substrates for
• 17:55proteases.
• 17:56And this ends up being
• 17:57a really important aspect for
• 17:58the drug translation.
• 18:00And then the surprise was
• 18:01that some of them actually
• 18:02got into cells.
• 18:04Right? And so on the
• 18:05left, I have a fluorescently
• 18:06labeled unstable peptide, and then
• 18:08on the right, a fluorescently
• 18:09labeled staple peptide.
• 18:11And all of a sudden,
• 18:12you know, these jerk out
• 18:13leukemia cells had fluorescent peptide
• 18:14in their little scan cytoplasm,
• 18:16and that was pretty surprising.
• 18:19But that really opened the
• 18:20door to be able to
• 18:20do cell biology studies and
• 18:22to use them as potential
• 18:23therapeutics and treat tumors in
• 18:25mice, and it really kinda
• 18:26opened the door. And so
• 18:28I had been doing this
• 18:30and trying to figure out
• 18:31if there were any b
• 18:32h threes that bound to
• 18:33bacs, but I had
• 18:35stapled b h threes that
• 18:37were actually folded in the
• 18:38way that they were naturally.
• 18:40And when I did these
• 18:41same fluorescence polarization assays, in
• 18:43my hands, a subset of
• 18:45the stapled versions bound to
• 18:46backs just as if it
• 18:47was an anti apoptotic protein.
• 18:49So I got very excited.
• 18:51And
• 18:53so, at that point, the
• 18:54question was, well, now that
• 18:55you have a binding interaction
• 18:57and you can measure it,
• 18:58that was the door that
• 18:59was open to doing structural
• 19:00biology research.
• 19:02And so I, at that
• 19:03point,
• 19:04was given advice by my
• 19:06mentor
• 19:06to find
• 19:08the world's expert,
• 19:10in,
• 19:11back structural biology. And that
• 19:12was actually easy because the
• 19:14person that had done the
• 19:15first NMR structure of backs
• 19:17and published it in Cell
• 19:18was Niko Chandra, NHLBI.
• 19:21And I told this story
• 19:22at dinner last night,
• 19:24that when I called Niko
• 19:25the first time,
• 19:28you know, he disclosed to
• 19:29me you know, as a
• 19:30young person entering the field,
• 19:31I viewed him as, like,
• 19:32one of the kings of
• 19:33the field. He solved the
• 19:34structure of BACS.
• 19:35And I remember him saying,
• 19:36well, you know, I actually
• 19:37saw the structure of BACS
• 19:38to honor my mentor, Ad
• 19:40BACS.
• 19:41And that was why I
• 19:42did that. And so I
• 19:43was like, oh gosh. And
• 19:45while I said, I have
• 19:45a ligand, you know, that
• 19:46that that binds bacs. And
• 19:47he, you know, said, well,
• 19:48so do a lot of
• 19:49people.
• 19:50He said, oh, and by
• 19:51the way, your ligand binds
• 19:52and activates bacs. You can't
• 19:54do NMR on a moving
• 19:55target where, you know, the
• 19:56whole thing goes from a
• 19:57twenty kilodome protein to an
• 19:58oligomer in five minutes. So,
• 20:00you know, no chance of
• 20:02this working. And if you
• 20:03wanna do anything, just go
• 20:04make it weak enough so
• 20:05that it could bind but
• 20:05not activate.
• 20:07So as a young person,
• 20:08I was like, well, that
• 20:09sounded like really good advice.
• 20:10So I,
• 20:12I don't know if he
• 20:13thought I was going to
• 20:13follow-up his advice, but I
• 20:15went back and,
• 20:16a year and a half
• 20:17later, I found I was
• 20:18able to iterate this to
• 20:19make the peptide weak enough
• 20:21to bind but not activate
• 20:22over, like, a two hour
• 20:23period.
• 20:24And so I went back
• 20:26and, we collaborated with,
• 20:29one of his staff scientists
• 20:30who was the post doc,
• 20:31who's the first author on
• 20:32that BACS NMR paper, Matoshi
• 20:34Suzuki.
• 20:35And we added this peptide
• 20:38to Matoshi's n fifteen label
• 20:40BACS. And there were very
• 20:41subtle changes,
• 20:43but they were very
• 20:45specifically localized. And so when
• 20:47we mapped those residues or
• 20:48those chemical shift changes onto
• 20:50the protein, they all lined
• 20:51up on one side of
• 20:52the protein.
• 20:54And what was interesting was
• 20:55that that traditional groove that
• 20:57I started out telling you
• 20:58all about is over here.
• 21:00So
• 21:01this chemical shift change perturbations
• 21:03that aligned on one face
• 21:04of the protein was not
• 21:05the famous part of the
• 21:07protein. It was not what
• 21:08we call the canonical groove
• 21:10that accepts b h threes.
• 21:11This was something different.
• 21:12And so what I did
• 21:13was I took that site
• 21:15and then I started, you
• 21:16know, painting it with the
• 21:18colors that,
• 21:19Steve Fesick and Craig Thompson
• 21:20painted the BCLXL binding pocket
• 21:23in their initial paper.
• 21:25And when I did that,
• 21:27I remember I mean, I
• 21:28remember the day
• 21:30that I did this in
• 21:31my, like, brand new office
• 21:33trying to figure out what
• 21:34the hell was going on.
• 21:35And I looked at this
• 21:36and I was like, well,
• 21:36there's that hydrophobic
• 21:38groove. That's nice. But what
• 21:39really blew my mind was,
• 21:40oh, look at the orientation
• 21:42of the positive charge and
• 21:43the negative charge in the
• 21:44hydrophilic. This looks like a
• 21:45very similar thing. Like from
• 21:47the upper left, blue, green,
• 21:48blue, red. That's the same
• 21:49thing going on on the
• 21:50other side. And then I
• 21:51got really excited. Like maybe
• 21:52this is an alternative BH3
• 21:54binding site.
• 21:55But then we had a
• 21:56problem because you can't solve
• 21:57a structure in two hours,
• 21:59by NMR. And so we
• 22:00had to come up with
• 22:01some alternative approach. And at
• 22:03that point,
• 22:04Everest Gavathiotis,
• 22:05you know, was giving a
• 22:06talk about how he had
• 22:07done some work on solving
• 22:09structures of weak protein protein
• 22:10interactions by NMR.
• 22:12And I said, you gotta
• 22:13come and help me with
• 22:14this and join the lab
• 22:15and be be a post
• 22:16doc,
• 22:17again, around the time that,
• 22:19Sam,
• 22:20also joined. And and Everest
• 22:21was like, well, we could
• 22:22do p r e NMR
• 22:23where you stick a paramagnetic
• 22:25label at the end of
• 22:25the peptide,
• 22:27and that should disrupt the
• 22:29pattern of chemical shift perturbation.
• 22:31And then you'll see at
• 22:32least orientation wise, like, where
• 22:34this thing is binding on
• 22:35one side of the protein.
• 22:36And then you could stick
• 22:37the label on the other
• 22:38side and, you know, figure
• 22:40out where it's binding on
• 22:41the other side. And then
• 22:42things that don't change are
• 22:43usually the ones that are
• 22:44kinda, like, right there in
• 22:45the middle. And with these
• 22:48distance constraints, you could calculate
• 22:49a model structure.
• 22:50And so that's what we
• 22:51did, and we got this.
• 22:52And what was super satisfying
• 22:54about this is that that,
• 22:55you know, all the electrostatic
• 22:57pairings
• 22:57were perfectly lined up, the
• 22:59hydrophilic pairings perfectly lined up,
• 23:01and it looked really beautiful.
• 23:03You know, that you had
• 23:03this very tight binding interaction
• 23:05from the hydrophobic face, and
• 23:06then it was reinforced by
• 23:08this complimentary charge charge hydrophilic
• 23:10network.
• 23:11And and that looked very
• 23:12much like the traditional b
• 23:13h three and group that
• 23:14was solved for BCLXL,
• 23:16except this site was a
• 23:17bit more shallow.
• 23:19So we had, of course,
• 23:20prove this. So we started
• 23:21out with two assays, an
• 23:22oligomerization
• 23:23assay, where you actually look
• 23:24to see whether your ligand
• 23:25could make BACS go from
• 23:26a monomer to a higher
• 23:27order species. And then also
• 23:29like a more, physiologic one
• 23:31where you could take mitochondria
• 23:33and do the experiment on
• 23:34a mitochondria and see if
• 23:35they start releasing cytochrome c.
• 23:37And so we had our
• 23:38positive control, you know, that
• 23:40we had started with with
• 23:41our peptide and our protein,
• 23:42and then we started mutating,
• 23:44like, key residues, you know,
• 23:46in this interaction network. And
• 23:47every time we hit one
• 23:49key interaction, now all of
• 23:50a sudden these assays did
• 23:51not work.
