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Peter Attia MD#09 – David Sabatini, M.D., Ph.D.: rapamycin and the discovery of mTOR
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CHAPTERS
- 0:04 – 5:40
Intro and David Sabatini's Path to mTOR
Peter Attia introduces his guest and why mTOR and rapamycin matter to him. Sabatini then describes his position at MIT and the Whitehead Institute and why he is unusual in staying with his graduate-school topic.
- •Sabatini is a professor of biology at MIT and a member of the Whitehead Institute
- •He discovered mTOR as a student and has kept working on it
- •Most academics change direction after graduate school and the postdoc, but he did not
- •Attia says his interest in mTOR comes from its role in nutrient sensing and longevity
- 5:40 – 11:58
Choosing a Project in Solomon Snyder's Lab
Sabatini recounts joining Solomon Snyder's lab at Hopkins and the freedom Snyder gave him in picking a project. Rapamycin was only a control drug for FK506 there, but Sabatini found it more interesting and Snyder had obtained a large supply of it.
- •Snyder told him only "we work on the brain," which left him free to choose his own project
- •The lab used FK506 to study calcineurin, with rapamycin as a control
- •Rapamycin was not commercially available, but Snyder had obtained a large amount from Wyeth
- •Sabatini defines a small molecule as roughly under a thousand daltons
- 11:58 – 16:10
Purifying mTOR from Rat Brains
Sabatini describes how he purified the target protein, first called Raft1, and that Stuart Schreiber's group found it independently. He explains the experiments and the long hours behind the 1994 Cell paper.
- •Raft1 stood for rapamycin and FKBP target 1, while Schreiber's group called it FRAP
- •Rapamycin binds FKBP first, and the complex then interacts with mTOR
- •Sabatini only learned of Schreiber's parallel work after his paper was accepted
- •He purified the protein from hundreds of rat brains, working very long hours
- •A silver stain band that appeared only with FKBP and rapamycin convinced him it was real
- 16:10 – 21:51
Cloning the Full-Length mTOR cDNA
Sabatini explains the molecular biology tricks used to sequence and clone mTOR, including working out the order of peptide fragments with degenerate oligonucleotides and PCR. He also describes the lucky recovery of a full-length 9-kilobase cDNA by letting slow-growing phage plaques grow longer.
- •Paul Tempst supplied peptide sequences without any information on their order
- •Degenerate oligonucleotide pools and PCR combinations established the order
- •The full-length cDNA made the phage replicate slowly, so plaques had to grow longer
- •The cDNA was about 9,000 base pairs and came out intact
- •This work became his PhD, and it would now be roughly figure 1AB of a student's paper
- 21:51 – 28:31
Starting a Lab at Whitehead and the Hunt for mTOR Complexes
Sabatini tells how he got the Whitehead fellow position and then struggled to run a lab while the field stalled. A postdoc's switch to the detergent CHAPS finally kept the mTOR complexes intact and led to mTORC1 and mTORC2.
- •He applied to three independent fellow programs and the Whitehead one was the last to respond
- •He knew from early cross-linking experiments that mTOR had a small partner protein
- •The common detergent broke apart the complexes, and Dos Sarbasov found CHAPS preserved them
- •Raptor and rictor were identified around 2001-2002
- •Several years of no progress were followed by rapid progress once the detergent was changed
- 28:31 – 33:47
What mTORC1 and mTORC2 Do, and the Link to Longevity
Sabatini explains that mTORC1 accounts for the effects previously attributed to rapamycin, while mTORC2 was more mysterious and acts via the PI3 kinase pathway and AKT. He recalls that the longevity connection became clear only later, helped by worm genetics.
- •mTORC1 regulates growth, translation, autophagy, metabolism and cell size
- •mTORC2 is part of the PI3 kinase pathway and regulates AKT
- •Some people criticized him for working on a "silly molecule"
- •A C. elegans mTOR mutant with longevity effects was a key paper, and one DAF mutant turned out to be raptor
- •Rapamycin does not get into worms
- 33:47 – 39:32
Why Inhibiting mTORC1 Extends Life: The General Contractor
Attia asks why mTORC1 inhibition can extend lifespan, and Sabatini says the answer is not known. He suggests autophagy is part of it and offers an analogy of mTOR as a general contractor for the cell, with the risk that affecting so many processes also brings downsides.
