Controlling the Machinery of Life with Synthetic Photoswitches | Dirk Trauner, NYU
AAllison DuettmannJul 19, 2021 at 12:18 pm55min
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00:00Speaker 1
Hello, everyone, welcome to the latest in our monthly seminars that we have here in the foresight molecular machines group. So for those of you who don't know me, I'm James Cooper. So I'm the chair of this group. And for those watching on YouTube later on also, welcome. So this evening, we have one speaker, which is Professor dog trainer from NYU, who is going to discuss controlling the machinery of life for photo pharmacology. So the floor is yours. Well,
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00:32Speaker 2
thank you so much, James, for your kind introduction. And thanks to the foresight team for inviting me. And thanks to all of you who are attending at what I would consider past my bedtime for some of you. So stay with me, I hope I'll keep you awake, with my take on molecular machinery, because I have a sort of fascination as all of us with the molecular machinery of life. And My take is that instead of building molecular machinery from scratch, which of course has made incredible advances in recent years and decades, I would rather hijack the evolved over billions of years molecular machinery of life and endow it with new functional properties put on new ignition elements, control elements, which is to be able to turn on and off and shape its its performance. And we have done this mostly with light, I should say that we are also interested in using magnetic fields, for instance, to do this in ultrasound, but this is not the topic of today's lecture of this talk, rather. And please interrupt me at any point to sort of Formula a bit off track. What I want to tell you about today is the use of synthetic photoswitches and their merger with some of the best studies and best understood molecular machines in sales in order to in order to control cell functions. But of course, we're also interested in the individual molecules themselves. What I like about this approach is that it's relatively easy to address these machines, right, because they're kind of wired up for expression in the male brain. And then you can interrogate them for instance, with electrophysiology and other methods. Let me see what I can advance my slides here. Yes. So here is a sample of the machinery we have been working on. We started our work some 1518 years ago, and I actually with voltage gated ion channels, which of course make these wonderful movements across the membrane in response to changes in voltage opening a gate letting ions flow. And then we have progressed from voltage gated ion channels. Over the years to ligand gated ion channels such as ionotropic glutamate receptors such as pentameric ligand gated ion channels, for instance, GABA a receptors nicotinic acetylcholine receptors. Today, I will tell you quite a bit about G protein coupled receptors, because they have been a wonderful platform for photo pharmacology where we put synthetic photos which is on I won't tell you much about arguably the best studied, most famous, at least, molecular machine at present phase some years ago, we published a small molecule photoswitchable molecule that functions like a photoswitchable range, and was able to control the f1 fO atpase with light in that way. But we've only done biochemical studies and the sort of mitochondrial targeting versions is still not ready for publication. So I want to wait a little bit before I can say show you some Sarah work. But we have also worked on, for instance, on exciter amino acid transporters. And we have actually very nice paper in development with Paul Neeson on zerker on the sarco, endoplasmic reticulum, calcium atpase, which we can also switch with a photoswitchable version of topical toxic algal control. And then we move over the years a little bit more into Sailele I have really fallen in love with the cytoskeleton with its highly dynamic nature and its fascinating movements, it allows and enables. And this dynamic instability, dynamic of the of acting and tooling for instance, we could control with photos which will molecules and we'll throw a little bit into there about an attempt to control a Kenny's in spindle Kaneez in called egg five with the photo switchover inhibitor, which is not published yet but which we are soon going to publish that allows us to control mitosis with light. And then it is a work on transcription factors, nuclear hormone receptors, a lot of work recently has been done on photoswitchable lipids because these so benzene photos, which is you guessed it, which I'm going to focus on today, they are absolutely a match in heaven with with fatty acids. There are lipophilic molecules that can have cysts, double bonds, if you treat them with light, that's what you'll find in lipids, for reasons and since I can impart this with light, we can do some pretty cool lipid physiology, but also some very nice membrane biophysics with photoswitchable lipids and there's a whole series of papers in there, already published, I can tell you that we have been able now to feed photoswitchable fatty acids to certain cells. And we find that up to 40% of the endoplasmic reticulum of the phosphatidyl
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05:17Speaker 2