• 23:53And so that got us
• 23:54very excited. And then the
• 23:55other thing we noticed was
• 23:57that every time we calculated
• 23:58a model structure based upon
• 24:00our data and we looked
• 24:01at Niko Chandra's structure where
• 24:03you have this protein and
• 24:04in in in magenta, I'm
• 24:06showing you the binding site,
• 24:07and then this green colored
• 24:09area is a loop, the
• 24:10alpha one alpha two loop
• 24:12that sits between the two
• 24:13alpha one alpha two helices.
• 24:14That was always in this
• 24:15what we ended up calling
• 24:16the closed confirmation.
• 24:18But when our peptide was
• 24:20in there,
• 24:20it was always in what
• 24:22we call the open confirmation.
• 24:23In other words, this loop
• 24:24was kicked out.
• 24:26And I showed you at
• 24:27the beginning that when this,
• 24:28you know, when the pin
• 24:29is pulled on this grenade,
• 24:30like, this protein totally changes
• 24:31its shape.
• 24:32And so we started to
• 24:34think maybe
• 24:35the the catalytic conformational change
• 24:38is this displacement of the
• 24:39loop from a closed to
• 24:41an open position.
• 24:42Now the one thing that
• 24:43got me even more excited
• 24:45about this was that at
• 24:46the time, if you were
• 24:47studying BAX, there was an
• 24:49antibody
• 24:50that everyone would buy,
• 24:52came from Richard Ewell's lab,
• 24:53also at NIH, and it
• 24:55was the
• 24:56confirmation specific
• 24:58activated form of BAX. Right?
• 24:59So if you had BAX
• 25:00in a cell or BAX
• 25:01in a test tube and
• 25:02it was inactive, this antibody
• 25:04did not recognize it, couldn't
• 25:05immunoprecipitate
• 25:06anything. But if you activated
• 25:08BAX in a cell or
• 25:09in solution, and then you
• 25:10put this antibody and now
• 25:12magically it it binds to
• 25:13activated bacs,
• 25:15the epitope
• 25:16that that bound to was
• 25:18underneath
• 25:19the loop and became exposed
• 25:21when the loop opened.
• 25:23So we're like, wow. Okay.
• 25:24Maybe this is actually we're
• 25:26sniffing something out here that
• 25:27makes sense. That the six
• 25:29a seven epitope that defined
• 25:31activated backs was covered by
• 25:33the loop in the inactive
• 25:34form and was completely exposed
• 25:36when the loop was opened.
• 25:39So we wanted to test
• 25:40this. So what we did
• 25:41was
• 25:42we
• 25:44installed two cysteines and made
• 25:46a disulfide
• 25:47tethered version
• 25:49of backs where the loop
• 25:50could not open
• 25:52because now the loop is
• 25:53literally covalently
• 25:55stuck into the binding site.
• 25:57And we did, like, very
• 25:58simple experiments. So we we
• 26:00we basically made this protein.
• 26:02We oxidized it. We added
• 26:04the ligand, and we didn't
• 26:05see anything.
• 26:06And we needed to prove
• 26:07that it wasn't just oxidizing
• 26:09VAX was the problem. So
• 26:10then we oxidized
• 26:11normal VAX, not mutated VAX,
• 26:13and it worked fine. So
• 26:15that was that was lucky
• 26:16for us that the oxidation
• 26:17of wild type VAX did
• 26:18not disrupt it. So then
• 26:20we did the experiment that
• 26:21we were really wanting to
• 26:22do is we took this
• 26:23disulfide,
• 26:24you know, closed version, and
• 26:26we just threw in reducing
• 26:27agent, and then all of
• 26:28a sudden it's back.
• 26:30Right? And then we had
• 26:31to show that if you
• 26:31just reduce wild type bacs,
• 26:33it doesn't just, like, auto
• 26:34activate it, and it doesn't.
• 26:35Okay. So clearly, when you
• 26:37allow that loop to open,
• 26:38it works. And when you
• 26:39don't allow it to open,
• 26:40now the protein is dead.
• 26:43So now we really started
• 26:44to
• 26:46think, okay, we're gonna start
• 26:47marching down that picture that
• 26:48I showed you of, like,
• 26:49what in the world is
• 26:50going on with this protein
• 26:51from start to finish? And
• 26:52that was kind of a
• 26:53that has been a project
• 26:54in my lab since I
• 26:56opened the lab. Like, step
• 26:57by step by step, what
• 26:58is going on with this
• 26:59protein? And so
• 27:01now we have the complex
• 27:03between the b h three
• 27:04and the and the protein.
• 27:05Then we had this loop
• 27:06thing happening,
• 27:08and then we thought, okay.
• 27:09But this protein goes from
• 27:11the cytoplasm
• 27:12to the mitochondria.
• 27:14How does that happen? And
• 27:16so we did some more
• 27:17short term NMR experiments,
• 27:19and we did
• 27:20a little bit longer and
• 27:21longer time courses up to
• 27:22two hours, higher and higher
• 27:24doses of our ligand. And
• 27:25what we saw was that
• 27:26there were chemical shift changes
• 27:28now
• 27:29in the c terminal helix
• 27:31of the
• 27:32protein. And the c terminal
• 27:33helix is the membrane insertion
• 27:35helix for BACS.
• 27:36So what we started to
• 27:37figure out here was that
• 27:39if you hit this thing,
• 27:40you know, on one side
• 27:41of the face, now all
• 27:42of a sudden the c
• 27:44terminal helix is allosterically
• 27:46popping out
• 27:47and probably responsible for it
• 27:48now going to the mitochondrial
• 27:50membrane.
• 27:51And so, again, that was
• 27:52a hypothesis based upon the
• 27:53NMR, and you could see
• 27:55these beautiful dose responsive changes
• 27:57in the shifts of all
• 27:58those residues in the c
• 27:59terminal helix.
• 28:00So, you know, our disulfide
• 28:02trick, you know, hit the
• 28:04payload the first time, so
• 28:05we thought let's just do
• 28:06it again. So we now
• 28:07installed the the the disulfide
• 28:09bond between the c terminal
• 28:11helix and the traditional groove.
• 28:13And we said, okay. Now
• 28:15is BACS unable to go
• 28:16to the mitochondria?
• 28:18So we did this very
• 28:19simple mitochondrial translocation
• 28:21assay where we took the
• 28:23the protein, again, oxidized condition,
• 28:25reduced conditions,
• 28:26see if it goes from
• 28:27the supernatant to the mitochondria.
• 28:29And the only time we
• 28:31saw it going from the
• 28:31supernatant to the mitochondria was
• 28:33when we reduced the protein.
• 28:35And then, of course, once
• 28:36you allow it to go
• 28:37there, the cytochrome c now
• 28:39leaves from the mitochondria and
• 28:40goes into the supernatant.
• 28:42So it seemed that this
• 28:44direct displacement of alpha one
• 28:46alpha two loop and binding
• 28:47at that site was now
• 28:48being transmitted to the c
• 28:50terminal face of the protein
• 28:51and dislodging the c terminus.
• 28:53So now that pops out,
• 28:54sends your protein to the
• 28:55mitochondria.
• 28:58And we wanted to prove
• 29:00this, of course.
• 29:01And one of the things
• 29:03that was also a major
• 29:04aspect of the mechanism was
• 29:06that
• 29:07the b h three helix
• 29:08of BAX, I showed you
• 29:09that in the initial slide
• 29:10with the anti apoptotic protein,
• 29:13BAX is blocked by the
• 29:14anti apoptotic coming in and
• 29:16grabbing onto the b h
• 29:17three. So that means that
• 29:19that b h three must
• 29:20be exposed at some point
• 29:22in this mechanism, or else
• 29:24you would not be able
• 29:24to block BAX.
• 29:26Okay? So we wanted to
• 29:27see in our system if
• 29:28we could detect for the
• 29:30first time
• 29:31this confirmational exposure of the
• 29:32b h three.
• 29:34So we used antibodies initially.
• 29:36And so first, we did
• 29:38this experiment with normal bacs,
• 29:39and what we found was
• 29:41that we could detect
• 29:42the six a seven epitope,
• 29:44but we could not detect
• 29:45the b h three.
• 29:47And so we thought, well,
• 29:48maybe that's because
• 29:49it pops out and goes
• 29:50to the mitochondria, and it's
• 29:52just so incredibly fast that
• 29:54we can't capture it. So
• 29:56we thought, okay. Well, we
• 29:56have a way of stopping
• 29:57mitochondrial translocation. We have our
• 29:59disulfide linked version,
• 30:01which doesn't allow the tail
• 30:02to come out. So maybe
• 30:03we'll trap this thing in
• 30:04some intermediate form. And so
• 30:06we tried it again, and
• 30:08there's your, you know, dose
• 30:09responsive induction of the six
• 30:11a seven recognition.
