Transcript for Diomedes Logothetis Talk
SPEAKERfairly recently on, in addition to just a lot of impressive work signaling and I and channels, we are about to go. They make him. They put him in charge of stuff. And so he's been a dean and he's run a couple of programs. And also everywhere he goes, he gets mentorship award, which is actually, it's true through his mentorship that I got to know him. So I'm very excited to have him show you what it does without further ado. I think you don't thank you for inviting me and thank you for coming. So today, I will introduce you do the work that we do, I guess. Is it going so, uh, this is either this in areas, signs and engineering complex. At least it's your building. And my lab is right at this corner and I say, share a stage with one year. And if he's now a distinguished professor at Northeastern, So the story that I'll tell you today centers around an example. what I will I hope that you get from it, is sort of chose You grab discovery. So what the second half of my talk will be sort of how do we go about to use a lot of these basic knowledge that we have learned that we acquired over the years to actually treating diseases. And we're doing, in a way, that part of the lab design process is not sort of one only for a specific target but kind of be the pipeline making multiple molecules for this particular guardian or for many more targets. So that's an ambitious goal. So let's see how far I take it. The first part will exclusive getting us aquainted with layers that molecules that I studied. It's a ion channel that regulated by signally through DBC arson probings. And so I'll introduce you to it. And the second important point in this sort of background presentation of the first part will be to realize that what is a controlling element in the activity of the science is in either option that the tunnel has, with the forceful meetings with the I do title impossible be forced. So I'm going to spend a little bit of time trying to show you how that is. How is that for school? Big controlling activity of this channel and the third part will be kind of the heart of the whole talk, developing occupational borders that describe dynamically the physiology if we are successful in doing that, then the transition to the second part, which is utilizing small molecules and making use of these computational models to tell us how these small molecules either activate or inhibit the target of the ion channel would be extremely valuable, because then we are starting to eat the molecules or restore the molecules to make them better. drugs better. molecules to regulate could give the channel. We have a new breed of every detail of what they're doing. So that's the goal. So let's start. I made a big deal about the PIP2 As I said, is this lipid molecule that you can't see very well in Oh, here it has basic leads. Claim to fame is that it can stick phosphates to the brains and positions for a time. And this PIP2 molecule is kind of central to many, many processes in many diseases, as I listed here from the heart to the brain as I will address a little bit in my talk. But not limited to those also in concert inflammation. So we all know inside products. In fact, the next slide kind of reminds you that the precursor of the PIP2, which is over here. is the I is the title obtains and I you stay aerial through basically phosphate bone to the ring that gets formulated by specific kind of disease. That are the phosphate of either position. Three. They still ring a position four or five to make. The model was for related species, three of them on. and of course, that corresponding biphosphates for space back down and to give you again. There's a set of kimazes that also recognized that model for invading species. and those are the PIP kimazes that recognize that, let's say PIP3 kimazes recognizes the PIP3 B in onset phosphate five. Position five. 35. We have similarily 34 and 45 and we have 45B2 is a key intermediate that gives rise to separate messengers. And if you activate phosphate lite enzymes, then you hydrolyze to right on these connection of facial things with head group to decorate IB Three. That now has phosphates have positioned one four and five and also they glyceriod and the lipid apart with two are big medicine. There's idea three either a calcium from internal stores. They as a literal ozone broking. Fine a C, another very important product of the PIP2 to ease another formulation event that position three to yield PIP3 And then you know that this is a growth stimulus that actually feeds down AKP pathway. And, uh, its importance as a drug stimulus is appreciated when we see that the phosphate for days will be, then that will take you from the PIP3 to PIP2 is effective and mutations this enzyme guild to its defective function. And when you cannot disformulate well, then grow. So the PIP2, therefore, is many things for quite some time now because of these three products that has that are very important. Signaling growth in concert so forth. But in the mid-nineties, it was appreciated also that it was important in its own right as a interacting molecule with memory proddings. So Don Killemon from UT Southwestern provided very nice data so that 2 membranes broad H one and I 80 p 64,000 and another sport there, the shoulder pass from exchanger were both dependent. for the interactions with PIP2 activity So, in fact, if you were to, let's say deplete the, you know you don't have the be some stain and, in fact, so very elegantly that if he stopped their function of, if you let's say, to degrate it you would see a very rapid drop down. This would explain the behavior off many ion channels as people studied them with parts down and put them in an environment that's excites the parts in an environment that didn't have ATB and these phosphateize were. very very are active in disformulating PIP2 to any any of the one of us for creating, or or the I so in the plasma membrane, the two molecules that refined are PI4P and PI5P where another membranes or other organelles some of the other dite was formulated that was formulated PIPs are existing. So, for