- •Autophagy is probably the most important process mTORC1 regulates, but it is not the whole answer
- •mTOR is likened to a general contractor that touches every major process in the cell
- •Slowing aging likely requires changing many things at once, and mTOR is a single control point for that
- •The downsides include effects on sperm production and heart function
- •Immunometabolism has grown into a large field, with mTOR central to it
- 39:32 – 42:57
Limits of Rapamycin and the Ideal mTORC1 Inhibitor
Sabatini explains why complete mTORC1 inhibition would not be tolerated and why rapamycin only partially inhibits it. He describes the ideal molecule and why his lab targets upstream regulators instead.
- •mTORC1 is probably required for the growth of any normal cell, and deleting raptor can be harmful in tissues such as gut epithelium
- •Longer rapamycin exposure also inhibits mTORC2, which could harm glucose homeostasis
- •Rapamycin occludes the substrate channel, so autophagy is only weakly modulated
- •Rapalogs behave much like rapamycin apart from PK/PD differences
- •mTORC1 and mTORC2 share a kinase domain, so the lab goes after upstream regulators instead
- 42:57 – 50:00
Nutrient Sensing: Insulin, Glucose, Leucine and Arginine
The conversation turns to how nutrients and hormones feed into mTORC1. Sabatini describes central versus local signals and the lab's discovery of leucine and arginine sensors, noting that mTORC1's key role may be to turn off when something is missing.
- •Insulin and glucose act through separate pathways that activate mTORC1
- •Cells may need both a central fed-state signal and local nutrient availability
- •Leucine seems the key branched-chain amino acid for the receptor the lab found
- •mTORC1 is on by default and turns off when any needed input is missing
- •Why evolution chose leucine and arginine is unknown
- 50:00 – 54:35
Sensing Across Species and the Methionine/SAM Sensor
Sabatini discusses how amino acid sensing differs in flies and yeast and then describes the lab's methionine pathway via a SAM sensor. He explains why methionine falls most during fasting and how animal models will test whether this sensor mediates methionine restriction.
- •Flies do not care about arginine but respond to other amino acids
- •Yeast can make amino acids and has no known amino acid sensors
- •The new sensor detects SAM, a metabolite made from methionine
- •Methionine is the amino acid that drops most in fasting mice
- •The lab is generating animal models to test whether the sensor mediates methionine restriction
- 54:35 – 59:40
Intermittent Dosing, Cancer, and the Balance of Anabolism and Catabolism
Attia asks how to dose rapamycin, and Sabatini says intermittent dosing makes sense, since both too little and too much mTOR or autophagy are harmful. They then discuss how rapamycin might affect cancer and other diseases, with Sabatini noting that it is not cytotoxic and may act on tumor growth rather than initiation.
- •Intermittent dosing lets the system induce autophagy and then rebuild
- •Genetically deleting raptor or deleting the tuberous sclerosis complex repressors are both bad, and both can cause muscular dystrophy in muscle
- •Transplant patients on rapamycin do not show the higher cancer rates seen with FK506
- •Sabatini bets rapamycin affects growth of existing cancer cells, not the mutations that start cancer
- •For cardiovascular disease, autophagy modulation might affect incidence of plaque
- 59:40 – 1:03:17
mTOR in the Brain
Attia asks about rapamycin and TOR in neurodegeneration. Sabatini says autophagy and mTORC1 activity matter in the brain, and the brain is protected from nutrient changes during fasting, so what regulates mTORC1 there is an open question.
- •Autophagy mutations in the brain cause neurodegeneration, and autophagy connects to lysosomal storage diseases
- •mTORC1 activity is needed for healthy synapses and for brain growth
- •The brain is spared when a fasted mouse's other tissues shrink
- •Neuronal activity regulates mTORC1, but other regulators are unknown
- •Attia offers his view that the appetite center may belong in the periphery
- 1:03:17 – 1:11:27
Thought Experiments: Rapamycin versus Lifestyle, and Dream Studies
Attia poses a hypothetical human experiment comparing rapamycin with lifestyle changes. Sabatini says mTOR modulation might capture many fasting benefits but perhaps not all exercise benefits, and he describes the dream experiment of mapping tissue responses over time. He ends on the goal of a pan-mTORC1 inhibitor acting upstream.
- •An intermittent rapamycin arm would ideally use biomarkers of autophagy and mTOR activity, perhaps in muscle and liver
- •Nutrients stay high with rapamycin but low in fasting, so effects may differ
- •Methionine restriction is easier to run in animals than caloric restriction and could be tested with rapamycin for synergy
- •The dream experiment is a time-resolved description of what many tissues do under different diets and states
- •The goal is an upstream-targeted pan-mTORC1 inhibitor that spares mTORC2
