The Peter Attia Drive - #09 - David Sabatini, M.D., Ph.D.: rapamycin and the discovery of mTOR — the nexus of aging and longevity?

Episode Date: August 13, 2018

In this episode, my good friend David Sabatini delves into his extensive work with the mechanistic target of rapamycin—better known as mTOR—and rapamycin. The compound rapamycin is the only known pharmacological agent to extend lifespan all the way from yeast to mammals—across a billion years of evolution. David, a professor of biology and a member of the Whitehead Institute at MIT, shares his remarkable journey and discovery of mTOR in mammalian cells and its central role in nutrient sensing and longevity. Fasting, rapamycin, mTOR, autophagy, gedankenexperiments: having this conversation with David is like being the proverbial kid in the world’s greatest candy store. We discuss: mTOR and David’s student years [4:30]; Rapamycin and the discovery of mTOR [8:15]; The connection between rapamycin, mTOR, and longevity [30:30]; mTOR as the cell’s general contractor [34:45]; The effect of glucose, insulin, and amino acids on mTORC1 [42:50]; Methionine sensing and restriction [49:45]; An intermittent approach to rapamycin [54:30]; Rapamycin’s effects on cancer, cardiovascular disease, and neurodegeneration [57:00]; Gedankenexperiment: couch potatoes on rapamycin vs perfectly behaved humans [1:03:15]; David’s dream experiment with no resource constraints [1:07:00]; and More. Learn more at www.PeterAttiaMD.com Connect with Peter on Facebook | Twitter | Instagram.

Hey everyone, welcome to the Peter Atia Drive. I'm your host, Peter Atia. The drive is a result of my hunger for optimizing performance, health, longevity, critical thinking, along with a few other obsessions along the way. I've spent the last several years working with some of the most successful top performing individuals in the world, and this podcast is my attempt to synthesize what I've learned along the way to help you live a higher quality, more fulfilling life. If you enjoy this podcast, you can find more information on today's episode and other topics at peteratia-md.com.

In this podcast, I'll be speaking with my close friend and amazing scientist, David Sabatini. David's a professor of biology, a member of the Whitehead Institute at MIT. He's also an investigator at the Howard Hughes Medical Institute, and a senior member of the Broad Institute, along with a bunch of other accolades that would take too long to get into here. This podcast was actually recorded initially as part of an interview series I was doing for Research Around My Book, and this was recorded in August of 2017. was actually recorded initially as part of an interview series I was doing for research around my book. And this was recorded in August of 2017. Maybe at some point we'll even just put the video up as

this was actually done as a video interview with David along with a number of his amazing postdocs and certainly some of those will probably make their way into the podcast as well. Now in this episode we talk about his amazing journey in science and the work and stuff that he's done around MTOR and RAPA MISON. And if you've been following the blog and or paying attention to stuff that I'm interested in, you'll know that MTOR and RAPA MISON sit kind of at the heart of it. Now, about four years ago, David and I were having lunch one day. And it was kind of the first time that he ever really told me the full story of his work

as a graduate student at Hopkins, where he was part of the first time that he ever really told me the full story of his work as a graduate student at Hopkins, where he was part of the MD PhD program. And I was just, you know, I remember sitting there taking notes on a napkin and thinking, God, this is such an incredible story of science. And I remember thinking, God, you know, one day we have to have this discussion again, but such that most people can actually hear it besides just me. So part of what we discuss on this podcast is actually that journey. And how as a young PhD, newbie grad student, David methodically went

after a problem that really wasn't even deemed particularly interesting at the time, which was to basically figure out how this thing called rapa mice and actually worked. And of course, through the process ended up being the first person to identify this mechanistic target of rapamycin in mammalian cells. Now, stuff that I found really interesting in this podcast is that David points out that he's from an academic standpoint, kind of an unusual bird in that he's one of the few people who has carried his work from graduate school into his career and that's actually

pretty unusual. He's incredibly thoughtful, and some of you may have already heard a podcast that David, myself, and Nav Chendell, another good friend who will also be on the podcast, recorded back with Tim Ferris on Easter Island back in the fall of 2016. We'll link to that here as well. And obviously, the reason we went to Easter Island

was as sort of a pilgrimage based on the discovery of the bacteria that ultimately led to rapamycin, a bacteria by the name of Streptomyces hydrocoficus. The interest I have in mTOR of course has to do with its central role in nutrient sensing, and of course it's I believe and many believe its central role in longevity. So if you are interested in longevity, if you're interested in fasting, if you're interested in rapa, Mison, you're really going to want to listen to this podcast because David is effectively mTOR man.

I don't think there really is a person on the planet. And I'm saying that without trying to be hyperbolic, but I don't think there's anybody on the planet who knows more about rapa, Mison and mTOR than David's abatini. And if you like this podcast, please make sure to check out the one that's going to be out soon with Matt Cabralin, which will take this discussion to another level as well, looking at Matt's work in dogs. So without further delay, here's my discussion and conversation with David Sabatini.

David, thank you so much for making time to sit down today and talk about what is potentially mutually our favorite topic of discussion. Before we jump into it though, maybe for people who don't know, can you tell us a little bit about how you got here and what it is you do specifically? Sure, sure. So thank you, Peter, for coming and for visiting and both of you and for wanting to talk to me. So I am a biologist.

I'm a professor of biology at MIT, and I'm also a for wanting to talk to me. So I am a biologist. I'm a professor of biology at MIT, and I'm also a member of the White Institute, which is where we are today. And I receive a lot of funding from the Howard Hughes Medical Institute, which is a key charity that works with biomedical researchers. I have studied this protein that I'm glad you like a lot.

It's my favorite protein, called the Emptore Protein, which is the protein through which this drug, rapamycin, which gets quite a bit of attention now, acts. And I basically worked on that from the earliest point. We discovered that when I was a student. And so my career is as an MD-PhD, never really following the clinical track though and staying on the research side. And finishing that and actually coming to the whitehead in a program that is quite interesting and very unique at the time, which is that you

could start your own lab after graduate school. So I did that and I eventually joined the faculty here and now I've moved up the academic ranks. And to some extent, I'm a little bit strange from an academic point of view because I've continued to work on not exclusively, but to a large extent, what I started in my graduate school. So most people, as you know, do something in graduate school,

they do something in their postdoc, and then they sort of morph along the way. I've kind of stuck with this emtor protein, and in many ways I was very lucky because we were there at the beginning, and it turned out to be such an exciting thing to work on. So let's go back to the beginning a little bit. You were an MD, PhD student at Hopkins, and after a couple of years of doing pre-clinical

stuff, you pick a lab. Exactly, right. So, you do two years of medical school, and then you pick a lab, and I was very fortunate to be taken by Salman Snyder, who was at the time the head of neuroscience. He had a very big lab, lots of MD-PhD a lot of MD PhDs wanted to go to his lab, and I was lucky that he let me go. And Saul is a really interesting man. He still has actually a really prominent lab at Hopkins, and in fact the department is now named after him. And he was that person who had a lot of very interest. So he was a neuroscientist,

but he was also a psychiatrist, and he was also a pharmacologist. So he really loved small molecules. And he liked, particularly potent small molecules, that is small molecules that act at low doses. How do we define in pharmacology a small molecule? What's the cutoff point? You know, some people say 1,000 Dalton's, which rapamycin is about that. Through a larger sense, it's sort of a non-peptide also.

So it's not a piece of a protein. In many cases, it's not a natural molecule, although in our case, it's made by microphones, it's not natural to our body. So probably thousands of adult. And so he had these set of interests, and when I went to his lab, I was actually really interested in neuroscience, so I'd had some classes in which I was sort of fascinated by some neuro questions. But when I got to his lab, I actually never did anything on neuroscience. And I often told the story that the most influential scientific discussion I probably ever had is when I went to talk to Saul. And basically, as a student, you need to pick a project. This is something that is quite challenging.