molecules of the endoplasmic reticulum contain these photoswitchable fatty acids. And therefore we can control for instance exocytosis with light in the forthcoming Rick, but today, as I said, really want to focus mostly on ion channels and gpcrs. And for a little bit in on, on Kenny's in, and then maybe, if we get around, also talk about aefi. Like is is these are the proteins that mark our proteins for destruction and we can turn on them we kind of honor of light as well. So, yeah, a little photos which occurred, I think, towards this audience, I don't have to emphasize what is wonderful, but so benzenes from my perspective, the fact that they switch so fast that there is no time to fall into triplet states. And therefore, there is no single oxygen production in biological systems, which actually distinguishes them from other wonderful switches, which are not as useful in biology because you do fall into prepared states occasionally, but a nasal benzene just flips within two Pico seconds and therefore there is no time for that. And as you know, we can greatly vary them some 12 years ago already, we made Hato suitcase of benzenes. So you can change the photophysical properties device to be referral base the beauty greatly you can change the photo stationary states but trying to pull the absorption spectra off this is in the transformer path as much as you can of course, many others named Don here have made amazing and made amazing contributions to that. Also, in terms of sine inversion, Rana haggis is daisies for us were born as well. These are the days of pains and derivatives the more stable feminine amicably in the dark in the beam form, and then you can push them towards these elongated form as opposed to regular or semi regular is amazing. And that of course allows us to pharmacologically sign invert in many cases and I have a whole series of papers in the world published but also in the making the this was really crucially they actually it's a beautiful way to sign in. But the logic often so benzene and rocks, there are some limitations in terms of actual spectra but other than that, they're wonderful focus which is and we have contributed to the synthesis as well to make them as synthetic accessible. Yeah, let's classify for the pharmacology the simplest form of photo pharmacology is some ligand. For instance, ad blocker shown here an ion channel blocker that changes its efficacy with light which for instance in the dark is a good blocker it crams itself into the into the poor, whereas upon photoswitching it becomes this dial Pentagon. This is my cartoni language. Now, a cute star means active Gao Pentagon means no means inactive and an ion slow. And there could be a photoswitchable blocker of ion channel for instance, who have made them and they had actually gone very far they are now evaluated in the company for vision restoration. But much earlier than that the summit earlier that we actually initially tethered the blocker to the channel of interest and that has one big advantage namely that the tethering and core requires some form of bioconjugation and this bioconjugation can put on a genetic control. The simplest form we can put the system in there and you do some system Olamide chemistry, but as you will see, they are much cleaner and more exciting ways to correlate Li attach something to a protein of interest or nearby. And in this very early version from 2004 we tethered an extra cellular block a tetraethylammonium ion to the surface of a voltage gated potassium channel and with that, we could unblock it if the ace of angels in the transformer block it when the ac urbaines was in this form. This is shown here more structurally this was this very simple molecule mulato ca le and that's a nice nomenclature I've become better scenes with acronyms. Malema quaternary ammonium and easily in between. And that could be tailored and engineered since then on the distorted loop of a voltage gated potassium channel and then could reach the poor or you could withdraw it. This was done together with rich Kramer in Berkeley and have to emphasize that this is the second paper in the history of optogenetics. So of course then the channeled options came along to kind of wipe this out. But conceptually, at least historically. This is a very early version of optogenetics. And we could make hippocampal neurons fire. excitable cells fire in a light dependent fashion by controlling these voltage gated machine with an extra light gate. So little bit of history here. It's long, long, long ago. And then over the years, we have refined this idea of tailored for the photo pharmacology, as opposed to simple photo pharmacology that is the parent one, the terror one requires some call it attachment and that typically are always because you can also do affinity labeling, but typically that requires some form of
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10:18Speaker 2
modification here, which you can put on a genetic control. So, of course, biologists love that word genetic control, in addition to light control. So here's the general scheme ketones came and he has some molecules here for instance, glutamate derivative, which which with which we control ligand gated ion channel, so called Yana tropic euromod receptor with light. These are truly fascinating molecular machines. They this catone. While nice, doesn't do it justice, you have this transmembrane domain, I'm sorry, this is a mistake, they should be TMD transmembrane domain here. And then you have a ligand binding domain, which looks like a clamshell like a venus flytrap it shows down on the neurotransmitter on the ligand on glutamate. And in doing so this is mechanically coupled with the opening of a of a gate through which then ions can flow in this case, so to mean and that, of course, depolarizes, the membrane leads to a firing of an action potential and that's the major excitatory neurotransmitter glutamate when you have it postsynaptic released presynaptic released on the postsynaptic side, it opens these channels. Through this mechanical event I just described them. And we couldn't resist tethering a version of glutamate to the clamshell to this clamshell domain It is called in this world in such a way that the local concentration changes dramatically from well as inaccessible to fitting right into the binding site. And then the clamshell closes and the channel can open. And this is also ancient history now, because this required system ultimate chemistry and you refine this chemistry somewhat, for instance, remain from this Ace of veins in the ASO aniline. To this is a means in which is a push pull system, which has a electron donating subsequent here and sort of neutral more or less neutral subsequent here, slightly eloquent, withdrawing perhaps. And then we've been doing so you can greatly shift the action spectrum from a lambda max of in the UVA range to one in the blue range. And in