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Discovery of Protein S-nitrosylation Leads to Creation of Oxygen and Nitric Oxide Exercise Monitor


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Dr. Daniel Simon (Host): Hello, everyone. My name is Dr. Dan Simon. I am your host of the Science@UH Podcast, sponsored by the University Hospitals' Research and Education Institute. This podcast series feature University Hospitals' cutting-edge research and innovations. Thank you for listening to another episode today.

I am happy to be joined by our guest, Dr. Jonathan Stamler. Jonathan is the president and founder of the Harrington Discovery Institute and an internationally acclaimed physician-scientist known for the discovery of protein S-nitrosylation, a global post-translational modification of proteins that is widely involved in both physiology and disease.

Jonathan also has a track record for innovation and entrepreneurship as a founder of institutes, biotechnology companies, medical societies, innovation platforms and impact investment funds.

I also have the pleasure of having worked with Jonathan now for nearly 35 years, so I know him very well when we trained together in Boston. Welcome, Jonathan.

Dr Jonathan Stamler: Yeah. Thanks, Dan. Pleasure to be here.

Dr. Daniel Simon: Great. So Jonathan, you discovered a mechanism of what we call protein S-nitrosylation, and that all classes of proteins can be modified by this, and that aberrant S-nitrosylation plays a role in diseases such as Alzheimer's disease and cancer and others. Maybe you could just tell us a little bit about how did you stumble upon this? What made you think that there was another way for proteins to be regulated?

Dr. Jonathan Stamler: So, you're taking me back a long way, Dan. It's actually a story you know pretty well. But I was in training, I guess, in medical school interested in what we call redox phenomena or oxidative stress, the rust that built up in systems as we age. In those days, it was thought to be specifically related to free radicals. And I somehow identified those free radicals with daffodils, which were very frequent in the fields in England and fell in love with that area.

I was also interested in cardiology. And then in 1987, papers were published in Nature on nitric oxide. And it's a magical moment in science when we learned that this gas, a free radical, an oxygen-free radical in fact, was produced by blood vessels, dilated blood vessels, and did so many amazing things, transforming fields. And I thought, "Wow, that's what I'm going to work on. I want to be a cardiologist. I want to work in free radicals. I'll work on nitric oxide." And in the ensuing two or three years, as I said, this magical molecule did absolutely everything, if you think about it, right? It was fighting cancer and improving memory, and improving heart function and erectile function, there was nothing the molecule didn't do, and the question was how. And in reality, no one knew at the time, and they had identified a mechanism for which the Nobel Prize would be given that made no sense for how it worked. It made sense in a very narrow exception to the rule circumstance, and that is blood vessel vasodilation, but not for the general action.

And I guess that's where I had some insights. I knew that we needed to attach that molecule, not to one protein, but to many, many, many proteins for the molecule to have many, many actions because that's just the way science worked. There was no chemistry for that at the time. And so, I needed to think through ways to do this. But ultimately, I was in search of what is known as allostery. It's sort of the second secret of life. The first secret is, of course, the gene sequence, right? The second secret of life, as Manod would call it, is allostery, the changes in shape and function of proteins that come about by attachments of small molecules. So, I wanted allostery, I wanted to attach nitric oxide to many, many different proteins. And that was the idea and I guess the foundation for the work that we would go on doing.

Dr. Daniel Simon: Well, I think that's an amazing story. And it is sort of ironic that we're sitting here today when we, I think, published that first paper in PNAS back in the early '90s that you could nitrosylate three candidate proteins, albumin, cathepsin and tPA, and sort of the world changed, I guess I remember it as being your technician and mostly bleeding my blood every day for about a month and became anemic because we needed so many supply of our blood to do these studies. But those are really terrific memories.

Now, one of the things that our listeners don't know, and maybe I can set the stage of how this really exploded on the scene and tipped the world upside down, was the most studied protein in the world is hemoglobin. And we all learn in medical school, right? It carries two gases, oxygen and carbon dioxide. So, it picks up oxygen in the lungs, dumps the oxygen in the tissues, picks up CO2, exits the lungs, and that's how we live. And all of a sudden, Jonathan Stamler published, "Well, guys, it's great that this is the most studied protein in the world and it carries two gases, but you forgot a third. It actually carries nitric oxide." So, tell us about that, because you’re most famous for that, and it's really probably one of your most impactful observations because it changes the whole way we think about how we bring oxygen to tissues. So, tell our listeners about that. It's a great story.