I see with my own students, they really get quite apprehensive of what they're projects. So I went to talk to Saul, and I went to his office, and I only met with Saul, like, I don't know, maybe five or six times during my PhD. So this was like a big deal. And so I went to talk to him, and he basically said, David, we work on the brain.

And I thought that was great, because I wanted to neuroscience, but then he didn't say anything else. So I was it. And so I remember leaving his office really anxious, basically I didn't have a project, but I realized now in retrospect what he did is he actually was giving me complete freedom to do what I wanted to do. And that was, as I said, probably the most important thing anyone's ever done for me, because it really forced me to come up with my own project. And I think was a key sort of foundation in becoming a scientist.

I have, and it's something that I try to foster amongst my own people. So anyways, I was in his lab and I didn't have a project. And at the time, they were actually working with this other drug called FK506, which is a lot of... It's an immune suppressant. Simulant suppressants used clinically still. Mechanism of action, although structurally it's very different than cyclosporin, and actually mechanistically works on the same target,

which is calcinaurin. And at the time, this is before we had a lot of the tools we have now, like RNAI or CRISPR. And so you need to control. So if you had to drug what you tended to use, was another drug that kind of looked like it, but didn't do the same thing. And so their control was rapid-mising.

And when I started reading, we're rapid-mising. And this is what year? This would have been in probably late 91 to 92. And it was clear to me that this molecule in many ways was much more interesting than FK56. And as you very well know, this had come from Wyatt Ayers, the pharmaceutical company by Soran Segal, the champion did. And there was a number of papers which at the time,

where actually a few papers were largely abstracts from meetings that show that had antifungal effects, immunosuppressive effects, anti-cancer effects. So it seemed like an interesting model. I had just come from medical school. We'd learned about immunosuppressants like cyclosporum, which at the time, we're really just coming on, and we're really seen as miracle, bro.

But your lab's interest in FK506 was not its immune-suppressive properties, but it's calcinerin inhibition. Exactly, because calcinerin, as the name implies, there's a lot of in the brain. And so in Sol's lab, they're basically studying the modulation of calcinerin in the brain using FK506 as a tool.

And they were looking actually at excitotoxicity in the brain. Things that at the end didn't lead, I think in the directions they wanted to, but they were using it as a protocol, as a probe, basically. And so I basically decided why not try to work on rap mice? And so that's what I did. And so we... Which was just a control that nobody particularly cared for.

Yeah, I mean, there were people in the world that were interested in that. But in your lab, this is But in your lab, yeah. People have no real problems studying rap mice. We had this great advantage, though, is that you couldn't buy rap mice at the time. So, rap mice and was a compound that Wyeth was developing clinically.

It wasn't clinically available. You couldn't buy it. But Saul, being a very prominent scientist and having this interest in pharmacology, had actually written Seren, Cigol gole. And actually he had sent us, probably without any of the legalese that happens now. Now if you try to get a molecule out of pharmaceutical company, the amount of paperwork and red tape is huge.

But he had sent us a very significant amount of rep mice in which I remember when it did start to be sold, which was incredibly expensive, I kind of back calculated. It was like millions of dollars. Wow. Now of course it didn't really have that value, back calculated. It was like millions of dollars. Now of course it didn't really have that value, but theoretically it was sort of millions of dollars. Which incidentally that tube follows me all the way here,

and then it's- You still have the original tube? No, at some point it was lost. It disappeared at some point, but I know it's a weird thing. So we actually had it, which was cool, so we could actually do experiments with it. And so I went on to try and try and find out what worked and eventually we purified this

protein that at the time we actually called raft one was the original name we gave it and eventually it was called raft. And raft one stood for? It was rapamycin and FKBTP target one. And the reason that we and also Stuart Shriver, who was at Harvard and when he was working on this, he was also at Harvard, also identified m-tore basically at the same time. He called it FRAP, which was FKBP, rapamycin, associated protein. And both of us were trying to accentuate the point that rapamycin actual, the co-receptor.

This protein FKBP, from that point of view, it's a very unique kind of drug where it doesn't directly bind to a protein target, but rather it first binds to one target, and now that drug receptor complex has a new surface on it, which now, in this case, interacts with MTOR. And we were really trying to get that point across. Eventually. But independently. We actually had no idea that in fact the only point where I found out that they were working on it was once our paper had been accepted, I got, and this was pre-lots of email internet. We got a fax from a journalist saying he was writing an article on our paper and another paper from Stuart Shriver and actually sent us Stuart's paper, which we thought was really unethical at the time. And so we actually, at that point,

contacted Stuart and said, hey, we got your paper. You should know we're working on this too, and here's our paper. So I didn't know it all. And in many ways, I was very naive. I was in this lab. Saul basically let us do whatever we wanted to. We had this drug. Unbeknownst to Saul, I started working on this thing. Stuart had had history of FKFile 6, mechanism action.

So it was a logical progression to what he was doing Saul was not it was funny He came from a world where people looked for the receptors for drugs So if you look at his history, he'd really looked for receptors for drugs for small molecules including the endorphins for example But he wasn't big on cloning what we call cloning a gene Which is where you have that get the DNA sequence. You almost thought you didn't need to do that. Once you purified it, you could study the protein. So I was one of the first people there to actually clone a CDNA as we call it in his lab. So it was a fun time because it was clear that we got this protein.

But you did this in a very short period of time because your paper, which was in cell, was 1994. 1994, yeah. I worked like crazy. Really like crazy. So, in that lab in general, worked like crazy. We were, it was very common to be there until one in the morning, and then I would usually show up at 7, 8 in the morning.

You know, we would sleep in the lab a lot. And once, you know, once things started to go, so we were purifying, I purified out of the rat, out of the rat out of rat brains. And so we killed hundreds of rats to do this. And my friends would help me kill them, take the brains out. There's a method in biology to visualize proteins called a silver stain, which is a very sensitive way

of seeing a protein. And the first silver stain I did, where I actually saw sort of a glimpse of MTOR on this method. I remember that really clearly, because at that point, I knew I could do it. How did you know it was MTOR that you were looking at? Well, I mean, I had all the controls, and there was this band on what we call a gel. They showed up just in the right place. So I was like, okay, there is a protein here that has all the properties that I want.

And at that time, what properties did you know? You didn't know it's size, did you? You didn't know it's size. Well, we know it bound to FKBP rapid-mice. But you didn't know that that exclusively bound to it, did you? We didn't. But we knew that it could be competed by FKF-06, based on some competition type experiments.

And so we had done that. So there was these features. It was mostly the specificity that it required rapid- micecentebine to FGVP. And that was crystal clear in the early experiments. When we had FGVP by itself, there was no band on the gel. And when we added rep micecene, there clearly was. And when we added FGVP by 6, it clearly went away. And so we knew that that thing had all the right properties.

But I remember very strongly feeling, OK, and at the time, now we have very, very sensitive methods to sequence proteins. Larger than mass spectrometry there, we didn't. And so from what I saw in that gel to actually figuring out what its sequence was, I knew it was hard, but I knew it could happen. That was like a very powerful feeling. It was the existence, for instance.

Exactly. So I knew the thing, like the kind of the enemy existed and I could get it, but then going from that initial glimpse on a gel to then having enough to actually sequence it, that's what took hundreds of rats to actually get to enough that I could purify it. And eventually we collaborated with this guy called Paul Tempts at a memorial songcaddering in New York and he was able to sequence enough of the protein that then through a whole variety of molecular biology tricks, we were able to clone it. And it was a really huge CDNA, which basically is the length of the DNA sequence that encodes it.