• 30:13And now all of a
• 30:13sudden for the first time,
• 30:14we got dose responsive recognition
• 30:16of b h three. We
• 30:17had never seen that before.
• 30:20And so now you kind
• 30:21of start to imagine
• 30:24these three very important parts
• 30:25of the protein undergoing a
• 30:26conformational change that's allowing it
• 30:29to go to the mitochondria
• 30:30and and go to the
• 30:31next step, which is, like,
• 30:32self association and membrane disruption.
• 30:35But there was
• 30:37a weird,
• 30:38missing link,
• 30:40and that was that this
• 30:41process was catalytic
• 30:43so that you could put
• 30:44in the tiniest amount of
• 30:46a staple BIM peptide or
• 30:48the tiniest amount of the
• 30:49native bid protein,
• 30:52not stoichiometric,
• 30:54and this thing would just
• 30:56fire.
• 30:57And it was not clear
• 30:59how that happens.
• 31:01But
• 31:02we were staring, you know,
• 31:04at these sequences, and we
• 31:05realized that the backs b
• 31:07h three sequence
• 31:08in the core homology domain
• 31:11area that defines what a
• 31:12b h three is was
• 31:13essentially identical to the triggering
• 31:16b h three sequence.
• 31:19You could see that here.
• 31:20And so we thought, you
• 31:21know, maybe the mechanism is
• 31:23that BIM comes in, literally
• 31:24lights the match, goes to
• 31:26that new site,
• 31:28activates the protein,
• 31:29backs his b h three
• 31:30pops out. And now backs
• 31:32his b h three can
• 31:33function like the initiating BIM
• 31:34b h three, and then
• 31:35it just activates itself from
• 31:37there on.
• 31:38That was the idea. And
• 31:39then we're trying to figure
• 31:40out how in the world
• 31:41we were going to prove
• 31:42this.
• 31:42And so what we decided
• 31:44to do was, like, a
• 31:45reverse complementary
• 31:46mutagenesis
• 31:47experiment
• 31:48because we knew from the
• 31:49BIM b h three BACS
• 31:50interaction that there was this
• 31:51very important k twenty one
• 31:53e sixty nine interaction.
• 31:55And we thought, well, if
• 31:57we could activate BACS in
• 31:58another way that didn't involve
• 31:59a ligand,
• 32:00right, we could see if
• 32:02maybe these two things would
• 32:03touch each other between two
• 32:04activated forms of BACS.
• 32:06And Doug Green at Saint
• 32:08Jude had published this great
• 32:09paper saying that, you know,
• 32:10heat activates backs, which makes
• 32:12sense because it's a confirmationally
• 32:14kind of labile protein. So
• 32:16you heat it up and
• 32:17you start allowing it to
• 32:18breathe, and then, you know,
• 32:19it would activate itself. So
• 32:20we thought, okay. That's gonna
• 32:21be the system we'll try.
• 32:23So we tried it and
• 32:24we just gave plain old
• 32:25wild type backs. We heated
• 32:26it up and over time
• 32:28it started to aligmarize, which
• 32:29is exactly what Doug had
• 32:30had published.
• 32:32And then we mutated one
• 32:33of these residues at a
• 32:34time. And if you mutated
• 32:36one of these electrostatic pairs,
• 32:39nothing.
• 32:40If you swap the position
• 32:42and did a reverse complementary
• 32:43mutagenesis, you were back to
• 32:44the beginning.
• 32:46And again, this was such
• 32:47a simple experiment. It was
• 32:49one of my favorite experiments
• 32:50from this paper because,
• 32:52you know, all you did
• 32:53was move the e to
• 32:53the other guy and move
• 32:54the k to the other
• 32:55guy, and then all of
• 32:55a sudden you restored wild
• 32:57type activity. So clearly, they
• 32:58were touching each other and
• 32:59clearly they could activate BAX
• 33:01could autoactivate itself and explained
• 33:03a really elegant paper done
• 33:04by David Andrews' group where
• 33:06they actually would take the
• 33:08BIDDH three, they put it
• 33:09on to BAX, you know,
• 33:11on a liposome, and then
• 33:12they would
• 33:13freeze stop the experiment,
• 33:15take away the supernatant. Right?
• 33:16They would do an immediate
• 33:17spin down, get rid of
• 33:18all the ligand, and they
• 33:19would have their pellet. And
• 33:20then they would take the
• 33:21pellet and just add more
• 33:22inactive BAX to it, and
• 33:24it always worked.
• 33:25Right? And the reason why
• 33:27it worked was because there
• 33:28was active backs in the
• 33:29membrane already, and then you
• 33:30added inactive backs and the
• 33:32backs activated itself. So it
• 33:34explained that.
• 33:36So here's kind of where
• 33:37we were.
• 33:38Hand grenade,
• 33:39totally quiescent, nothing going on.
• 33:41You have a ligand in
• 33:42response to stress.
• 33:44It pops the loop. It
• 33:45changes the confirmation. Alpha nine
• 33:47pops out. Alpha three pops
• 33:49out. B h three pops
• 33:50out, goes to the mitochondria,
• 33:52and now it can start
• 33:53and activate more and more
• 33:54and more and trigger this
• 33:55chain reaction
• 33:56to the point where you
• 33:57have a critical mass of
• 33:58backs at the outer membrane
• 33:59and you get permeabilization.
• 34:01So that's where we were.
• 34:03Canonical pocket
• 34:04was not accessible
• 34:06to b h threes because
• 34:07the alpha nine stuffed in
• 34:09it. So that made a
• 34:10lot of sense. And so
• 34:11the actual trigger site was
• 34:13on the opposite side.
• 34:15Now here's a very kind
• 34:16of in the weeds subtle
• 34:17point.
• 34:18If the b h three
• 34:19is bound to anti apoptotic
• 34:21proteins,
• 34:23why wouldn't they bind to
• 34:24this other site? I mean,
• 34:25it's sitting there. I drew
• 34:26it for you in orange.
• 34:27And it turns out the
• 34:28difference between the two sites,
• 34:30and you can see this
• 34:30with your own eyes right
• 34:31here, the red site is
• 34:32very deep.
• 34:34The orange site is shallow.
• 34:36And it turned out that
• 34:38a prefolded helix
• 34:40was needed to bind to
• 34:41the trigger site, but you
• 34:42didn't need a prefolded helix
• 34:44to bind to the canonical
• 34:45one because it was deeper
• 34:46and the, you know, induced
• 34:48folding was much more powerful.
• 34:50And so it just so
• 34:51happened that having a prefolded
• 34:52helix
• 34:53was what you needed to
• 34:54detect that other site because
• 34:56there wasn't enough
• 34:57interaction to cause induced folding
• 34:59at that site.
• 35:00But t bid, the ligand,
• 35:02is a highly structured protein.
• 35:03The b h three is
• 35:04already structured.
• 35:07So then at the same
• 35:07time, we're like, well, this
• 35:09could be a druggable binding
• 35:10site for turning on backs
• 35:11in cancer.
• 35:13But, again, we had a
• 35:13lot of limitations. And the
• 35:15limitation the biggest limitation was
• 35:17you couldn't make a ton
• 35:18of backs. And like I've
• 35:19been saying, it's not the
• 35:19most stable thing. So if
• 35:21you're going to do a
• 35:22drug screen, you know, with
• 35:23and you need a ton
• 35:24of ton of backs, it's,
• 35:25like, very difficult because it's,
• 35:26now you see it, now
• 35:27you don't. And so
• 35:29we later conquered that problem.
• 35:30But but at the beginning
• 35:32days, we're like, well, we're
• 35:33gonna have to do in
• 35:33silico screening. Now look at
• 35:35the date. So the until
• 35:36you know, the paper was
• 35:37twenty twelve, which means, you
• 35:38know, we probably started this
• 35:40and we started this pretty
• 35:41much right when we had
• 35:42solved the complex, I would
• 35:43say, two thousand and nine,
• 35:44maybe.
• 35:45We started doing in silico
• 35:47screening to try to figure
• 35:48out if we could find
• 35:48something. And and the tools
• 35:50back then were not like
• 35:50they are today. That's for
• 35:52sure. But,
• 35:53I think we got very
• 35:54lucky. And and and Everest,
• 35:55who's the post doc doing
• 35:56this work, had actually spent
• 35:57time at a company in
• 35:58England,
• 35:59before he did his post
• 36:00doc doing,
• 36:01in silico screening using novel
• 36:03methods. And so that was
• 36:04a very useful skill that
• 36:06he brought to the table
• 36:07here. And so he did
• 36:08that in silico screen,
• 36:10and we found a bunch
• 36:11of compounds. And we just
• 36:12did a competitive binding assay
• 36:13against our fluorescent b h
• 36:15three peptide to see which,
• 36:17if any, of them could
• 36:18actually displace.