Dr. Jonathan Stamler: So, it's interesting that you introduce hemoglobin in that way because I didn't come to hemoglobin, because it carries nitric oxide. But rather, coming back to those three proteins that you and I worked on together, tPA, cathepsin B and albumin. Albumin was a general case for me. My point being here that they were chosen for a reason. tPA, which was prevalent in the lab, had a cysteine in an allosteric site; cathepsin B, the cysteine was in an active site. So, I wanted to demonstrate that there was a general function on proteins by stabilizing nitric oxide and giving activity to it, that was albumin cathepsin because the modification would be on the active side of a protein; and then, for tPA, it was an allosteric regulation. In fact, a central station increased the activity of tPA.

But at the end of the day, I was aiming for allostery. And the most famous example of allostery in science is in fact hemoglobin. It turns out, unbeknownst to virtually everyone, there are only three amino acids strictly conserved in hemoglobin, absolutely strictly conserved. Two of them are required for carrying heme for oxygen binding. Without them, you can't carry oxygen, and it makes complete sense as to why you absolutely have to have those two amino acids. The third is a cysteine, not talked about, but essential for life. It's invariant in every mammal, absolutely invariant. And Perutz, the famous Perutz, would ask a question as to why that cysteine is needed and, moreover, why its reactivity and function appeared to be linked to oxygen binding. There was no answer for that.

But I knew, lo and behold from reading about hemoglobin and thiols, the cysteine residues to which nitric oxide would bind just generally, that this was the best possible example of allostery. If I could attach nitric oxide to that cysteine and hemoglobin, by definition, it would have to change oxygen binding, and I would've proven, I thought, to the scientific field that S-nitrosylation, the binding of nitric oxide to proteins to cysteines, and proteins in fact, could work through allostery, and I would've solved this problem of how nitric oxide actually works. Little did I know that that would be so far from the truth.

In actuality, I was able to put that nitric oxide onto cysteine, show that oxygen binding changed and prove that point. And there were major ramifications to that because in fact, at the time, the Nobel Prize had been given for the idea that hemoglobin consumed and eliminated nitric oxide. And I was showing that you could attach it to a protein and change protein function…that caused controversy in its own right. But the greater problems arose because I decided that having demonstrated that I could change function and had proven that S-nitrosylation, that nitric oxide could work through an allosteric mechanism, I then went on, as physician scientists will tend to do, to try and work out the physiology and that would then, I think, get me into more trouble. But the reality is that, today, I think we know that in fact hemoglobin does carry three gases, the nitric oxide on that cysteine. Really important to understand that the genetic basis for nitric oxide binding and its importance generally is as important, as well established genetically, as it is for oxygen.

Dr. Daniel Simon: So, tell me just a little bit about when we exercise, and we're trying to increase delivery of oxygen to the muscles. How does that happen? What is nitric oxide actually doing? Are we carrying more oxygen or are we just increasing blood flow?

Dr. Jonathan Stamler: Okay. Well, a lot of things are going through my mind here, because there are a lot of interesting stories and implications in answering your question. But first to understand, we have a lot of oxygen and we have very little nitric oxide. But the amounts of nitric oxide that we have are tremendously powerful, tremendously potent. So even though it's true that S-nitrosylation of hemoglobin, binding nitric oxide to hemoglobin, will change the binding of oxygen, okay, that's a fact…it's oxygen coming on and off that ultimately releases or binds nitric oxide to hemoglobin that's important physiologically.

So when oxygen goes on, nitric oxide goes on. When oxygen comes off, nitric oxide comes off. That's first to understand. And what nitric oxide does is... it opens little blood vessels in tissues so that oxygen can go in. Moreover, since oxygen only comes off in one form of hemoglobin, and hemoglobin assumes that form only in hypoxic tissues, nitric oxide is only coming off in hypoxic tissues, and the implication of that is that nitric oxide matches blood flow to oxygen requirements. Where oxygen is very low, hemoglobin is releasing oxygen, releasing nitric oxide, opening those blood vessels proportionate to demand. So, the long and short of that is nitric oxide is regulating blood flow to hypoxic tissues.

Now, the scales have really been removed from my eyes when I learned that the physiology of blood flow in the respiratory cycle, the cycle that delivers oxygen to tissues, had not been integrated well with the ideas in textbooks on oxygen delivery. So when we read textbooks on oxygen delivery, everyone just thinks you pick it up in the lungs and you deliver it to tissues. In fact, the importance of oxygen in all of this is small in comparison to the role of blood flow. You know, Dan, better than most as a cardiologist, that the game is blood flow when it comes to low oxygen. You can increase your blood flow in an exercising limb by a hundred fold, you don't really change your oxygen levels.