It was very big, sort of in the eight, nine kilobases, which is very hard to work with, particularly at the time. And I got very, very lucky in the sense that I did a bunch of tricks and I got the whole thing at once, which is also was kind of unheard particularly at the time. And I got very, very lucky in the sense that I did a bunch of tricks, and I got the whole thing at once, which was also kind of unheard of at the time. How did you do that? Yeah, so back in the time, what people would do is they would get pieces,

and then they would sequence them, and they would just- We're overlapping. They would overlap and stitch them together. But what I did when I screened, what we called libraries at the time, for these pieces, I would get some pretty big pieces, but I knew, when I would sequence it, I knew the front of the protein was missing.

I was missing it, and I couldn't get it. It would never, I could never get beyond a certain point of the protein. And so then what I did, which really turned out to be like incredibly lucky, so what we would do is we would screen libraries of phages. And so this was basically people would take CDNA, complementary DNA from RAPRIN, and they would clone it into these bacterial virus-gal phages. And so now every little CDNA was in a virus,

and you'd have hundreds of millions of this library. And you would plate it out on these plates, and the phage would make little plaques, and then you would screen those plaques. And so you'd have dozens of these plates each with thousands and thousands of these little dots on them. And so what I decided to do is that I would screen this library with a piece that I knew as far towards one end and as far towards the other end. And so I screened it with both

and I looked for plaques that hybridized both. And in fact, when I first did it, I got nothing. It was really disappointing. I got plaques, I had one piece and I had plaques and I had another piece, but I didn't have any plaques to have this thing. And then what I realized, and this was really key, I realized that the so-called

full-length CDNA was so big that it was going to make the phase replicate slowly. Because basically their genome was so much bigger now that to replicate, it would take longer. And on what order of times? It was probably two to three times more that it would take. I got it. So you could have been missing it. I could have missing it because the plaque they would have this would be incredibly small. And so what I did is I went back and redid it and now I let the plaques grow longer and I re-screened it. And in fact I got one plaque. There's a tiny, tiny little plaque that hybridized with both, both probes. And when I looked at what was in there, it turned out to be the complete full length CDNA, which was amazing because it was unheard of that these libraries would give you something

like 9,000 base pairs, but it was. When I see it was literally the intact thing from one end to the other. So I got very lucky because that would have been pretty hard to assemble at the time. You knew at the time, had Michael Hall's work in Yeast been published yet? It had been published sometime during this period of time, but you didn't know anything. You didn't know even what the yeast form of this program was. No, no, we started, we were in doing it, and in fact, when we first started getting sequenced, there was no sequence out there.

And the yeast protein, really only the kinase domain is concerned. And so most of the peptide sequences that we had, the Paul TEMPS had sequenced for us, we didn't know at all where they were, right? And so these are kind of fun things that used to happen in the past. You used to collaborate with a person who did protein sequencing, and they would give you back a series of peptide sequences.

But you didn't know what order they went in. So let's say you gave you back, I think Paul gave me, Paul was amazing, because he would give you, let's say, 15 peptide sequences. He'd say, look, your protein, these 15 peptides exist in your protein. And then some... Or without overlap in those peptides.

No, overlap. These are short. Mathematically, it's impossible to, by chance, figure out, like, you need more clues to figure out the order, because it's combinatorially impossible. Yeah, you have no idea what the order is. And so what you end up doing is a cool thing. Paul was really cool because he would actually, in the sequence of the peptides, he also had uncertainty.

Sometimes he'd say this amino acid. Plus or minus. Could be this or could be that. And he would tell you what he thought it was. And it turned out he was so good that when I actually figured out the sequence, everyone that he said it could be this or that, he was right, his prediction. But so what you do is you have these peptide sequences. And what you could do now is design,

we know the, obviously the code, the amino acid code. So we can predict what the DNA sequence would encode. But as you know, the DNA sequences degenerate. Right, so one peptide sequence can be encoded at the DNA level. You don't know what the extrinsed entrance look like. I don't know anything, right? But each peptide can be encoded at the DNA level. You don't know what the extrins and introns look like. I don't know anything, right? But each peptide could be encoded potentially

by thousands of oligoneuclidides. And you don't know the order of the peptides. What you would do is you would make a degenerate pool of oligoneuclidides that had thousands of different ones. And you'd make them in both orientations. And now you'd do PCR between them in all combinations, and you would find which ones worked. And that would define the order of the peptides.

And this is before you had real-time PCR. Yeah, real-time PCR usually is for quantitation, but we had PCR, and so we would take these all-ago libraries, and we'd mix and match them all combinations in all orientations. And if you got a band, it means that you got, you know, basically for how you can. And then you could take those fragments and go and screen the libraries. And so it's funny, because now, you know,

with my students when we discover a new protein, all you do is you look up at the database because we have the whole genome sequence. I always tell my students that my paper, which was the discovery of M-TRA, which at the time to be fair, we did not realize how important M-TRA would be. My paper basically is like figure one AB of their papers.

Because my whole paper is about discovering the protein, sequencing, and all this kind of stuff. Was that paper effectively your PhD? That was my PhD. So you went back to finish a couple of years of med school, obviously decided not gonna do a residency, I'm gonna become a full-time scientist. And then you basically have been at MIT since or affiliated with MIT since.

Saul's lab was big and I was very independent. So the people said, why don't you do one of these fellows positions where you can store your own lab? And at the time there was only three. There was the whitehead one, there was one at Carnegie Institute, which is in Baltimore, and there was one at Colesbury, Harvard, New York. And I applied to all of them. And I got accepted pretty quickly, although after I graduated to Colesbury, Harvard, and to Carnegie. But I didn't hear anything from a whitehead, like nothing. And only like basically once I graduated and I was kind of unemployed at that point, I

was technically a postdoc in Sal's lab. But I hadn't taken like the boards, which Hopkins, you know, Bar Hopkins didn't make you take the boards, the medical boards graduate, which was a nice thing. My mother was like you're gonna starve, you know, have a job, you can't do residency now because you didn't apply, you didn't take the boards. And then I got a call from Whitehead, actually inviting me to interview and I did. And then it took again a lot of time to like hear back. And I remember they called me and said,

look, we're gonna offer you a position, but you need to understand you will never ever stay here as a faculty member, ever. I was like, okay, I realized I was applying for this white head fellow position, not a faculty position, but then eventually I came and eventually I did stay. And then when I look actually at history,

it's they do keep about a third of the people who come through, but they give you this sort of speech that you will never ever have. That's the expectation. And incidentally many of the people named David have stayed. So that's a good thing to be named. Actually our current director was a whitehead fellow, his name is David. One of the other faculty members in name is David. So I didn't know the time, but now I realize that David was a big advantage.

So how was your work evolved? I mean, you came here in the late 90s, right? In the late 90s, I think so. A rapamycin would go on to be approved by the FDA in 1999 as a frontline treatment as part of the double or triple cocktail for patients. That's rapamyun, right? Right, that's rapamyun, along with often prednisone, cyclosporin, or MMF. So now you're here, and I mean, we're gonna get into much more detail,

but effectively, you've never looked back. You've never really left this space. I got here, and I was in Crowley night. I realized at this point how I thought, you know, I knew a lot, I thought I knew how to run a lab. I had been very independent on my own, that was a mean that I was sort of independent from like running a lab.

Behind the scenes in Saul's lab, I was the entire finance. I had written grants and things and entire finances organization, but there was a lot. I could be independent me, but then the lab is a different thing. And so that was a hard transition to run,

even though it was a small lab, to run a lab. And it was clear that at that time, I felt that this field had kind of plateaued. There had been the discovery of Amtore, but we weren't getting very far. People were using rapamycin to look at lots of things. And Amtore by implication through rapamycin was being connected to lots of different things. But one of the things that was obvious to me, and I think to others as well, was that MTOR had to act with partner proteins. And so we set about trying to identify what we now know are these MTOR containing complexes,

MTOR-1 and MTOR-2, MTOR-1 and 2. But yeah, it was really hard. We failed for years. It was again one of this field that has had a series of just like little things that until you figure them out, you make no progress. So we would purify emitter and we'd look for other proteins. We would continue to work with Paul Tams and we just wouldn't find anything. To be clear, you knew that you had discovered the gene for torque.