• 36:19And it turned out that,
• 36:21you know, most of them
• 36:22didn't work, and a few
• 36:23of them did.
• 36:24And, you know, we then
• 36:25went ahead and tried a
• 36:26dose responsive experiment. And most
• 36:28of them that did actually
• 36:29compete were quite weak except
• 36:31for one.
• 36:33And, you know, we had
• 36:34gotten this from a library
• 36:35that came with, you know,
• 36:36tons and tons of compounds.
• 36:38So to make sure about
• 36:39the integrity of this thing,
• 36:40we resynthesized it ourselves, repeated
• 36:41the experiment,
• 36:42and the compound was legit.
• 36:45And that's what it looked
• 36:46like. Pretty greasy compound.
• 36:48And we went ahead and
• 36:49did the NMR. We repeated
• 36:50all the experiments that we
• 36:51had done with the b
• 36:52h three, but now with
• 36:53this molecule, and most of
• 36:54the results were identical. So
• 36:55here's, you know, the
• 36:57the chemical shift perturbations that
• 36:58occur. Again, alpha one, alpha
• 37:00six.
• 37:01You see where the the
• 37:02docking suggested that it was
• 37:04disposed, that k twenty one
• 37:05residue, which we found was
• 37:06very important with the natural
• 37:08interaction, again, was very much
• 37:10part of how the molecule
• 37:11interacted,
• 37:12at this site.
• 37:13And then we did a
• 37:14oligomerization assays and showed that
• 37:16you could get, you know,
• 37:17dose responsive
• 37:18triggering of BAX in a
• 37:19liposomal release assay.
• 37:21But if you had a
• 37:22ligand that,
• 37:23looked a lot like the
• 37:24triggering one but did not
• 37:26do anything in this assay,
• 37:27it didn't do anything either.
• 37:29And then if you mutated
• 37:30that lysine twenty one on
• 37:31VAX, which doesn't have any
• 37:33endogenous activity, you also saw
• 37:35much, much blunted
• 37:36activation by the small molecule.
• 37:38So it looked like it
• 37:39was obeying a lot of
• 37:40the same criteria,
• 37:42that we had established for
• 37:43the peptide.
• 37:44And then, you know, among
• 37:45the more exciting results is
• 37:46that if you took cells
• 37:47that had BAX in it
• 37:49but no BAX, you saw,
• 37:51dose responsive,
• 37:52induction of cell death. But
• 37:54if it didn't have BAX
• 37:55in it, then the cell
• 37:56didn't respond. And if it
• 37:57didn't have BAX or BAX,
• 37:59of course, it then didn't
• 38:00respond either as the ultimate
• 38:01negative control.
• 38:02And, again, you could look
• 38:03at this under the microscope,
• 38:06and so satisfying to actually
• 38:08just like
• 38:09I I have to say,
• 38:10by the way, histology and
• 38:11I'm not saying this because
• 38:12of the crowd because you
• 38:13can valid you could verify
• 38:14my comments here. Histology
• 38:16was always my favorite subject
• 38:17in med school. I TA
• 38:18histology and histopathology
• 38:20at Hopkins for years all
• 38:21throughout grad school.
• 38:23And a lot of times
• 38:24and and so everyone in
• 38:25this room really enjoys seeing
• 38:27things with their own eyes.
• 38:28But a lot of times,
• 38:28people don't look, you know,
• 38:30at their experiment. Like, you
• 38:31know, they'll do a ninety
• 38:32six well plate or something.
• 38:33It's like, did you look
• 38:34at the cells? What do
• 38:35they look like? Oh, I
• 38:35didn't no. I just put
• 38:36it in the plate reader.
• 38:38And when this was happening,
• 38:39I was like, you know,
• 38:39it'd be kinda nice to
• 38:40look at this and see
• 38:41if it looks like apoptosis
• 38:42as opposed to God knows
• 38:43what else.
• 38:45And so for me, I
• 38:46love this experiment because I
• 38:47just went to the, you
• 38:48know, microscope and, you know,
• 38:49face contrast microscope and started
• 38:51taking pictures. I was like,
• 38:52that looks like really nice
• 38:53apoptosis, like, right out of
• 38:54a textbook, which was very
• 38:56reassuring.
• 38:57So, you know, I don't
• 38:58have to convince you a
• 38:58picture's worth a thousand words,
• 39:00you know. Yeah. I mean,
• 39:00that that it really is.
• 39:04So, you know, my message
• 39:05for this part of the
• 39:06talk, you know, what is
• 39:07that,
• 39:09you know, a lot of
• 39:09basic science, you know, went
• 39:11into figuring, you know, these
• 39:13steps out. And, you know,
• 39:14we kinda walked along from
• 39:16discovering the trigger site and
• 39:18then figuring out what all
• 39:19the downstream steps were and
• 39:21then figuring out if that
• 39:22site could be drugged. And
• 39:23then my postdoctoral fellow is
• 39:25now full professor of biochem
• 39:26at Albert Einstein, went on
• 39:27to work on this more
• 39:28and took it into animals
• 39:29and worked a lot on
• 39:30the pharmacology and the optimization.
• 39:33And that gives you an
• 39:35entire
• 39:36other arm of the cell
• 39:37death pathway
• 39:38to think about drugging. Right?
• 39:40There's
• 39:41billions and billions of dollars
• 39:42focused on the anti apoptotic
• 39:43side, but there's a whole
• 39:45another side to this pathway,
• 39:47and that's the pro apoptotic
• 39:48side. And, you know, we
• 39:49talked about this at dinner
• 39:50last night as well, but,
• 39:51like,
• 39:52you might have something that
• 39:53you're very excited about, but
• 39:54that doesn't mean that anyone
• 39:55else is going to get
• 39:56excited about it. Right?
• 39:58Youth may think you have,
• 39:59you know, great mechanism, great
• 40:01target, great prototype therapeutic,
• 40:03but then the the the
• 40:04bigger job and and sometimes
• 40:06the job that never pays
• 40:08off, not not literally, but
• 40:10figuratively,
• 40:11is, like, getting someone to
• 40:13give it a shot because
• 40:14giving it a shot is
• 40:15incredibly expensive. And so, you
• 40:17know, I felt like my
• 40:17part was to kind of
• 40:18write a review and say,
• 40:19hey. VAX is a great
• 40:20target. You know?
• 40:23And and so I think,
• 40:24you know, there's a lot
• 40:24of, you know, sociology
• 40:26around how do you go
• 40:27from new discovery to new
• 40:29drug, and it may or
• 40:30may not happen. But, you
• 40:32know, I'm I'm one who
• 40:33believes that you just gotta
• 40:34keep trying. Right? If you
• 40:35just keep trying, you just
• 40:36keep trying. You know, you're
• 40:38destined to fail so many
• 40:39times, but if you give
• 40:40up with your first, second,
• 40:41or third failure, like, you
• 40:42know what the numbers are.
• 40:43You know it's a ninety
• 40:44nine percent failure rate. But
• 40:46there's a lot of blockbuster
• 40:47drugs. And, like, we started
• 40:48out talking about venetoclax. Like,
• 40:50that could so have easily
• 40:52been on the cutting room
• 40:53floor
• 40:55years ago,
• 40:56but it was or human
• 40:57beings that that kind of
• 40:59forced that to keep going.
• 41:01So, anyway,
• 41:02don't get discouraged.
• 41:05So then, you know, a
• 41:07big part of science also
• 41:08is, like, knowing what other
• 41:09people are doing.
• 41:10Right? You can't live in
• 41:11a like, I love facts,
• 41:13but, like, I can't just
• 41:15live in a vacuum. And
• 41:16so, you know, you read
• 41:17and you talk and you
• 41:18go to meetings. And,
• 41:19and we had come across
• 41:20this beautiful paper in cell
• 41:22by, Doug Green's group. Jerry
• 41:24was the first author. He's
• 41:25at Mount Sinai, a full
• 41:26professor.
• 41:28And they had found this
• 41:29interesting thing where, like, it
• 41:30seemed like certain aspects of
• 41:31sphingosine lipid metabolism,
• 41:34was relevant to BACS activation.
• 41:36And they did this study
• 41:37showing that, you know, he
• 41:38literally took every byproduct of
• 41:40singamyel and metabolism and showed
• 41:41that only one of the
• 41:43byproducts, this this lipid hexadec
• 41:45e now,
• 41:48like, sensitized release of, you
• 41:50know, cytochrome c when you
• 41:51threw Bax on there. None
• 41:52of the other lipids did
• 41:53that.