example, in lysotones the memory of lysotones we have the five, you can think of this possible lipids is kind of zicphones that many drawings kind of end up in that particular ziphones whether it's the plasma membrane or membrane or any membrane because proteins have specific, residues that bind quite specifically, some of these different. And so these shows you a 12-year-old basically a table here of all of the different ion channels and some transporters that are depending on you for their activity. And, you know, like these views You made any more than this listed here, but this is kind of readable. concise people picture that a lot of different ion channels it's more the rule rather than the exception that you have a ion channel that there's no that depends if do is the exception that you find one that you can figure out whether we do is involved. The ones have been a focus today. Are this yellow one? The KIR3s These are in the family of inwardly rectifying potassium channels, and the GIRK3s, in particular, are the ones that I studied. thoughout my career it involve gated ones. So before I go to them, let me give you the basic idea how to gate ion channels and the conceptual model. There is a closed channel here, confirmation of the channel Besides that the radiation potassium is number BC in this confirmation of the molecule But then when we do the negatively starts phosphates a position four and five interact with basic residues in the sequence of the protein. Then confirmational change happens that opens to the permeation pathway. Now I don't feel becomes permissive See? Okay, so this is how you should think of it in fact is there evidence for that? Yes. In 2011, Rod McKinnon's lab in Rockefeller worked on a provided a structure for crystallized one of these potassium channnels. gated by Holdings together with the PIP2 It's the time with you you can see that this is thought to be one of the gates of this channel and you can see that on the other side of the ski mix the PIP2 that is coordinated by several basic. So this is absolutely ski for controlling Alice. Seriously this gate and we'll see other gates as well, So Ion channels are a family of them that basically are made or four subunits making an ion channel are wasted here over predominantly red on green, some blue some purple. You can see that they're potassium channels . Are you know, half this large family and channels that will rectifies and I'll talk to you about is the simplest kind of family. Basically, they have one of the four subunits that I mentioned. He's so here. Two great domains, these units and it's the simplest possible get for a hole in the memory where the ions are going through. And so these are the channels that will be talking about. And in fact this is there another way of showing this diagram from here, they're relating. And the ones that we're gonna focus on this KIRs KIR3s are basically. Another name for them are GIRKs Deep rotating, inwardly rectifying, potassium channels. There are 4 of them and in the brain. We find expression of get two or four for polymers on. And also I want to get one and two, get one and four get two and three. I don't form whole respect themselves. They have other either two or four. In order to make viable channels in the heart, we have get four homers and get one for General Motors. So get to its fine from predominantly in the brain and your own elevation. Okay, So back when I was a student, I had the privilege of working in David Problems Lab. What time was the Brigham and Women's Hospital and Harvard? And we use the pots. Come pick me that. I'm sure you have heard about to study these channnels. Understanding the question off. What? How are these? Activated by deep proteins of units. At the time, there was no example of the debate of housing units opt, imitating an ion channel that this was the first example of such an effector of the potassium channel So the way we did the experiment was to plug away, a little parts away from the cell that had a team signed surface facing the bob so we could perfuse to refine some units of deep roading and we perfusion units. Or reactivated the opposite units that make up of units the primate broteins and see whether either of those activated the channel. And to our surprise, we found that the gamma and the activated the alpha. The classic example that we all learned signaling was daily sideways and that the blast was probably waiting. They designate enyzme of the conversion of A and B. Where did the alphaize you are a beautiful is in giving that. But the major gammas units that time had no role, they fall to be their spot. So this was the actual first example. So that made betagamma a role in activating. And so when we think about how signally works normally the primer associate with the receptor can even do it better as GTP-binds and starts to get activated. So we're not going spines. conformational state of the receptor allows the alpha. Now you need to exchange his GTP, which makes it to GDP. No way. And now the interaction. The confirmation teams of the accepted gets meeting to confirmational change of their on the opposite unit, that allows it to lower its affinity for betagamma and to come apart presumably and made betagamma. And when the GPDs hides the GTP now all pervading betagamma increases and more beats away from the channel, to rebind it and go to again to the state. So this is kind of the mechanism that we got from vision from 1987 that this work was Bob Lee's to. 12 years later, or 13 years later, in 2000 in my own lab we were able to find that there was another very important element in the regulation of this channel So if we express in general ago, cells must already like two receptor and activated the channel, with several cooling, maybe the receptor with a single bullet, we would see the channel activated in a way that could. The activity would be produced by a second challenge of several polling a few minutes later. However, if we poor express the most burning like two receptor, which is in the eye coupled with the most Granic Type one receptor, which is a receptor you probable