You suspected that this thing exists in different complexes. And I already knew that there was other proteins because when I was doing the M-TRA original purification, the way that I was following M-TRA was with a funky cross-thinking assay, where I was cross-thinking a radioactive FKBP to the putridif target. And there was always two bands on the gels. There was the protein M-TRA, which I eventually purified, but there was a smaller one, which I could never get. either because it was just low Abundance I couldn't do to I don't know what but that little protein which at the time I called raft two actually

Basie remained unidentified So I knew that those so basically the first version of M-Tro complex one was Tor and the version of M-Tro complex two was raft two No, no, no that protein actually now that we found it turns out to be in both complex. Oh, I see. But what I knew was that there was an associated protein with emtore. I knew from, I didn't know what his identity was, but it was very clear on all my experiments that there was a small, emtore is very big. It's around 300 kW, there's a big protein.

This was a little protein, it was around 30. So it was about 10 times smaller. So from a technical point of view, it's about 10 times harder to get because there's about 10 times less peptides in that protein. So I failed to get it. So when I got here to the whitehead, I knew there was another protein to find. And we kept trying to go after this protein. And others, we knew it had to work.

It's a really big protein, big proteins work with friends. And it turned out, this is again, these little things, it turned out that the detergent, so when you, when you work with mammalian cells, you have to lice them, you have to break open their membranes, you typically use a detergent. It turns out the detergent we were using, which is by far the most common detergent that every lab in the world use, breaks apart these complexes. Just by bad luck. Just bad luck. And I had a postdoc, his name was Daw Saurbosov, who figured this out.

And he found this other detergent called Chaps. They kept them together. When you think back here at career, and you're like, well, what are like these key inflection points? His discovery of that detergent was key. Because once we did that, we purified all the interacting proteins.

And that eventually led to M-Turk 1 and M-Turk 2, and eventually led to all the proteins associated with those. Basically, that was the key to all the biochemistry. There was several years of nothing, and he found that, and then everything has sort of... From that point on, we've sort of marched along and figuring out that all the components of this pathway. We still don't know why, things are sensitive to Triton We still don't know why things are sensitive to Trident.

We don't know why they're incentive to solve the detergent, but it's the kind of happenstance of science that I guess makes it interesting. So when, roughly by year, where are we when we have a, we meaning that the world is a result of your discoveries in the lab, where are we when we sort of know that now we have MTOR complex one around Raptor, MTOR complex two around Richter. This is... It's around 2002, right? So when we're doing that around 2001, published around 2002, it's in that range, it's in the early 2000s. Although as I said, we knew there was complexes even back in 94. And at this point in time, your thought was these two complexes control what or sense what or are important for what.

Right, so it was very clear early on that MTORC 1 was doing most of the things that we had connected before to MTORC. So, you know, we'd had RAPAMISON, and to RAPAMISON, in a way, it had allowed us to know a lot about mTORC 1. We now realize that otherwise we would have known. Because we didn't have really genetics, we didn't have easy ways of modulating mTORC, but we had rapamycin. And so there was a body of knowledge acquired by many different investigators about what was so-called downstream of mTORC. What did mTORC do?

We had some ideas. It was a growth regulator, a regulatory translation, a regulatory, it's off a G, a regulatory, it's many, many metabolic pathways, we regulate cell size. We knew that largely through the use of rapamycin. And so now when we discovered MTOR I, which, the first part we discovered was a protein called Raptor, we now could go and say, well, is Raptor matter

for all those things? And it turned out it did. So it was very clear that M-Torq 1 must be doing the things that we have described to RapidMyson. M-Torq 2, therefore, remained very mysterious for a long period of time, because it wasn't doing those other things. And only later did we find that it was actually part of the PI-3 kinase pathway in a regular AKT.

And that clarified lots of things. And in many ways, MTORQ2, you could actually even say, and we've written papers arguing this, that it's almost like upstream of MTORQ1 because the PI3 kinase pathway is one of the inputs into MTORQ1. In many ways, MTORQ2 is less important than MTORQ1. I mean, you can modulate it more and still survive more.

So we've really focused largely on M. Torkwan. And when I first got here, you sort of asked me, OK, well, what did you end up doing, right? And I was pretty worked up when I got here. And I had to realize I was sort of running a lab and unclear exactly what I was going to do. And I ended up working on M. Torkwan, or M. Torkwan. I should say largely because I didn't know anything.

So I basically had to work on something. And I remember some people here were pretty critical of me working at Rabbis and they were like, why are you working? That's silly molecule, okay? Now you have the target. And the truth was, that's what I knew how to do. Even at the time you didn't appreciate what you do now, which is that effectively MTOR sits at the center of the universe for at least some of the things that we care

a lot about, including potentially longevity. We did not. When did that become clear to you? Is this... That became clear. When we started understanding the connection to nutrients and the fact that the caloric restriction had been connected to longevity, we started thinking, okay, we actually tried doing experiments on worms at the time with rap mice in terms of rap mice doesn't get

into worms, but there was really some important paper in worms where there was a mutant in the C.E.L.A.L.A.G.N version of MTOR that had longevity effects. I would say that was sort of the key paper. And this is unrelated to DAF2. Unrelated to DAF2. Although, interestingly, in the screens, they gave the DAF mutants. One of the DAF mutants, in retrospect,

one of the ones actually had never been identified what the gene was, was simply a mutant that had a mutation. Turns out to be raptor. I think it's DAF 15, I don't quite remember. So there was a 16. I don't remember, we could look it up. But so it was interesting, there was all these DAF mutants that had these interesting phenotypes. And once we found a raptor, someone went back and found that one of the DAF mutants was actually raptor.

So that connected again to M-Torque 1. Now not only were there mutations in M-Tor itself, the C-L-A-L-I-N-M-Torque, but also in the C-L-A-L-M-Torque, but also in the C-L-L-L-M-Raptor that connected it to it. We did not realize that, you know, of course, our paper was published in cells, Stuart Fryer's paper was published in nature, Emma Nature wrote a news and view, so people appreciated that the finding of M-Torque mattered. But I think more from, okay, this is a new signaling pathway, this is a new component. I don't think we realized that it really, we certainly didn't, at the center

of so many important processes as we do now. People sometimes joke and say, well, you know, amtard has everything, right? So if something does everything, at some point, okay, how interesting is it, right? And so it's a funny line. Not a lot of people studying oxygen these days. Exactly, or like from the ribosome, we all appreciate the ribosome makes proteins and so it's a funny line. Not a lot of people studying oxygen these days. Exactly, or like from the ribosome.

We all appreciate the ribosome makes proteins, and so it's important for everything. But you don't study it as a sort of something that's regularly able, although now we realize ribosome is very, very regular. So it starts to fall into that category. But luckily we have enough of these sort of regulatory systems that clearly shows us that it's a very regularly processing

myself. But today, MTOR and by extension, RAPA-MICEN and its analogs are really interesting, not just in your world, but in mine. So, the plebes over here, out in the peanut gallery, this is super interesting, right? This is potentially a molecule that could make people live longer, at least if what it does in yeast, flies, worms, and mammals is any indication. So why is it that rapamycin or asked another way, why is it that the inhibition of mTOR, specifically mTOR complex one, is you'll probably elaborate on, can extend life.

I find that a very interesting question, and it's a question that I'm often asked. And I think we should say up front, we don't know the answer to that question. One way of addressing it is that you can eliminate many of the things that M-Tore one does, and then ask, well, now I'm why M-Tore one, do I still get life-span effects?