• 41:54And it was really interesting,
• 41:55and it suggests that there
• 41:56was crosstalk between the endoplasmic
• 41:58reticulum and the mitochondria
• 42:00and that, you know, lipids
• 42:01made in the ER were
• 42:02being shuttled to the mitochondria
• 42:03and it's sensitized.
• 42:05And he did beautiful work.
• 42:06You know, if you shut
• 42:06down this pathway, BAX doesn't
• 42:08activate as well in the
• 42:09mitochondria.
• 42:10And so I had a
• 42:11chemistry grad student,
• 42:13in the that joined the
• 42:14lab, and he read the
• 42:15paper, and he looked at
• 42:17the hexadec enal. And there
• 42:18was no structure,
• 42:20you know, and there were
• 42:22no there was no you
• 42:23know, you had to be
• 42:24like a chemistry person to
• 42:25kind of look at this.
• 42:26He looked at this, and
• 42:27in one second, he said,
• 42:28oh, that's a lipid electrophile
• 42:29off of beta unsaturated aldehyde.
• 42:31That is super reactive.
• 42:33And I was like, okay.
• 42:34He's like, oh, I bet
• 42:35you that, you know, any
• 42:36cysteine in BACs is gonna,
• 42:37like, fire and covalently modify.
• 42:40And so I was like,
• 42:41alright. Well, go check it
• 42:42out. So, you know, he
• 42:43comes back with this mass
• 42:44spec, and look at these
• 42:45doses. I mean, you need
• 42:46it a lot.
• 42:47But he was able to
• 42:48completely convert inactive backs in
• 42:50solution
• 42:51to fully,
• 42:53you know, what what I
• 42:54don't know what the right
• 42:55word is. Hexadec
• 42:56e analyzed,
• 42:57you know, derivatized
• 42:58backs.
• 42:59And then if you reduced
• 43:02that and delivered, you know,
• 43:03not alpha beta unsaturated
• 43:05aldehyde, then it it didn't
• 43:06work at all.
• 43:08So, clearly, this was a
• 43:09chemical reaction that was happening.
• 43:12And the question was, you
• 43:13know, was this actually what
• 43:14was happening,
• 43:15in in in in cells?
• 43:17And so, you know, we
• 43:18put it through the system
• 43:19and show that when you
• 43:20add more and more of
• 43:21this, hexadec e now, you
• 43:22get more and more oligomerization.
• 43:24If you use the reduced
• 43:25form, you don't.
• 43:27We then went ahead and
• 43:28did NMR,
• 43:29and
• 43:30completely different area of the
• 43:31protein was was modified as
• 43:33opposed to all the other
• 43:34NMR that I've showed you
• 43:35today.
• 43:37And a lot of the
• 43:38action was around,
• 43:40cysteine
• 43:40one twenty six.
• 43:43And you can see the
• 43:43residues that underwent chemical shift
• 43:45change here. They were all
• 43:46at the core,
• 43:48of the protein and and
• 43:49the in and the residues
• 43:50that it interacted with circumferentially.
• 43:53And, again, I kept I
• 43:55keep saying, like, grenade, but
• 43:56part of the analogy of
• 43:57the grenade is that, like,
• 43:57alpha five is kind of
• 43:59like the pin. Right? That's
• 44:00the helix that kind of
• 44:01traverses through the whole protein.
• 44:02It's the one helix that
• 44:03interacts with every other helix,
• 44:05in the structure of the
• 44:06protein. So we thought, well,
• 44:08that's kind of interesting.
• 44:09You know, how do we
• 44:10prove this? So we have
• 44:11this liposomal system
• 44:13that has fluorophore in it.
• 44:14You add the protein. You
• 44:15add the ligand. It triggers
• 44:17backs to translocate, and then
• 44:18you just measure the fluorophore.
• 44:20Sometimes the simplest assays are
• 44:22really the best assays because
• 44:24it's just like here, it's
• 44:25like, does the fluorophore get
• 44:26released or does it not?
• 44:28And so you can detect
• 44:29this, you know, and you
• 44:30can see that inactive,
• 44:32BACS, you know, has this
• 44:33little background release because, again,
• 44:35you know, because pure as
• 44:36you make it, there's always
• 44:37some activated in there, so
• 44:38you get a little tiny
• 44:39bit of release. But then
• 44:40you put in the staple
• 44:41peptide, and you get nice
• 44:42release.
• 44:44When you make the liposomes
• 44:45with these lipids, which was
• 44:47what we did, if you
• 44:48take the reduced version of
• 44:49the lipid, it was no
• 44:50different than not having a
• 44:51lipid added at all.
• 44:53But if you take the
• 44:54hexadecenal
• 44:55version in the liposomes, now
• 44:57all of a sudden, the
• 44:58BAX alone starts to activate.
• 45:00And then when you add
• 45:01the BIM in there to
• 45:02trigger BAX, it activates way
• 45:04better.
• 45:05Now this starts to sound
• 45:06a little bit like what
• 45:07Jerry Chipick was saying in
• 45:08his cell paper that this
• 45:10lipidation,
• 45:12right, this covalent lipidation that
• 45:14we discovered was somehow
• 45:16sensitizing BACCS activation.
• 45:19And so the next question
• 45:21was, you know, which cysteine?
• 45:22Right? So we kind of
• 45:23did the same experiment,
• 45:24but we took out cysteine
• 45:26one twenty six, on the
• 45:27left, and we took out
• 45:28cysteine sixty two on the
• 45:29right. And you can clearly
• 45:31see that cysteine one twenty
• 45:33six is your cysteine. Right?
• 45:34Because it looks just like
• 45:35wild type, you know, with
• 45:37no goosing from the lipid,
• 45:39on the left. But on
• 45:40the right hand side, when
• 45:41you get your cysteine one
• 45:42twenty six back, now it
• 45:43looks just like, you know,
• 45:44triggering and sensitizing with the
• 45:46lipid. And And so we
• 45:47did this in mitochondria. Again,
• 45:49we saw the same thing
• 45:50that we got sensitization just
• 45:51like wild type on mitochondria,
• 45:53which has native,
• 45:54you know,
• 45:55ligand in there. Here, we're
• 45:57just taking advantage of the
• 45:58native hexadecimal
• 45:59or whatever else is in
• 46:00the outer membrane. And when
• 46:02you mutate that c one
• 46:03twenty six, much, much, much
• 46:04blunted,
• 46:05mitochondrial cytochrome c release when
• 46:07you add the triggered back.
• 46:08So that was reassuring.
• 46:10And then we did some
• 46:11reconstitution
• 46:12studies where we put into
• 46:13double knockout
• 46:15cells wild type and then
• 46:16c one twenty six a
• 46:17mutant back and then treated
• 46:18with the pro apoptotic stimuli.
• 46:20The double knockouts are very
• 46:21resistant, not surprisingly, when you
• 46:23give them a combined,
• 46:25BCL two and MCL one
• 46:26inhibitor.
• 46:27When you reconstitute wild type,
• 46:29you restore killing. And our
• 46:30c one twenty six a
• 46:32mutant was
• 46:33somewhere in the middle. It
• 46:34was just not
• 46:36all of what wild type
• 46:37bacs was before when you
• 46:39took out that cysteine.
• 46:40And so what we discovered
• 46:41was that there's this non
• 46:43enzymatic
• 46:44lipidation that occurs when BAX
• 46:46ends up at the outer
• 46:47membrane where c one twenty
• 46:49six gets modified by this
• 46:50hexadec enal,
• 46:52lipid electrophile, and that sensitizes,
• 46:55BAX activation.
• 46:57So that led us to
• 46:58the next thing, which was,
• 46:59well, cysteine one twenty six,
• 47:02covalent inhibitors, covalent activators. That's
• 47:04in. Right? Like, ten years
• 47:06ago or whenever that was,
• 47:08if you mentioned a covalent
• 47:09inhibitor. I had a grad
• 47:10student in my lab that
• 47:11was gonna work on a
• 47:11covalent inhibitor. Her DAC said
• 47:13absolutely not. Only focus on
• 47:15noncovalent inhibitors. You know, covalent
• 47:17inhibitors and activators are passe.