receptor. Now the Bader became very different. We got rapid activation, but also a inoculation or the desensitization as we termed it because, in fact, we gave the second possible settle holding. Now we didn't get any spots. We would have to wait until this desensitization phenomena was reversed, somehow to get basically the full response again. And we would show that if we inhibiting with blogger like the postal like they see like a C, then then the activity this inactivating or desensitizing went away so we could get the same response just like the first. So the user of the must Ernie wanna receptor and activation of the jukebox way he'll see had somehow was leaving as this guy, and that sort of brought the idea that is. Show here in this figure that M2 or M4 type receptors that signal through G. I activated a betagamma where M1 and 3 or five receptors co existing lets say in a cell, along with them receptors in the heart selling 80 cell would signal PLC and, as we discovered through a lot of work, be able hydrolysis was what was the cooperate for this desensitizing. So this you pathway PLC occupation was desensitizing doing. It was absolutely meeting in order for the debategamma to activating effect or commending, activating. So these two that pathways were integrated at the level of the channel with no light that I showed you a rapid activation and rapid desensitzation. So over the years, we've found that other investigators of the channel existed, for example, high for secular levels. And we found that the 50 for activation of the channel by sodium intracellular sore giving the order 40 million older, um, was was sufficient. You must have maximal activation and so experiments that way. So that production or the presence of people doing it was very much important. But intercelluar showed you would activate as long as P2 was intact Now you do way itself. Could know that today or hydrolyze of AP not activate But it was absolutely essential to have war grows may be present for so you or betagamma to do their job. So when we put both of that soul, you and Beta Gamma together and also additional effects, we could see that the activation we got showed you, which was slide. It was more with beta gamma.. But then when we put them together, we got great synergy. So you together somehow donating their time. I point you, you know, sort of the dates of these papers. This'd is basically 1998 the next one 1999 So maybe years. Okay. Where 2020. So over 20 years that we have known these. So the person is how exactly do these molecules you showed you the beta gamma you debating about that? They do come together to gate. And so these wait natural information. And it was not until 2013 that we had a crystal statue of the ion channel again from Rod McKinnon's lab. Working with McKinnonr gave the crystal structure of GIRK2 your own on issues with the data and you can see also show on you and you can see to sort of now we knew these results structure where each of the players was resigning. And at the same time, we also probably is a very similar structure based on computational work, that my student showing the one only six weeks, very similar place where they were to. So can we now learn that we have a statue? How this is comes together from the snap soul that we get from every cell structure, its not possible course gain a dynamic picture how small to open the channel. So we've had more these, and so you can see here in a better depiction where we have only 2 of the four subunits so that you can see the permeation clearly that the ions are going to this middle path way, and there are two major constrictions that are relatable. One is the helix bundle crossing at the bottom of the membrane and another one in the signers on these deals gates on the gates from ST and revenge going to and in the upper part there's a big demonstration. This is the place where the ions get selecting lets people passing on the street over there water and be passing through were sold you although its smaller no history by from its water energetic principles because of the size of the field there. And so what else can be let through? But I can show you. But the important thing is that these two restrictions although they are not quite strange as this in a community feel there are the ones that are regulated and look at where these gates are. So there, you know you can protect. This is approximately the same place. The helix bundle proceed, as I said, is that the border of the membrane and the cider solely to gauge is a little big into the side. Look at the position of a big to do is right between those gates. So right breaking between right with the level of the movement of the memory is working to resides. So it was over here in the purple circles and beta gamma on the side. So we put this system in a five year some here and performed molecular dynamics simulations to gain an idea of how sold your marketing and how made in the presence of so we run five systems alone, the channel will be and then channel. We need to ensure the time with this way. So human made a C, we would soothe synergies small, and we learned exactly what goes on. And so we would see here in the depictions of the of yellow and the green in two different confirmations, one blows one open. We can see sure of what changes, and we can see that the greens subunits when way have made gamma binding. They open up the kind of field outward. They have a rocking movement, and so we could study these details and see exactly by how much they do it. And then if we zoom in, we would see what exactly is changing and how it's there. interactions changed. So as this Cyrus Olek made rotating this very important element slide that is part of the membrane seems to be changing its orientation and the specific options that they surprising to say that the interactions that control the membrane gate where effective when betagamma was present. But when soldier was present, it was decided solely gate that was affected actions with that opened this Akrasanee gate. So through these small bills, we gave that we could learn only what happens when you have sold you