If you do that and look at many different processes, probably you'd vote autophagy is the most important thing that it regulates, which as you know, autophagy is the self-eating process where the cell breaks down some of its own components and presumably has to remake them. And so in a kind of naive way, you might imagine that what you're doing is throwing out the old and making new and again naively you might think well that's

going to rejuvenate yourself although none of that is of course proven. So that would be a simple answer but he clearly is not the whole answer. So my answer to your question why mTOR modulation has these longevity effects and yet many other pathways that in some ways are as complicated and as important for a variety of other things don't. And this is the way I think about it. I think about it, like I try to analogize it a little bit to like a building, right? So if I wanted to take a building like this one and make it younger, rejuvenated, you

know, I can't just get a plumber, or a electrician, or a painter, right, or a carpenter, because the building has many different features of which all of them have aged. Well, you really need as a general contractor, right? Who's gonna then bring in all of those? The subcontractors. And fix all the subsystems. We look at an old building.

An old building has lots of things that are messed up from it, from the electrical systems, to the windows, to everything. And to some extent, MTOR is like the general contractor for the cell. I don't know of any other pathway that does as many things. MTOR basically has a finger

in every major process in the cell. And so I think another way of thinking about your question is, what's the simplest way to manipulate a cell so that lots of things are changed? And the answer to that is a modulate emitter, because all these other pathways will, you know, maybe someone will regulate transcription, maybe some will do translations, some are going to change the shape of the cell. But if you got to do all those things, plus more,

the only way of doing it with like a single hit is to go after emitter. It is like the thing, it's like the brain of the cell which then has all these subroutines that do all these things. And so to me that's the simple answer is that to impact the state of a cell, to rejuvenate it, to slow the aging process,

you can't do one thing, you can't do two things, you can't do three things, you can't do ten things, you can't do a hundred things. And the only way you can do all of those things with one button is to go after empty. Now in biology, that tends to be a two-edge sword, right? Because presumably, if you have one switch that controls so much, if you have the wrong general contractor or if the general contractor does the wrong thing, the effect is much more noticeable. So when did it become apparent to you

or how is it apparent to you that this isn't just a linear relationship between signal and response? This is a very good point, right? So you could say, well, it's a general contract. There's a lot of things, and so not only is anti-aging one of the things it does, but how you sort of frampyl sperm production, which is a potential target, a heart function, right?

All these things require it, and so, okay, you might get the anti-aging effect, but you're also going to get all the downsides. And I think that is certainly true, and that's the major issue with targeting umptory. And so that's- Because at the time you really kicked your efforts off here, people thought of rapamycin and emtory as a one trick pony, which was, you give this drug every day, your immune system, specifically your cellular immune system,

doesn't work as well. And at least for that subset of patients who had foreign organs in their body, that's a reasonable thing to have organs in their body, that's a reasonable thing to have. And incidentally, you know, there is now, so funny, rap bison started as an immunosuppressant, the interest in emtory and the immune system pretty much was unexistent. And now there's an entire field of so-called immunometabolism of which emtory is probably

50% of that whole field. And so it's emtory, one emtory,, in different immune cells, T-regs, right? T-helpers. How much of this came out of the Novartis work from three years ago? Was it this perceived that or? Well, that is perceived that. I mean, the Novartis work was the first sort of work

in humans, right? They clearly showed beneficial modulation immune system. But in terms of studying which immune cells are most affected by rap mice and what the role of M2R1, that's come out of the academic world by a number of groups that were heavily enabled by the discovery of Raptor and Richter because now you could genetically inhibit each of those. And one of the things that my lab we've really tried to do is to put our mice out there. And so people use, for example, our Raptor mouse, or flocks, so-called flocks, Raptor mouse a lot.

But this question of, in a way, what you're saying is, how much can we sort of tolerate an M-tor modulation for beneficial effects versus non-beneficial ones? And again, I don't think we have the answer to that. To some extent, RAPA-MISIN is not a complete M-torque-1 inhibition. We know that.

And complete M-torque-1 inhibition is probably not tolerated. And so RAP-MISIN might be as good as you can get. You can get some modulation. Well, say a little bit more about that. So you're saying if we could wave a magic wand, Bobby was very eloquently spoke about why inhibition of M2R1 leads to inhibition of M2R2

and what the temporal relationship of that might be. But I don't think we got into this issue, which is if I could wave a magic wand and completely inhibit MTOR complex one, not lay a hand on complex two, why wouldn't that be a good thing? Because MTOR one is probably required for the growth of any normal cell. So to make for a cell to basically make its organelles,

to make its proteins to divide, MTOR one is probably an essential. So, at that level, it would start to mimic a crude chemotherapy agent that modulates it. It becomes 5FU at a ridiculous dose. Or something that's going to basically sluff off epithelial cells. It's actually going to get a false out. It's called basic atrophy of everything, antigroth, and probably salt death.

And in fact, in many tissues where new delete raptor, it can be quite bad. That's the phenotype. Yeah, like in that pothelium. And then God, at least when we've looked, that's what's coming in it. So I don't think there's two issues going on here as Bobby Shirley told you.

Rapmyson will also, with a longer time, when inhibiting M2 or two. And that is potentially bad for glucose homeostasis. The other issue is that rapamycin doesn't fully inhibit Emptr1. So in ideal world, you'd like to have, and what I mean by that is that Emptr1 probably has dozens of substrates, and rapamycin only effectively inhibits some of them and not others. Including, for example, autophagy is relatively weakly modulated by rapamycin. Why is that? Because the substrate, what rapmycin basically does is sort of

occlude the substrate binding channel and antiracone, and it's physically, physically occluding. And depending, probably, we don't, you know, this is somewhat hand waving, but there's some evidence for this. Probably the size of the substrate, if it's smaller, am I getting easier, and it's not included, if it's bigger, it's getting blocked. And so probably the key substrates in the autophagy pathway simply are not as affected because they get into the kinase domain of M-trop 1 still. By the way, is this issue different

for any of the rapologs? No. They're all basically producing the same effect as rapamycin. Some people might argue differently from that, but in my experience of them, they are basically as rapamycin. Some people might argue differently from that, but in my experience of them, they are basically like rapamycin with maybe different PKPD properties,

but from a mechanistic point of view, I wouldn't expect differences. And I haven't seen those differences. But so in an ideal world, you might want a molecule, they would inhibit all the substrates of Amtrakwan, not touch AmtTurk 2. But then, let's not do it constitutively. But also maybe not to 100% inhibition.

Right? So I'm not sure I would use that molecule to wipe out M-Turk 1. I would use it to bring down all the M-Turk 1 activity of all towards or to some extent, leaving M-Turk 2 intact. I think that's going to be very hard to do by targeting MTOR one itself, because MTOR one MTOR to share the same kinase domain. And so you can go for the ATP binding site, which is most kinase inhibitors, MTORs as a kinase,

protein kinase, like Klevec, for example. They all go for the ATP binding site. So run, I'm going to do it for that. And so our view is that the way to accomplish that is not to go after M-Truck 1 itself, but to go after its upstream regulators. And the big benefit in my view of doing that is that you should be able to have something now that modulates all M-Truck 1's substrate.

And you can also start to get tissue specificity because these regulators vary in importance across tissues. The aspect of this pathway that's kept our attention for two decades at this point, is that M-TRO1 is basically regulated by everything. Anything I do to the cell, whether I change nutrients, oxygen, pH, growth factors, osmotic, what's the direct effect of glucose and or insulin

on M-TRO1? It obviously plays an enormous role on complex two. It seems to activate them, right? So through independent pathways, there seems to be a pathway through which insulin acts, and there seems to be a pathway through which glucose acts. And even the glucose pathway probably has several sub-ranges to it.

I see, which again, teleologically makes sense because if it's a nutrient sensor, it should be activated by nutrients, but it becomes very complicated now because you have the same nutrient acting in completely different areas. And that's probably because you're looking at, in the cells that we use in culture, we can get both of these sensing systems.