• 47:19Now, of course, they're all
• 47:20the rage. So so when
• 47:21this happened, you know, people
• 47:23were getting excited about covalent
• 47:25modifiers again. So we're like,
• 47:26okay. Let's see if we
• 47:27can get a covalent modifier
• 47:29of the cysteine. And Jim
• 47:30Wells' group had published his
• 47:31disulfide tethering strategy, and they
• 47:34used that to develop, you
• 47:35know, KRAS inhibitor,
• 47:37with Kevan Shokat. And so
• 47:38we thought this would be
• 47:39a really cool technique to
• 47:40apply to back cysteine one
• 47:42twenty six. So we gave
• 47:43it a shot, and the
• 47:44grad student who discovered the
• 47:46alpha beta unsaturated lipid, you
• 47:48know, we shipped him to
• 47:49Jim's lab,
• 47:50for two months, and it
• 47:51was he loved that because
• 47:52he was from San Francisco,
• 47:53so he got to go
• 47:54home and work in Jim's
• 47:55lab to do the screen.
• 47:57And then he brought the
• 47:58compounds back,
• 47:59and it turned out that
• 48:00this screen was successful that
• 48:01we found, binders that were
• 48:03very effective at covalently modifying
• 48:05cysteine one twenty six even
• 48:06in the presence of, you
• 48:07know, lots of BME.
• 48:10And you could then go
• 48:12ahead and say, okay. I'm
• 48:13back to my liposomal experiment.
• 48:15We call the compound covalent
• 48:16BAX inhibitor one, And you
• 48:18could see that, you know,
• 48:19it really didn't affect,
• 48:21BACS alone other than actually
• 48:23get rid of your background
• 48:24BACS activation. So that was
• 48:25kind of like a nice
• 48:26thing. That little background BACS
• 48:27activation that you always see
• 48:29with pure BACS, it suppressed
• 48:30that.
• 48:31But then when you trigger
• 48:33BACS with the normal ligand,
• 48:35you got this big release
• 48:36and the compound seemed to
• 48:37be blocking that to some
• 48:38extent. And when we repeated
• 48:40this and did dose responsive
• 48:41work, I mean, it looked
• 48:42very real.
• 48:44So we went ahead and
• 48:45then had to go
• 48:46back and do the same
• 48:46thing, which cysteine. Let's make
• 48:47sure it's the right cysteine.
• 48:49So we did, our mutagenesis
• 48:51work, and we showed that
• 48:52cysteine one twenty six. You
• 48:54you take that out, and
• 48:55there's now no longer derivatization.
• 48:56So it's very, very selective
• 48:59to the to the cysteine
• 49:00one twenty six. Didn't bother
• 49:01cysteine sixty two at all.
• 49:04So then we went to
• 49:05the NMR. Like, how does
• 49:06this look like libidation? And
• 49:07it turned out it kinda
• 49:09looked a lot like libidation.
• 49:10Again, it was, you know,
• 49:11causing chemical shift perturbations
• 49:13in and around cysteine one
• 49:14twenty six.
• 49:17But it's an inhibitor,
• 49:18and I've been telling you
• 49:19that libidation is an activator.
• 49:21And so
• 49:22what's going on?
• 49:24Right? And if you talk
• 49:25to Jim and you talk
• 49:26to the folks that do
• 49:27these studies with, you know,
• 49:28doing modification,
• 49:30they will tell you that
• 49:31they always get both sides
• 49:32of the coin in their
• 49:33compounds. They'll get activators and
• 49:35inhibitors. It kind of really
• 49:36depends on the protein and
• 49:37depends on the screen. And
• 49:38the difference between an activator
• 49:40and inhibitor is, like, mechanistically
• 49:41and structurally very interesting.
• 49:44But we were very intrigued
• 49:45that now we had ourselves
• 49:46what looked like an inhibitor
• 49:47even even though we were
• 49:48going after an an activator
• 49:49site.
• 49:50And at the same time,
• 49:51we had another grad student
• 49:52working on the kind of
• 49:53like the pin in the
• 49:54grenade and studying what residues
• 49:56were very important
• 49:57at keeping backs inactive, and
• 49:59it ends up being these
• 50:00four residues here. If you
• 50:02take them out one by
• 50:03one in combination, as, you
• 50:05know, two or three or
• 50:06four, you could completely create
• 50:08hyperactivated
• 50:08forms of bax just by
• 50:10playing around with those four
• 50:11residues in the core.
• 50:13So we thought, wow. This
• 50:14is a good assay for
• 50:15us.
• 50:16Let's take a hyperactive
• 50:18semiautoactivated
• 50:19form of bax and see
• 50:20if our molecule
• 50:21could restore
• 50:23the inactive state.
• 50:25And so we took the
• 50:26f one sixteen a single
• 50:27mutant because that was our
• 50:28most active single mutant. You
• 50:30could see I'm putting it
• 50:31on,
• 50:33the liposomes with no stimulant,
• 50:34and it just fires on
• 50:35its own.
• 50:36And then you put in
• 50:37the the small molecule, and
• 50:38we saw dose responsive
• 50:40inhibition.
• 50:41And we saw this in
• 50:42the mitochondrial experiment as well.
• 50:44And that was super exciting
• 50:46to us. And then we
• 50:47decided to, years ago, initiate
• 50:50the application of this really
• 50:51cool technique,
• 50:52to b c l two
• 50:53family structural analysis, which is
• 50:55hydrogen deuterium exchange mass spec,
• 50:58which,
• 50:59we started a collaboration with
• 51:01John Engen's group at Northeastern
• 51:02and Thomas Wells, and they
• 51:04were really world experts in
• 51:05this. And it's a really
• 51:06simple in principle,
• 51:08very technically difficult to do
• 51:09in terms of all the
• 51:10machinery and everything. But the
• 51:11concept is very simple, which
• 51:13is you have a protein
• 51:14that's got n h bonds.
• 51:16The h is exchange with
• 51:17water. If you put it
• 51:18in deuterated water, the h
• 51:19is will exchange with deuterium
• 51:22if those h is exposed.
• 51:24Right? So things on the
• 51:25surface, things that are flexible
• 51:27will exchange quickly.
• 51:29And then you allow that
• 51:30to happen over time if
• 51:31you'd like and do a
• 51:32time lapse photography style experiment.
• 51:34And then you could quench
• 51:35it all, digest everything, and
• 51:37throw it on the mass
• 51:38spec, and you're asking a
• 51:39pretty simple question. Which of
• 51:40my peptides gained weight?
• 51:43And you can watch certain
• 51:44peptides gain weight, and then
• 51:45you can watch certain peptides
• 51:47never gain weight if they're,
• 51:48like, in the core of
• 51:48the protein and never have
• 51:49access to the d two
• 51:50o.
• 51:51And then you can plot
• 51:52this out, and you could
• 51:53say, okay. Here's one that
• 51:54exchanges over time.
• 51:56And then you can look
• 51:56at all these pretty like
• 51:58fragments of your protein.
• 52:01I love this technique because
• 52:03it's not easy to do,
• 52:04you know, how to proteins
• 52:06change their structure in a
• 52:07membrane environment over time.
• 52:09And this gives you, like,
• 52:11essentially a movie
• 52:12of what's happening over time
• 52:13with your protein.
• 52:15And and so when we
• 52:16mutate the f one sixteen
• 52:17and I told you that
• 52:18it makes the protein more
• 52:20loose,
• 52:21you see now all of
• 52:21a sudden you're getting anything
• 52:23that's going up like a
• 52:24mountain is deep protection or
• 52:26or faster,
• 52:28conformational change and exposure than
• 52:29the normal one because everything
• 52:30is a subtraction here. So
• 52:32I'm showing you how does
• 52:33f one sixteen a move
• 52:34compared to wild type? Well,
• 52:36in those regions that are
• 52:37looking like mountains, those regions
• 52:38are magically moving more. And
• 52:40where are they? On the
• 52:41right hand side, oh, they're
• 52:43right where that phenylalanine
• 52:44used to be. So that
• 52:46aromatic and all those interactions
• 52:47around it are now gone
• 52:48because it's an alanine, and
• 52:49those areas are not being
• 52:51kind of tethered down anymore.
• 52:53And they're moving, and that's
• 52:54a problem because you get
• 52:55back starting to move whether
• 52:57by this or by heat,
• 52:58and it's gonna fire.
• 53:00When you add in the
• 53:01molecule
• 53:02to the f one sixteen
• 53:03a and say, what does
• 53:04it look like now when
• 53:05I add the molecule
• 53:07f one sixteen a hyperactive
• 53:09version of ax, it is
• 53:10a mirror image.
• 53:12It completely
• 53:13eliminates
• 53:15all of the hyperactivity
• 53:17structurally
• 53:18of what you started with.
• 53:20And this is the type
• 53:21of experimental data that you
• 53:23can get from,
• 53:25HDX, and then you can
• 53:26go and map it onto
• 53:26your protein and kinda see
• 53:28where it is. And, again,
• 53:29it's so satisfying because, again,
• 53:30look where the c one
• 53:31twenty six is on that
• 53:32picture. It's that red cysteine,
• 53:34and look what's protected all
• 53:36of a sudden.