or beta gamma about. But when you have to do together, we show the most activity and what I mean by activities that we would give you a little electric field that drove the arms from one side to the other. And we got a little bit of activity in the sodium a lot more in the beta gamma. And then when we have them both together, there was synergy was way more unity than being are beginning to activate. And so this type of analysis is in a very strong sense of. What I have been telling you we have a model that reproduced and basic activities that these natural activators sold with these. so in summary from this first part. I like walk away thinking that this potassium that is activated by settled holding in the heart weak polling day initiated the heart. And when we do anything in places where we control the subunits ourselves, we pulled in GIRK1 and GIRK4. So this involves direct, proteins, proteins interactions with data. Thomas of units and this beta gamma interaction activated the channel activating opening the gate. the membrane by strengthening channel be doing that actions that are permanent keeping the gate of the membrane open. I wrote. This is a big two that is absolutely needed to maintain activty underlying that desensitization. So you and so that when you have the vagus nerve releases a little bowling in the heart and you slow the heart. Okay, activate this potassium channel, and you pull the memory pencil. Polarizing levels at the delivery activity slows down the cell that fire more with that. But you don't want to do that for too long. So this building membranes off at the same time, probably in the same signal. Several bowling working through and humanity through masquerading receptors. That's the way to relax and let go. That information is very important. Stay alive, right, so we can't afford are so so you get the job done. But then you desensitize to say make now so you can also activate the channel. We describe these models that we develop. How does it on? Basically, we see that the sanitation now is on the side of slowly gate. No memory. Hey, but Cyprus only game and we can see that the deal that goes on something like that to be open. And finally the dipping A called this rocking movement. The sodium kind of dissent. I recognize movement and we can see precisely waiting their actions are responsible or stablizing the gates now in the open state. So you might say this is nice and good. So where you gonna do with it? And so in 2016 I go back to Northeastern, full circle from where I started studying started my studies now as a professor and I wanted to take advantage of their needs environment and pharmaceutical science ease and work very closely with to go into a drug. I figured since I had a very good hold off how this really works and I had here models that I continually they're heaving with life. Why don't make his life ends now? Be small molecules that either work saying when Hans style between actions or work saying the Greystone, actually, in the first days later, in the second occasion, having what we do these. And so the second part is going to tell you the story of how small molecule channel gating layers work, exhausting channel interaction. So before, let's just fine. Think a little be right way. Want to do that? First case would there be a practical application? And so this is a table that was put together by Kevin Whitman also was a student of David Glover fully me on. He has been instrumental, exciting animal models, the channel where the samples are actually good. He's produced no pounds off, eats off the subunits that they're wanting for you, too, the three and has blue in many places to find out what they do. So the system that we just finished describing was in the heart and cardiovascular physiology. So if you make it all miles looking out of the Air Force units, these arresting cardia reports the minds of very high heart rates start with. So the effect is not impressive, and the activity of these channel addressed without having lying bully coming heart is no big, so it's a more this know quite as large. But where so if you stimulate in the US, but then exist them vagus nerve, and you take away your before you have a vague agrees, dropping response. Now there other tiles that respond to the virus. Pathetic regulation, but indirectly. But the main one that causes heart slowing is gone. Perhaps the most impressive field I was. What What? It's cold heart, brain day of the and so in Monte that when the vagus nerve Thompson central pulling the heart, the heart slows and does not extremely fast the system is really like. Now imagine that the vagus, which has a bone tonal level of relational falling decreases that, lowers. Now I'm gonna get a heartbreaking. You can go both ways any summer. Also, by the action of the segment they exist. To go from very low heart rates to very high heart rates. And that's anything good, heart doctor, believe me. And they made it hard to adapt with the manager of ranging heart rate. People have very high marathon runners, you see, you know that a lot of that they ability is controlled by the vagus nerve. It's very fast, so runners that have lowered our heart rates, you have very good, you know, grains of heart rates there you're leaving that he's that, he said. Any mix of when you go outside, it's either two months or even worse. No, bring me then that you don't have the ability to talent is that will rewire your heart rate change or regulate fresh. So what we see then you said we know how the year before we see that heart rate variability in the mind goes flat. Who will be no rains dates unable to so many are paid. And so these calls for the importance of this channel. Other things are meeting us on way. See both atrial relations as well as the uni syndrome and hypertension. Also four picks. We have expression of your four and you get out posterity's when you know how here before. So all these very rural effects in the brain there also quite a few effects I think giving effects upon broke out. The most clear ones are with pain. So if you know about the channels, you can see that you are hyper rounds