We're probably in vivo. There's tissues that are going to care more about the insulin arm. There's tissue that can care much more about the glucose arm, and there's some that are going to care about both. So if you think about being a peripheral tissue, let's say you're a cell somewhere in your leg and you need to make a muffal cell. Let's say a muscle cell.

You need to decide whether you're an anabolic state or a catabolic one. So clearly there's things of use and all that. But let's say just in response to nutrition, you kind of want two pieces of information, right? One, you want to know that the organism that you live in as a whole is in the fed state. You want to know that the organism that you live in as a whole is in the fed state. You want to be a good member of the community. And that is reflected

by things like insulin, which basically tells you the pain. It's a global metric, right? Pancreas saw glucose. We sent out insulin. Yep. And the other one is you actually want to know that you have the nutrient that you need. You could have like central command telling you, hey, I got glucose, but if you don't have glucose, you can't do anything. It's a local issue. And so you really want like the central signal

and you want the local signal. So I think one can interpret that the pathway senses both the nutrient. So the amino acid can be a local, right? The glucose molecule itself. It's a local one. If you're sure, we know it is.

Whereas the larger peptide can be sort of the central command. And now you can extrapolate that to there are many signals that are secreted in response to food, right? Insulin just being one of them. And then there are many local nutrients. And now you can start to see the enormous complexity of the problem, right? And now you add a temporal component to it. And now you actually have a concentration. And then you make things tissue-specific. So our view has been, if we can find the sensors of the nutrients, and that's what we focused on, so we focused a lot on amino acids,

but we're also working on glucose. And we could find those sensors by definition, they'll have small molecule binding pockets, right? Because they bind nutrients which are small molecules, although they're small, small, small molecules, compared to drugs. We should build a drug. So in 2015, in the fall, you had these two papers which are small molecules, although they're small, small, small molecules compared to drugs. We should build a drug.

So in 2015, in the fall, you had these two papers that came out that looked at losing, of course, huge interest, but also arginine. Losing an arginine can get into a cell very easily. Did they passively diffuse in? There's transport. There's relatively straightforward. But there's a high capacity transport. Okay.

In the cytosol, these amino acids bind to receptors that then downstream result in the activation of TOR. I'm sure. Specifically, M-TOR complex one. People have long talked about how branch chain amino acids are important for building muscle. Specifically, to be consumed in a workout was always sort of the rhetoric,

presumably because that's a very catabolic time for muscle. It now seems that that makes sense, at least in the presence of what LUCIN's doing. Do we think that the other two branch chanemet amino acids are having any effect? In our, at least when we look at the receptor we found for LUCIN, and then we look at the concentrations at which it might bind the other branch

in amino acids. We don't think those affinities are relevant. Particularly valine, it's way too low. I still leucine maybe in some situations could act through the receptor, but unlikely. So in our hands, again, looking in a very molecular point of view, it really seems like leucine is the key one. And I would think, you know, from talking to bodybuilders

and looking at bodybuilding products out there, it does seem like leucine is the key one. And I would think, you know, from talking to bodybuilders and looking at bodybuilding products out there, it does seem like Lucine is the one that people have focused on more than you and then the individual ones. And tell me, the difference between Lucine and Arginine then with respect to the signaling is what? One way of sort of conceptualizing Amtrakwan is it wants to drive a novelism.

And what is goal is to detect when something's missing for that. So we tend to think of the pathway like when we turn it on, but probably it's really key function is to turn off when something's missing, right? Let's say you're building a house, all of a sudden you'd run out of concrete, you want to turn off, all of a sudden run a wood, you want to turn off.

And so this path... So the default is on. The default when everything is there is on, but it's built, it's organized in such a way that the removal of anything can traditionally turns off. Now this is going to vary obviously between different tissues and so the pathway evolved that it needs to detect Lucene and it detect needs to detect originate. At least in most tissues. Now why is that? They're both amino acids. If you think about this during the course of evolution, you're an animal that ate another

animal, so you ate its muscle, you got protein, why do you need to sense two different amino acids? And they're very structurally different, right? They're about as structurally different as you could get in terms of amino acids. We don't have an answer to that. Why did evolution do that? Pick these two amino acids. I mean, that's a phenomenal question.

I don't know enough about amino acids to know what the evolution of amino acids looks like. I mean, a billion years ago, I assume we didn't have the same amino acids that we did. No, I think we did. We did. There's almost all forms of life have problems. So basically from the beginning of when we had DNA to RNA to protein, we had the exact same amino acids. So then it's even more of a mystery. Why?

And the fact that we... Exactly. Part of people in the lab that I was really encouraging to look at other organisms because the sensing part of the system is probably evolving quite quickly, because different organisms live in different environments. And so for example, flies, we know already, don't care about Argeny. They care about Lucene, and it turns out they care about a whole bunch of other amino acids that we don't care about.

What about yeast? So yeast in many ways is the most mysterious, because yeast, so we don't know any sensors in yeast, and none of the sensors we have found are in yeast. And that's because yeast can make amino acids. So that makes sense, yeast is very primitive. You give it nitrogen, you give it carbon, it's going to make every amino acid. So things like losing which are essential to us are not essential to yeast. They can make it.

And what state do yeast cease to activate tour only in the absence of the essential elements? So regulation of tour is not as well studied in yeast because it's harder to detect the output. And so typically what people do is they change the nitrogen source, so they change the carbon source. And so my view is that yeast has to have a sensor of nitrogen, whatever that means, right? It's not so easy to understand what that means. And a center of carbon. But not a center of individual amino acids. And as we find more sensors, so we now have, we've now connected the path to the methane sensing. Yes. And we have a receptor for that. That yeast doesn't have that either.

And so I've actually also tried to encourage people to look for what might be a nitrogen sensor in yeast for ammonia, or just a simple form of nitrogen. Maybe that's what's sensed. Maybe acetate is what's sensed for carbon. But we don't know. So say more about methionine because in the protein restriction literature, certainly one argument is that methionine restriction specifically could be beneficial if one believes that low IGF is beneficial and we can talk about whether that's causally the case or not,

not even getting into the IGF binding proteins, where does methionine fit into tour? Right, so methionine actually is a very interesting one. As you said, there's an extensive literature on what's so-called methining restriction having quite beneficial effects from glucose homostasis, actually to quite very reasonable fly span extension effect as good as chloric restriction. And there are some papers in flies, genetic papers, that suggest that some of the methining restriction effects go through the torut pathway and flies. We got to this basically through the protein. We found a protein of unknown function and we tried to figure out what it did and it turned out to be a

sensor of this metabolical Sam as a dentosilmethionine which is basically made by methionine. So it's actually quite interesting. People supplement with the variant of SAMe. Exactly. Right. SAM actually has some pretty interesting clinical effects. Actually, it's some quite convincing data antidepressive effects of SAM out there. So the sensor here is interesting, because the other sensors we have directly

by and losing directly by and originating, this one doesn't bind directly to methionine. It binds to a metabolite made by methionine, which is SAM, which SAM, many things can feed into SAM, so it actually can integrate lots of signals. So this sensor basically behaves like the other ones. As soon as methion levels go down, SAM levels go down, this sensor therefore inhibits this pathway.

And so SAM would not be a longevity agent by the oversimplification that excess SAM would be akin to excess methionine would be akin to failing to inhibitor. Exactly. So methionine restriction could be personally rescued by giving SAM. And we actually know in the pathway that we've built in cells that that's true. You can bypass methionine simply by giving SAM. So a molecule that could basically trick this sensor into thinking that Sam was not there,

would be quite an interesting one. I think methionine is probably the most interesting of these amino acids because if you fast an animal, methionine is the amino acid that drops the most. And the reason for this is- And you looked at all of the amino acids in mice. Okay. So we should do some of this in humans.