So, hemoglobin's job in all of this is actually to regulate blood flow. It's not really to control oxygen levels. And when you're sick and your oxygen levels drop to maybe 90%, we'll come back to this in a moment, you've still got tons of oxygen. And when you die, you've still got tons of oxygen. What's going wrong is blood flow. And what's going wrong is nitric oxide release.

Now, what's the physiological role in all of this? It's exercise. And when you exercise, you use up oxygen in the tissues and nitric oxide comes off to increase blood flow. So bottom line, we cannot exercise. And our exercise capacity is fundamentally dependent on release of nitric oxide from hemoglobin. Mice that cannot release nitric oxide from hemoglobin cannot increase their blood flow.

Dr. Daniel Simon: Yeah, and I think that, you know, this is just absolutely an incredible thing because we all studied Guyton, that was our textbook in medical school, and there were just so many holes in trying to understand how this all happened. So, this actually brings up a very interesting observation, you know, which obviously you've made. So, we all know about the problem in medicine of old blood, blood stored in a blood bank that's old. And as you went on to show, that's because it's depleted of nitric oxide. And then, when you give that old blood, it impacts oxygen delivery.

And so now, I think what's really exciting is that you've taken these discoveries to a new level. And you have a device from a company that you co-founded called NOx, that actually allows one who's exercising to monitor their nitric oxide and their blood flow. And it has tremendous implications for everything from brain health to cardiovascular health and exercise performance. Maybe you could tell us just a little bit about what that device can do and how eventually, probably we'll all have it on our Apple watches.

Dr. Jonathan Stamler: It's an opportunity for you and I, also to share the importance of exercise. I think, you know, first and foremost, in the community, there's really nothing we can do that is more important than exercise for our health, health span, lifespan; the single most important activity, perhaps with the exception of companionship, according to that recent Harvard study, in our lives. But companionship aside, exercise trumps virtually anything else. Now, we have to understand what does exercise actually do. And we know that it improves all-cause mortality, cardiovascular mortality, cancer mortality. I mean, it's truly remarkable. The question is how.

The fundamental behind exercise is blood flow and oxygen delivery. That's actually the foundation of exercise. When you exercise, your blood flow goes up and your oxygen supply nourishes your tissues, heart, brain, and muscle. And at a molecular level, we understand that. As cardiologists, it's blood flow that prevents atherosclerosis and prevents inflammation and prevents clotting. And it turns out that blood flow is also exceptionally important for cancer because cancer likes tissues that don't get oxygen. So if you get blood flow and you oxygenate, you're protected more from cancer, I'm talking simple terms. And blood flow to the brain protects against Alzheimer's. And if you don't have it, you get dementia. So, things sort of fall in place. If you have blood flow, your sensitivity to insulin is improved. So, bottom line is… it's blood flow and oxygen supply.

So now, we ask how do people measure effective exercise? They use heart rate, which is in actuality pretty ridiculous. So, it is true that if you exercise more, your heart rate goes up. And if you look at populations as a whole, higher heart rates mean more exercise. So, you're fitter and you can look at higher heart rate and then assume you are in some way fitter. You could even say that if you have a higher heart rate, you have a higher VO2, volume of oxygen consumed, which is a measure of fitness and health. Yeah, so there's some relationship. But if I ask you, Dan, a cardiologist, your heart rate's now 96, are you fitter? Your heart rate went to 106. Was that better exercise? Of course not. It's almost meaningless on the individual by individual level. It has no meaning.

So, we need real measures of effective exercise. And those measures are, your fuel gauge, what is your oxygen level in your tissue; and your power supply, how much blood flow are you actually getting in your tissues. Well, your fuel gauge is your oxygen, your power supply is your NO, and we've built a device that can measure both in real time. So, I can now tell you in real time whether you have effective exercise. Effective machines are using a lot of fuel and they're powering that system with a lot of blood flow. And so, it's really amazing. But you exercise, your nitric oxide goes up and you can see in real time that you're powering heart, brain and muscle. And you can begin with these measurements to consider and direct one's own exercise for efficiency and performance.