easy. Okay, so I also your response to different regulators of pain goes down. So, for example, your opioids that work through giving this channel and kind of winding things down you have to. Greece is easier. So you do need me receptors or tunnels to be there in the ability of off by senators. Lott made them in order to have effects. Also, they're involved in reward circuits. And again, you look at they get one too. Move, you know, die. You see that you have enhanced basil and booking induced reward in this time of sounds so energetic you don't express them. So they're involving reward. They're involved in a number of other things. For example, anxieties one, uh, Caesar's epilepsy and so only control. So and the year after the review, also, they were so do in if you have to get to know you have depression resistant behavior. So in at least where you do is express alone. No, you're going to do you can see that. Basically, you need this a little depression on if you don't have it, your resistance. So that way thought a state of pulling this morning. All they do that has been shown even here to work like many titles, thes type of journals. And let's see if we can study the mechanism of my wings and does it whether we can find new name, so do exactly what we want. And so first we wanted to have a change this ever 97 Morley, which is a really based scuffle in has it Laura Faneuil side When is here saying one and has to eventual rings Mental ring here and here's old rating on this position. And so, by optimizing these with our collaborator organised one of them additional damage collaborating. He was able to make a microwave reaction getting very, very short time. All right, human, it's being made these molecules. And so he could change any of the science and work by Vanderbilt University under Bill that had first from 97 amnesty or had so that different parts of the polling where Hillary what we ended up substitutions in different parts of this molecule would be tolerated. So so you examples, But what we found and also you evidence for that is that two of this substitution. You know, when we place these dying Laura Faneuil group with Roma order way not specificity in the way that this small family war. So this was our screaming off you know, sort of initial chemistry way of changing the modeling in many with many different speed winds. And we focused on site. When you scold here airs, I want that Why vulnerable? And so we maintains that all these values stabilise and testing. And so what we were looking for in our tests where here purple lines are the basal activity. Either you're all get 12 or the hard time 14 And when we have the drop, then we went to the Purple line for the control wins is 97 months a group or the brother Fire group was basically so here, 15 08 It was very similar to the 97 but the good thing that we were looking for was differences between the want to get one four. And so we saw that although the bell denying seven on TV cardio tunnel, this stated that we had no they and so was the gauge with natural substance. So we looked at least a little more carefully and in fact, teens the scientist, from from letting the known substituted no role in this case, we would see that we would still like to make both the car near red in the brain blue. If we made the college in less than in size problem like we can see here Floro Floro that are smaller than Bram Oh, or larger I die. We can see that the effect is not as cleaners with Graham on the Bronco doesn't reactivate cardio. It serves a nice activation off the off the brain. So the medicine will, As I said here was also effecting But it so sound in addition of the cardio, in fact, so we would refer them roll And here is the working Dale parts from records to show you that the Bronco was out affecting the cardio. But it was mating very well. The brain here is the particle. Mom was giving us a little bit of guardian activation which made it sort of any appropriate only before the drop. And here's the 15 when you are also know activating cardia, automating the brain but less well in Rome. Okay, so we have no ineffective multiple. Does he do it? So Liberators of the University of the Deana tested these in fear extinction. So in this paradigm, they rolled in here is trained in in books where the animal is so right after gold and you can see that within five bearings off. Don't off with. So you are a liar freezing here, which is a fear behaviour measured in these so 24 hours later. If you go now and that's the fear became your the animal in the absence of salt dust with Don't you see that the memory of the off, the fearful behavior consolidating and now you frieze, he each the logo. It's not followed by the sounds of Babylonian provisioning basically taking place on then the third parties day after that going in the either don't without salt on you can see that behavior is starting to fade. The lock singles here. So US fate. It's extinguished with no, and the green ones are green is a 10 milligrams per kilogram off £15 the only green is with 30. And so you can see that you get really good extension of the behavior. And so a number of other tests basically tested negative, giving us hope that this may be a good time. molecule to pursue further. So how is this working? And if we can learn how it's working. And if we use this combination all models how these Small molecule is doing what it's doing way need optimizing for it's a distribution is you know, in any status toxicity, then can we use our borders to the fine tuning? As supposed to, let's say, starting from scratch again, making a lot of molecules and testing them in the in people. So what, then? Uh uh, getting Whitman's lab. So in 2014 when they discovered these 97 they so that there were two amino acides in any position 1 37 and an aspirin, a position 1 73 that were critical to the activity of this molecule Of the molecule, this amount 97. So these two residues were extremely important. you dated them. If you made a lot of searing, you lost any of the block. If you change the house part dating started again. Dr. Working, Um, by, uh, So if you if you know