But it kind of makes sense. I can volunteer with you, man. Well, we? In mice. Okay. So we should do some of this in humans. But it kind of makes sense. I can volunteer with you. We could definitely profile. The reason probably is that arginine you can make some, right? Your liver can make it. And then lucine is an amino acid that's an essential amino acid, but to some extent it's only used to make protein. That's it.

So when you fast, you start to break down your muscle and release lucine. Methining is not only an essential amino acid that you use to make protein, and remember, the first amino acid of all proteins is the methanine. Yep. So by definition, every single protein has a methanine. But, it's also incredibly metabolically active through SAM, and the so-called methanine cycle. So when you fast, you probably just can't generate enough methanine

by breaking down your proteins to keep up with methion demand while you can for losing. So if you look at the blood of an animal that's fast, methionine is the number one dropped amino acid. Do we think that's true in autophagy in general? We may not have. If we put an animal into a state that induces autophagy independent of chloric restriction, so for example, yeah, yeah, would we see the drop in methionine as a readout?

You know, you might expect it to go up, actually, right? Because autophysi is going to break down protein and you might methionine. Yeah, if you're not recycling. If you're not recycling, it depends. If you induce, in the state, for example, post-exercise, I don't know what we know about the use of methionine and SAM, right?

Are you doing a lot? So SAM is used for methylation reactions. And there are hundreds of methylation reactions. SAM is the second most common co-factor in enzymes after ATP. Everyone knows about ATP, and it's in ATP, and then it's used in many, many, many reactions for phosphorylation. But SAM is the second most common one. So there are literally hundreds of proteins that use Sam.

So maybe after exercise, a lot of Sam is used, I don't know. It's an interesting question, right? But with fasting, methionine definitely plummets, Sam definitely plummets. And so we're now generating the right animal models to ask whether the sensor we have is involved in the effects of methionine restriction. So we can basically knock it out and then do methanium restriction.

And if the animal doesn't have the health benefits of methanium restriction, it means that this sensor and by extension, mTORQ1, are the key mediators of methanium restriction. So we'll see. So coming back to RAPA mice in specifically, and all of its limitations, so we've established that you can't just take RAPA mice in all day every day, because that experiment's been done, that's the clinical utilization of it. Certainly, the animal data have suggested, and the human data have suggested

that an intermittent dosing of rapamysin could produce a beneficial phenotype with respect to longevity specifically and also with respect to immune function. So, if you had to guess, based on triangulating these data, assuming no new drug came along that was going to selectively do some of the things that, you know, we've discussed, how would one dose an animal or a human for that matter, wrap a mice in to increase the odds in favor of longevity and against a harmful side effects, which presumably the most obvious ones would be immune

suppression and or glucose, homeostasis disruption. Yeah, and also epithelial through a toxic. Yeah, particularly the GI epithelium. So I think the intermittent approach is definitely the one that makes sense, because if you buy the idea that you want to induce autophagy, which you know a lot of people, of course,

like yourself, who study the effects of fasting, also view that that's one of the goals of fasting is to induce autophagy, which, you know, a lot of people, of course, like yourself, who study the effects of fasting, also view that that's one of the goals of fasting is to induce autophagy. So, if we basically want to chemically induce autophagy without fasting, I think the intermittent dose is what makes sense, is you basically let have an induction autophagy, a relatively weak one with rabbi mice, but then let the system rebuild. It's clear that both M-tore, you need just right amounts, right? You can't have too little, it's toxic, you have too much, it's toxic. The same thing with autophagy. If you remove autophagy, it's really toxic. If you have too much autophagy, it's really toxic. The cycling and abilism, catabolism might be the single most important thing to do.

It might be, right? And I think it's hard for us to know, but those intermittent dosing strategies, every other day feeding strategies, all point to that. And the genetics where too much is bad and too little is bad also point to that, right? So if you genetically inhibit this pathway by deleting raptor, if you genetically activated by deleting these repressors called the tuberosclerosis complex, both are bad.

Both in fact, in many tissues, like the muscle give the same output. They get musculoskeletal. Yeah, I was just about to say, there's an overlap with muscular dystrophy here, isn't there? Yeah, exactly. So this may be a theoretical question, but when we think about the life-extending properties

of rapamycin, do we believe that it is a result of delaying the clinical onset of disease? Let's use a disease where that tends to be more binary like cancer. But obviously cancer spends probably 70 to 80% of its time undetectable, but due to the just the law of growth, it becomes detectable only at the end. So do we think that in as much as, say, taking these agents would allow you to live longer by not dying from cancer at the same period of time, does it delay the time it takes for cancer to become clinically detectable and or delay the demise of the

animal once it has that cancer? Yeah, I think it's specifically, in the case of cancer, rapamycin is there are some situations where it has some decent activity, but in general, it's not a cytotalker agent, right? It's not going to kill a cancer soldier. We're going to say... Once an organism has cancer, do you know if it's doing anything to prevent the development of cancer?

We don't know that well. The only, there actually has been some epidemiological data where people have compared cancer rates in transplant patients. Identical patients who are with and without rapids. FGF6 versus rapamycin. And it's actually quite interesting because, as you know, immunosuppression in general is associated with higher cancer rates, right?

The idea that you have less immune surveillance, that's not seeing a rapamycin. So it is seeing a FGF6, it's not seeing a rapamycin. And the argument has been that rapamycin itself has cancer cell autonomous independent of the immunomodulation problem. So you're presumably getting less immune surveillance because it's immunosuppressant although of course that's not proven. But you're mitigating that. Yeah. By now directly. And they've canceled each other out. They canceled. And you know

the size of the effect from the FK506 cohort. Exactly. And other immunosuppressant things like this before. I have also been looked at that. So my bet would be that in the case of cancer, you're not going to, you're not going to cure cancer once you've got it. But I also think you're going to modulate the incidence, like the mutational frequencies that are giving you cancer, right? So if you think of cancer in a way is easier to think about when it starts, because you say, well it starts when you have a cell that has all the requisite mutations to be a cell that has uncontrolled growth. So if that's the point it starts I think we're not going to

affect that. But once that cell exists and now has to start growing and and also escaping the immune system, I do think that's probably what you're going to affect. In other diseases, like a couple cardiovascular disease where you could imagine things like autophagy could be quite modulatory, I think you can imagine that you're also being affecting the incidence,

at the exact point at which you'd say, okay, this is an atherosyrotic plaque or not. What do we know about rapamycin and tour in the brain, especially with respect to neurodegeneration? Yeah, that's a really interesting one and that probably is a really important question for the future. So we know a topology matters a lot in the brain. If you delete a topology and really in Mitsushima was the person who kind of made a topology interesting to lots of people. And it was a word of the Nobel Prize. No, he wasn't wasn't. Oh, he wasn't. He was for every year. But he didn't share.

He didn't know that much. I think was a bit of an oversight in my view. But anyhow, he basically studied atophagy in the brain, made mutations, showed you got neurodegeneration. So that was a really important find. Connects up to lysosomal storage diseases, which, you know, atophagy, basically, auto-fagosomal fuses with a lysosomal, so now you have that connection.

So I think like in all tissues, it's a bit of a double edged sword. You clearly need M-Troke 1 activity to maintain healthy synapses, certainly during brain growth, if you make mutations around boy and growing animal, you basically don't have a cortex. Yeah.

Right. On the other hand, you clearly need to be a modulated M-Troke 1 to have some level of autophagy to keep the system healthy. Now you could debate, is that in neurons, is that in glia, it's probably in both people have made mutants in, certainly in neurons, we suggest it's both, but then some of those promoters a little bit dirty. But the real question in the brain is what modulates M-Tort One?

Because it's not probably nutrients. Because they're so constant, you mean like, exactly. Your brain, your body, basically. Your brain prioritizes nutrients in the brain over it. If basically protects you guys. So if you take an animal and you fast it for two days and mouse, it loses a lot of weight, 25% of its weight.