Dr. Daniel Simon: Yeah, that's really amazing. And I know that this is moving beyond the prototype stage and is being used for not only the notion of improving brain health and cardiovascular health, but also for high-performance athletes and returning to the playing field, which Dr. Voos and our Browns team is using on professional athletes. So, we will follow that very closely.

I think our listeners are going to be really happy to hear that you've told us really two very important things about our lives. First is that companionship is most important, and second is exercise. So now, we've learned that you and I have to exercise. Kathy and you need to go out and exercise, and Marcy and I need to go out and exercise and if we do that, our lives will be much better and I think they'll be happy to hear that and I know they'll be listening to this podcast.

Okay. So, last topic to talk about. You and I are physician-scientists. We spend our lives working in the research lab with the hope of making a difference to patients. And many of our discoveries, most of our discoveries, as we say, stay on the bench top or get used in mice, but don't ever make their way to humans. And one of the most important things that you've done as the president of the Harrington Discovery Institute is to bring a total new mechanism of drug discovery and developments, both on the non-profit and the for-profit side. Just tell us a little bit about what exactly is that doing. How does it do it? And tell us about some of its successes so far.

Dr. Jonathan Stamler: Before I do, I just want to echo your comments and to double down on thanks to James Voos and Lee Ponsky as they develop devices and look to improve sports performance and men's health. But turning to Harrington Discovery Institute, I think your question was how do we do this? How do we go about creating cures, next generation cures? You know, I think you and I have the privilege of working in a truly noble profession that looks to make discoveries that will impact the future of human health and to apply that information to the next generation of cures. And we live in health systems that support us and allow us to do this. At the same time, individuals can only do as much as they can do and single health systems can only do as much as they can do. And you and I know really well as we've worked together on this, that when you get sick, you really don't care about one individual's discovery or one health system's capability. You care about the best medicine to cure you.

So, we go about, first and foremost, of bringing in the very best discoveries in the nation. Of course, we give a leg up to our own institutions because community is essential and you can't do anything without support in community. But at the end of the day, we live by excellence and by opportunity to make the very best medicine. So, we bring in the best science across the US, Canada, United Kingdom. We evaluate by high rigorous scientific standards, but also by drug development standards. We know that we have short periods of time to advance things in a meaningful way. So, we bring in the best evaluated by both pharma drug opportunity and by scientific excellence.

And once this selection is made, and this is through calls for RFPs for the very best science and technology, we give every one of our scholars across the US, Canada, United Kingdom, a team for two years of drug developers, business development support and program manager that will run basically a small little company, non-profit, to value inflection, to critical milestones that create value. And we do this against unmet needs that you and I think about and decide are the most important things that we should be focusing our resources on. We then bring in these scholars within portfolios so that we have many shots on goal. We are basically working to create 14, 15 technologies in any area of unmet need, putting resources progressively into those that are best, those that are making the greatest progress. And then, ultimately, we will begin to invest in those discoveries because the reality is that investment money, the change in discipline from non-profit to for-profit, is essential if we're going to see drugs affecting patients. So in short, we bring in the very best, we develop drugs using drug development principles and portfolio management principles and, ultimately, begin to invest in these discoveries so that they can be put on a route to commercialization.

Dr. Daniel Simon: You know, it's really exciting, and I think that people need to realize that University Hospitals and the Harrington Discovery Institute is widely regarded as sort of the number one drug development program in an academic medical center. You've funded now nearly 165 scholars. You've founded, I think, nearly 35 companies and you have 15, 16 drugs now in clinical trials. So, it's really working. And I think both of us are really hopeful that you're moving all the way through phase III clinical trials that very soon. The Harrington Project and the Harrington Discovery Institute will have its first FDA-approved drug, which would be just absolutely remarkable.

You know, this has been a tremendous short session with you. We could go on probably for hours. It's been truly an honor and a privilege to work so closely with you these past 35 years, and especially to have recruited you to University Hospitals from Duke nearly 12 years ago now.

Dr. Jonathan Stamler: And thank you, Daniel. I feel the same way. The privilege is mine, working with you for 35 years. And as I sign off here, I get this opportunity, you're a busy guy, I didn't have the time to tell you this morning that it looks like we have another deal with Takeda. They're going to take another drug. So, I told you about one last week. There's another one this week they look to be taking. So, make that 14 drugs licensed to pharma.

Dr. Daniel Simon: Excellent. So great. Thank you so much for taking the time to speak with us today, Jonathan.

For our listeners interested in learning more about research at University Hospitals, please visit uhhospitals.org.