• 53:37The residues
• 53:38of those peptide fragments that
• 53:39are staring at the cysteine.
• 53:42Right? So that molecule attached
• 53:44to the cysteine
• 53:45is now replacing what the
• 53:47phenylalanine did and is interacting
• 53:49with those areas and sucking
• 53:50it in to the point
• 53:51that now BACS is inactive
• 53:53again.
• 53:54And even beyond wild type.
• 53:56So we looked at, okay,
• 53:57how does the pattern look
• 53:59of the molecule bound to
• 54:00our hyperactive BACS versus just
• 54:02plain old BACS?
• 54:03And you see that there's
• 54:04even more protection
• 54:06than what you started with.
• 54:07And where is that more
• 54:08protection?
• 54:10Right across the street from
• 54:11the molecule that just derivatized
• 54:13your cysteines. It's super intellectually
• 54:15satisfying.
• 54:17And then the other part
• 54:18of this, which was a
• 54:19little bonus prize, was that,
• 54:22not only did it confirmationally
• 54:23constrain and everything I just
• 54:25showed you was stuff in
• 54:26solution,
• 54:27but I told you that
• 54:27lipidation is sensitizing and that
• 54:29occurs at the mitochondria. Well,
• 54:31if I take that cysteine
• 54:32out
• 54:33of the game
• 54:34and you're essentially, like, alkylating
• 54:35it. Right? Now all of
• 54:36a sudden, the lipid can't
• 54:37go there anymore, and it
• 54:39can't sensitize backs at the
• 54:40mitochondria anymore. So now you've
• 54:42done two things. You've confirmationally
• 54:44constrained the protein and you
• 54:45prevented it from interacting with
• 54:47the site that actually would
• 54:48stimulate its activation. And so
• 54:50we wanted to prove
• 54:50this, and so we took
• 54:52a reagent that reacted with
• 54:54free aldehydes.
• 54:55And when you form this
• 54:56covalent reaction, you still have
• 54:57your free aldehyde. It's a
• 54:59psi five labeled reagent.
• 55:01And you can see that
• 55:02when you add the vaccine
• 55:03with the,
• 55:05lipid,
• 55:06the lipid, you know, goes
• 55:07crazy and it modifies BAX,
• 55:09and then BAX becomes a,
• 55:10you know, heterogeneous
• 55:12oligomer and ladders like this.
• 55:15And when you add in
• 55:16the molecule, you get dose
• 55:17responsive inhibition of the laddering.
• 55:19And in fact, at the
• 55:20bottom right hand corner, you
• 55:21actually get your backs back.
• 55:23Right? Your your monomeric protein
• 55:25that, like, disappeared into this
• 55:26oligomer, you restored actually what
• 55:28you started with.
• 55:30So that kind of led
• 55:31us to this, you know,
• 55:33explanation that this cysteine one
• 55:36twenty six as a drug
• 55:36target
• 55:37can confirmationally
• 55:38constrain VACS,
• 55:40but could also competitively,
• 55:42inhibit VACS.
• 55:43And so that dual mechanism
• 55:45is kind of exciting to
• 55:46us. And if you remember
• 55:46at the beginning, I showed
• 55:47you this dial and you
• 55:48can block survival,
• 55:49you know, and do good
• 55:50in one area of diseases
• 55:51and you could block death
• 55:52in another area of diseases.
• 55:53And so we're really interested
• 55:55in this because, potentially, you
• 55:56could use this to, like,
• 55:58I don't know, infuse something
• 55:59that blocks back in the
• 56:00midst of a stroke or
• 56:01a heart attack or a
• 56:02nerve injury and temporarily
• 56:04arrest
• 56:05cell death. And we know
• 56:06that that's important because there's
• 56:07tons of mouse models out
• 56:08there and backs knockout mice
• 56:10and back knockout mice that
• 56:11you inhibit them
• 56:12genetically and do a, you
• 56:14know, induction of cardiac arrest
• 56:15or induction of stroke, and
• 56:17there's less death, you know,
• 56:18in the mice that don't
• 56:19have backs or backs. So,
• 56:20I mean, the the genetics
• 56:21part of this has already
• 56:22been proven, right, to be
• 56:23a potential benefit. Can we
• 56:25do something pharmacologic
• 56:26now with with this site?
• 56:29Okay. So what I've told
• 56:30you today is,
• 56:31we've spent a lot of
• 56:33time looking for ways to
• 56:34activate,
• 56:35BACs,
• 56:36and all of this I
• 56:38can't say it enough. All
• 56:39maybe I should say this
• 56:40at congress. All of this
• 56:42came from basic
• 56:45science, as basic as you
• 56:46possibly could be. Where does
• 56:48a peptide or a protein
• 56:49bind a protein
• 56:50target? Or where does oh,
• 56:52a lipid binds to a
• 56:53protein at the outer mitochondrial
• 56:54membrane. Oh, and that led
• 56:56to this whole idea about
• 56:57how to inhibit VAX. Like,
• 56:59connecting those dots. It all
• 57:00starts, you know, here. Right?
• 57:02It all starts in academia
• 57:04where you have the time
• 57:05over twenty years to flesh
• 57:06these things out so you
• 57:08can kind of figure out
• 57:08the biology, figure out what
• 57:10you could possibly do about
• 57:11it. Okay. In the last
• 57:13one minute, I'll leave you
• 57:14with, like, what's next.
• 57:16The last step.
• 57:18Okay?
• 57:20So, again, we've been kind
• 57:21of obsessively going through every
• 57:22step. The last step is
• 57:23how in the hell does
• 57:24this actually come together to
• 57:26dissociate, disrupt the membrane, and
• 57:28the answer is nobody knows,
• 57:29really. Okay? There's no structures.
• 57:32No one knows. So this
• 57:33is this has been called
• 57:34the holy grail of apoptosis
• 57:35research.
• 57:36Like, what does this look
• 57:37like?
• 57:39I showed you ladders of
• 57:40backs throughout this whole talk.
• 57:42I told you about how
• 57:43Niko Chandra said, how do
• 57:44you study moving target? That's
• 57:46the problem.
• 57:47And by the way, this
• 57:48isn't a membrane. And so
• 57:49we figured out that the
• 57:50only way
• 57:52to to think about this
• 57:53is to somehow create an
• 57:54oligomeric species that is homogeneous
• 57:56and stable,
• 57:58which seems
• 57:59like god bless these two
• 58:00grad students, Hausman and Harvey.
• 58:01They took on this crazy
• 58:02project for their thesis. They
• 58:03actually did it together,
• 58:05to try to figure out
• 58:06how you take the most
• 58:07unstable thing and make it
• 58:08stable.
• 58:09And so they did a
• 58:10detergent screen, and they took
• 58:11inactive backs, and they took
• 58:13oligomer,
• 58:14different detergents and looked at
• 58:15what it did. And, you
• 58:16know, some of these detergents
• 58:17gave you a peak that
• 58:18looked fairly, you know, reliable,
• 58:20consistent. You run it out
• 58:21on a native page. It
• 58:22was like one band. That
• 58:23was exciting.
• 58:24Well, maybe it's this detergent
• 58:26doing something. Who knows? Repurify
• 58:28it without the detergent.
• 58:30And what was there that
• 58:31was induced actually survived after
• 58:33you got rid of the
• 58:34detergent, and there the band
• 58:35was still there again.
• 58:37So we said, okay. What
• 58:38what did we make? What
• 58:39is this?
• 58:40And so we showed that
• 58:42just like t bit triggered
• 58:43bacs that translocates to membranes,
• 58:44this thing you throw it
• 58:45on there, we call it
• 58:46Bax o for Bax oligomer,
• 58:48automatically goes to the liposomes.
• 58:49So it's behaving like the
• 58:50ligand triggered Bax.
• 58:52We showed in our liposomal
• 58:54release assay that just like
• 58:55everything I've showed you multiple
• 58:57times,
• 58:58liposomes alone, VACS alone, ligand
• 59:00alone. You add the two.
• 59:01You get this nice release.
• 59:02You throw in oligomeric VACS.
• 59:04It looks just like TBID
• 59:05triggered VACS.
• 59:07Now you go to the
• 59:08electron microscope because we'd like
• 59:09to you know? Again, let's
• 59:10go look at this thing.
• 59:11Here are your liposomes. Here's
• 59:13your TBID triggered BACS liposomes
• 59:14with these nice holes in
• 59:15them. The BACS oligomeric form,
• 59:17nice holes. Looks just like
• 59:19the panel in the middle.
• 59:21Well, what about mitochondria?