And now you take every single tissue and you weigh it, every tissue has shrunk. Some like the thymus are shrunk ridiculously. The kidney shrinks, which you wouldn't expect. The heart shrinks. The brain, nothing. Now clearly, probably if you, in amounts you can't do that extreme a fast. And so the body protects the brain from a nutrient point of view.

Yet, M-TRIQ-1 activity is high there. Clearly we know that we have to modulate autophidies. So something must be inhibiting M-TRIQ-1. I mean, this is my peripheral argument for why. And I'm in the huge minority here. I do not think the brain is really the appetitive center. I think it's the modulator, but for that exact reason, I think it wouldn't make sense for evolution

to put our appetite center in our brain. It should be in the periphery. It should be in the liver, I think. I think the liver should be the problem. But people who argue that the things that are hypothalamus are in the periphery, right? Because they're not protected. They're a part of your brain like the hypothalamus.

And the point is, I think it has to be your appetite center needs to be regulated to something that senses very rapid. Outside of the brain. For sure. Yeah, for sure. And exactly where it is, you know, in the bottom line is probably. But I never thought of it through the lens that you just explained it, which was the implication of that for TOR is enormous.

So does TOR look different in the brain? Or, obviously, the protein won't, but do the co-factors around it look different? So, really, we keep talking. We have never done framble biochemistry out of the brain. It's something that would be really interesting to go and do now. I think now, it's something we talk quite a bit as a lab to do. We haven't quite done it at all. But then what actually regulates,

it's very clear that neuronal activity does. But are there other, like as you're suggesting, maybe neuronal specific factors are regulated? I think that's a completely open area. I've tried to get some of my students interested in that. My brother's a neuroscientist. He's argued we should really do some work there. We just haven't.

Maybe when we run out of sensors in the periphery, we'll go to the brain. And that where I purified M2, it was out of brain. So there's a ton of M2 in the brain. And I did that not because I was like whatever. I basically measured how much there was, and it was clear the brain had the most. One of the challenges of studying biology and humans is that you can't do the same experiments, you can do in animals. If we had a G'donkian experiment where you could take a sufficiently large number of human subjects and divide them into groups, and you had a control group.

These guys were going to do everything that the standard American does. You had a group that you could give, wrap a mice into, in any way shape or form you decide. Then you had a group in which you could manipulate their behaviors, and they would behave as animals. They would do anything you want with respect to how they would eat or how much or when exercise would every like. First question is, how would you design arms two and three? To have the best outcome with respect to longevity.

And then I'm very curious to know what you think the difference between groups two and three would look like. So I think as we spoke before, I mean, that M-odulator arm would probably be an intermittent type dosing one. We're hopefully we'd have biomarkers of that. And you and I have spoken on the past, a biomarker for autophagy, for example, biomarker for M-Traumodulatory activity, and yet to decide what tissues you cared about. Probably the muscle would be one that you'd want to focus quite a bit on and perhaps deliver. Now, I don't think that M-term modulation on its own is going to give you all the benefits of good lifestyle modulation. So it might give you lots of the

benefits of the dietary manipulations, the fasting manipulations, although clearly there's differences there. But I'm not sure if I'm going to give you all the benefits of the exercise modulation. And so if then the lifestyle side, which you obviously know better than almost anyone, what you'd exactly want to do, there'd clearly be an exercise component to an on top of a dietary component. I think M-Tore modulation will give you a subset of that. I see. And so let's simplify the experiment then. Let's assume that everything but food is the

same in the groups and the RAPA group gets the intermittent doses you've see fit, and the other group now can fast or do any sort of CR mimicry that you want. Do you think that normalizes the playing field? I think it gets a lot closer for a simple reason. So if you give an M-Tromocellator versus a fast room, there's one really important difference is that nutrients in the M-Tromocellation case are actually still high. Yeah. Because first of all, in fact, if you actually look in cells, they can actually even be higher because the cell

thinks it's starving. So it does all the stuff. So it shuts down processes that would accumulate them. Yeah, and it's more intelligent. And so we've looked in cells, so actually they tend to go up. Versus a fast where things are going to be lower.

On the other hand, if all those nutrients are eventually doing their stuff by communicating through amtore and you've sort of inhibited downstream to those downstream processes, things look the same. It doesn't matter. We have a lot of nutrients here and very low nutrients here, so the modulation of amtore is what matters. So I think to answer that question, we really need to understand whether all these nutrients

are still there in the fed state have a lot of other signaling effects. And it would be naive to think that they don't. They do, we know they do. Now do they matter and do they matter a lot? I don't think we know. And I think a lot of the genetics and pharmacology would argue that within the range that we can actually manipulate lifespan, it could be that those fasting regimens and rapamycin are somewhat similar. And certainly in the mice, it appears to be at least similar if not better in favor

of rapamycin. Exactly. And that's why I'm particularly excited about the methanine restriction work, because you know, caloric restriction is not only hard to do in people, it's hard to do in animals too. It's really hard. You have to weigh the food, pair-wise feeding. It's a real pain, and it's a real restriction to doing lots of experiments.

While methyl-resistant, it's a lot easier. Yeah, just by methane and free-chall. Yeah, and not totally free, but lower, right? And so there we can do these kind of experiments where you could do a methyl-resistant plus-repe mice, right? And actually ask, do you get synergy, do you not? So I think that's going to be an intervention that's going to be a lot easier for us to play with.

So if resources weren't constraint, what are the sort of dream experiments or what's a dream experiment that has been on your mind that you want to do, but it's either technically we're not there yet or it's just economically a challenge. Yeah. I think what I want to know, and this is I think the challenge for anyone who does what we call signal transduction in a dish like we've done for a long time, is to really try to understand in each different tissue in a temporal fashion in response to a variety of different diets and nutritional states what those tissues are actually doing. Right now we have these little time points in the liver,

in the muscle. We don't really have a deep, sort of, kinetic understanding of what the actual physiology is doing. We'd really like to know. Because even in the mouse, you know phosphorylation in one moment, you don't have an end to grow. In one tissue. We don't have that.

And we don't even have, it's just their expensive and hard experience that do. That's it. I really wanted to take mice, fast them, and in all different tissues, and ideally tissues are complex, right? Now, with all the single cell sequencing, we're seeing much more complexity. So even in tissues like the liver that we tend to take a chunk and say it's liver, we know that it's an amazing complexity, right?

And so in ideal world, we'd like to have a description of all these different tissues are doing over time, and then you'd like to do it on your different diets, under whether they were obese mice, whether they were exercise mice, and so the matrix becomes ridiculous at that point. But I think that's the future of signal transduction. People like me have done a good job of finding all the pieces in some random cell line in a dish. And clearly, the systems have all these pieces

because it allows them to communicate in vivo to many, many different upstream signals. And now the challenge is, how do we go back? And actually see that happening. And that's going to teach us, OK, which tissues actually matter? We've talked a lot about longevity. Do you need to impact all tissues?

Is it the muscle? Is it the liver? Is it the brain? Maybe that you need to impact. You know, people debate still how much rap mice and gets in the brain. Are you actually affecting the brain? I don't think those are open questions, to some extent. So it would be a complete description of what these systems are doing over time across many tissues under many different states. Well David, we're pretty much out of time, but is there anything else that we should at least take advantage of while you're here? I think we already touched upon it when we talked about targeting M-Truck 1 or other things. And so I think to me, and this is why we sort of had commercial interest in this regard,

how do we go and target other things upstream that might be more amenable to giving us sort of more this dream molecule of a Pan-M-Torque-1 inhibitor and no M-Torque directivity? David, thank you very much. Thank you very much. It was awesome. You can find all of this information and more at pterotiamd.com forward slash podcast. There you'll find the show notes, readings, and links related to this episode. You can also find my blog and the Nerd Safari at peteratia-md.com. What's a Nerd Safari you ask? Just click on the link at the top of the site to learn more. Maybe the simplest thing to do is to sign up for my

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