• 59:23Same story. You got your
• 59:24TBID triggered VACS. You add
• 59:25in your dose response of
• 59:26VACS oligomeric
• 59:28species, gives beautiful dose response
• 59:29of cytochrome c release.
• 59:31And then the last funny
• 59:32thing that I will tell
• 59:33you, which again is why
• 59:34I really believe, like, you
• 59:35have to just
• 59:37sometimes think do multiple things
• 59:39that seemingly have nothing to
• 59:40do with one another,
• 59:42and you may get an
• 59:43intersection that you never would
• 59:44have come up with. And
• 59:45this was a grad student
• 59:47coming to my lab and
• 59:48said, oh, antimicrobial
• 59:49peptides are very important in
• 59:51antibiotic resistance. And guess what?
• 59:53There's hundreds of alpha helical
• 59:54ones, and they're very, very
• 59:56toxic, you know, to membranes,
• 59:57and they're really good drugs
• 59:58against antibiotic resistant bacteria. Oh,
• 01:00:00but by the way, they
• 01:00:01they nuke every membrane.
• 01:00:03And I thought, well, that's
• 01:00:04great. Like, we're trying to,
• 01:00:06like, deliver staple peptides to
• 01:00:07cells, cancer cells, so they
• 01:00:09don't disrupt membranes. Maybe if
• 01:00:10we just study how they
• 01:00:12actually nuke membranes,
• 01:00:13something will come of this.
• 01:00:14Somewhere in the middle, what
• 01:00:15we learn about nuking membranes
• 01:00:17and preserving membranes will find
• 01:00:18us something interesting.
• 01:00:20We had no idea we'd
• 01:00:21end up landing on backs.
• 01:00:23Okay? And so we published
• 01:00:24this paper about how to
• 01:00:25make antimicrobial
• 01:00:27peptides
• 01:00:28selective for bacteria
• 01:00:29and not disrupt
• 01:00:31mammalian membranes, a story for
• 01:00:32another day. But what what
• 01:00:34we've learned from that was
• 01:00:35that these were hydrophobic
• 01:00:38faced with cationic charges on
• 01:00:40the other side, and the
• 01:00:41mechanism how they work is
• 01:00:43they form
• 01:00:44toroidal pores. They cause negative
• 01:00:46Gaussian curvature to membranes.
• 01:00:48And when you scan bacs
• 01:00:51for hydrophobic
• 01:00:52and cationic
• 01:00:53sequences,
• 01:00:55guess what?
• 01:00:56Alpha six, if you didn't
• 01:00:58know anything about anything and
• 01:00:59you're an antimicrobial
• 01:01:01peptide professor, you would say,
• 01:01:02oh, that's an antimicrobial
• 01:01:04peptide
• 01:01:06as the alpha six helix
• 01:01:07of BACS.
• 01:01:09And there they are sitting
• 01:01:10on the surface of BACS,
• 01:01:12and you look at an
• 01:01:13anti apoptotic protein. Oh, they
• 01:01:14don't have those arginines on
• 01:01:16the surface of their alpha
• 01:01:17sixes. Oh, and I could
• 01:01:18put them on liposomes, and
• 01:01:19alpha six pops those liposomes
• 01:01:21all by itself in BAX,
• 01:01:23but the anti apoptotic one
• 01:01:24does not do that.
• 01:01:26And then I could start,
• 01:01:27you know, doing reverse polarity
• 01:01:29mutagenesis, and all of a
• 01:01:31sudden my BAX doesn't work
• 01:01:32in a liposomal assay as
• 01:01:33well, in the cytochrome c
• 01:01:35assay as well. If I
• 01:01:36do the double mutant experiments,
• 01:01:38even worse,
• 01:01:39less activity.
• 01:01:40And then the the most
• 01:01:41important experiment for the reviewers
• 01:01:43is show me that it
• 01:01:44works in cells, and you
• 01:01:45can reconstitute
• 01:01:46cells with wild type acts,
• 01:01:48in a DKO background and
• 01:01:50then take out just those
• 01:01:51two arginines, and bacs is
• 01:01:53now no longer working very
• 01:01:55well at all.
• 01:01:57So, you know, there's been
• 01:01:59a lot of work trying
• 01:01:59to figure out what BAX
• 01:02:01is doing and is it
• 01:02:02a poor and this and
• 01:02:03that. But, honestly, I,
• 01:02:05have come around to the
• 01:02:07idea
• 01:02:07that BAX is a membrane
• 01:02:09disruptive
• 01:02:10protein
• 01:02:11and that you get it
• 01:02:12on the mitochondria and it's
• 01:02:13self associating, and it's seeing
• 01:02:14those alpha six cationic
• 01:02:16hydrophobic
• 01:02:17amphipathic peptides there that look
• 01:02:19just like antimicrobial
• 01:02:21peptides, and it is lysing
• 01:02:23the membrane.
• 01:02:24And so, you know, of
• 01:02:25course, we're super interested in
• 01:02:26understanding what those structures are.
• 01:02:28We have sacks
• 01:02:29of our,
• 01:02:31stable,
• 01:02:32Bax o, and it looks
• 01:02:34like a finger. You know,
• 01:02:35you've got, like, what looks
• 01:02:36like
• 01:02:37three dimers there attached to
• 01:02:38one another, and you can
• 01:02:40kinda think about on the
• 01:02:41right hand side those holes
• 01:02:42that we showed in the
• 01:02:42liposomes. And you can kind
• 01:02:43of imagine these linear structures
• 01:02:45of these oligomeric dimers sitting
• 01:02:47around and just
• 01:02:49disrupting the membrane and causing
• 01:02:51deformation so that it leads
• 01:02:52to rupture.
• 01:02:53So lots more work to
• 01:02:55do on this part,
• 01:02:57but I feel like we're
• 01:02:58kind of inching closer to
• 01:02:59really trying to understand how
• 01:03:01backs and back really
• 01:03:03cause cytochrome c release by
• 01:03:05disrupting the outer membrane. So
• 01:03:06I know I'm right at
• 01:03:07the minute there, so I
• 01:03:08will stop.
• 01:03:09Thank everybody in the lab
• 01:03:11that has done it
• 01:03:13in recent years, that has
• 01:03:14done it like Sam in
• 01:03:15prior years, our amazing collaborators.
• 01:03:17And I just
• 01:03:18have to end
• 01:03:23with
• 01:03:24mentorship.
• 01:03:28You know, there is
• 01:03:31no chance
• 01:03:33that I would be standing
• 01:03:34here
• 01:03:35telling you any of this
• 01:03:36if it wasn't for him.
• 01:03:38Changed my life. And although
• 01:03:40many of you know that
• 01:03:41he died way too young,
• 01:03:43the age of fifty four,
• 01:03:45the impact that he has
• 01:03:46had on so many scientists
• 01:03:48and the reason why no
• 01:03:49matter how
• 01:03:50far away it is from
• 01:03:51the time of his death
• 01:03:52in two thousand and six,
• 01:03:53I will never be able
• 01:03:54to talk about him without,
• 01:03:56emotionality.
• 01:03:57But
• 01:03:58the impact
• 01:03:59that your mentors,
• 01:04:01the young ones here today
• 01:04:02that will be mentors,
• 01:04:03you know, it is
• 01:04:05that relationship.
• 01:04:07It is that cultivation.
• 01:04:09You know, that's part of
• 01:04:10what we're trying to preserve
• 01:04:11literally today. That's at threat.
• 01:04:14Right? That continuity, that legacy,
• 01:04:15that chain reaction of the
• 01:04:17senior person inspiring the younger
• 01:04:19person who's then inspiring the
• 01:04:20younger person. And that is
• 01:04:22what our science is all
• 01:04:23about. And so we have
• 01:04:24to guard it and advocate
• 01:04:26for it and keep doing
• 01:04:27what we're doing, that we
• 01:04:28know that what we're doing
• 01:04:29is important. And at the
• 01:04:30same time, it's not only
• 01:04:32focusing and being persistent
• 01:04:34about our science, but we
• 01:04:36have to lead now too.
• 01:04:37We have to make sure
• 01:04:39that we get out there
• 01:04:40and somehow
• 01:04:41use what we think is
• 01:04:42so special about our institutions
• 01:04:44and what we're doing scientifically
• 01:04:45and medically
• 01:04:46and teach people that probably
• 01:04:48don't wanna listen and somehow
• 01:04:50convince, you know, elected folks
• 01:04:51that this is the lifeblood
• 01:04:53of health,
• 01:04:55you know, in America. Right?
• 01:04:57And so that's kind of
• 01:04:57a big new challenge that
• 01:04:59we've never ever had to
• 01:05:00think about before.
• 01:05:02So, anyway, thank you so
• 01:05:03much for staying. I know
• 01:05:04I'm five minutes over.