fact, when we look at the action spectrum, so it is an occurrence plotted as a function of light or darkness, because this thing also turns itself off automatically. Since it's a thermally unstable. One, it's fast relaxing. So benzene, you can see that we have very nice action spectrum. And I like this because this action spectrum pretty much matches the action spectrum of the blue calm in, in the retina. And in trichromatic vision, it's pretty much almost identical actually. And yes, indeed, these things have been used to restore vision, the systems, but not for very long, because something better came along. And there's something better I want to illustrate with the metabotropic glutamate receptor. The metabotropic glutamate receptor now is a G protein coupled receptor. But it's time Eric. And it has also this extra sailor, Venus flytrap we're like ligand binding domains LBD, which is actually quite similar to be one in your topic receptors. This is one of these motifs in nature, that has been picked up again and again and again. This sort of chewing down on it on the ligand has been adapted to a wide variety of, of receptors, ion channels, gpcrs, also receptor tyrosine kinase is transporters. Some ABC transporters have this motif. It's a typical case of Mr. Mason matching evolution, I believe, that leads to giant
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14:11Speaker 2
leaps in, in functional sophistication, changes at least. So and it's time Eric so there's there's the seven helix transmembrane domain and then the ligand binding domains actually linked together by a dicer offer so it's actually dimeric GPC is a family CJ PCR, if you want to classify it and that shows down on glutamate and then this leads to intracellular signaling, but not flow of ions rather dissociation of a heterotrimeric G protein, but ions to flow in the end, because you also activate correct channels G protein coupled in with rectifier channels and starches is one of the major modulatory metabotropic glutamate metabotropic receptors and we have initially banned some c pharmacology not so interesting. We've done some closely tailored for the pharmacology not so interesting because this system Malamud chemistry works nice on sales, and in some simple tissues perhaps also actually also in the retina, but it is due to the hydrolysis of of the maleimide. Not great chemistry for bioconjugation as shown here, so, this is the bioconjugation. But it turns out that this Malema is of course, not indefinitely stable in serum. But there's some a much better way to do this. And this is what I want to mostly focus on. This is bioconjugation through bioconjugation tags. These are essentially suicidal enzymes, proteins that self label that are engineered to correlate the attach electrophile with the cleanliness and the speed of an answer elven enzymatic reaction. And this electrophile is not advised directive does not react with water or with the firearm. It's a very poor electrophile. But once it's captured by this enzyme, it will lead to suicide or in some labeling, so to speak. And the arguably the most famous ones are the snap tag, and the clip tag in the halo tag, which racked with pain, slick warnings, Article highlights, respectively, and have been greatly engineered and can be fused to all kinds of proteins when nearby place nearby proteins to now allow for the corporate attachment of some tethered molecule. And as opposed to the previous closely tailored version, this terror has to be pretty large and flexible. Because these are not so small, it's a sort of 20 to 24 kilodaltons these domains. And therefore, you don't really change the local concentration much with the photos, which you move the photo switch into the pharmaco for as indicated here. And then you change the efficacy of the pharmaco for but not so much the length of the table and the orientation of the data. Because this is a long polyethylene glycol chain, we call this portlaw for photoswitching orthogonally remotely Terra laggin. You see we got a little bit better with our acronyms too. And it turns out this portal concept is indeed very broadly applicable. We'll see a lot of varieties in a moment. But the important feature really is that the switch is in the cannot pharmaco for all of us compare this with a dog on a leash, where the leash kind of restricts the action radios of the dog and tailors it to an owner of interest, but bioactivities in this nap, right? That's what happens as opposed to free diffusible talk. This by the way was in May 2020. Just to give you an idea about New York look like in these early days of the pandemic was eerily beautiful in a way and we have been able to apply this portal concept now to have a very broad variety of, of receptors. We've done it with ion channel now this is unpublished we've done it with glutamate receptors, we've done it with dopamine receptors we've done it with serotonin receptors, you'll see a little bit about this also unpublished. So family a Chico protein coupled receptors and family c G protein coupled receptors. And here's the metabotropic glutamate receptor version
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18:31Speaker 2
that will be n terminally extend the ligand binding domain with a snapback or some tech and then we attach the photos which on a leash. And if the photos which gets activated with light it activates first the ligand binding domain and this is mechanically coupled to change an interface here, this dissociation of the heterotrimeric G prothane. And this can be an itchy alpha if for instance in the glutamate receptor metabotropic glutamate receptor inhibitory itchy Beta Gamma then travels and and activates G protein coupled imod rectified potassium channels which also have an inhibitory effects. So, these are mostly inhibitory receptors. And, and that actually worked beautiful a little bit of chemistry. This is the table of glutamate. Here's the photos which here's the glutamate with a specific stereochemistry here configuration router here should be precise. And here we have the long model as long flexible data we have also long around shorter ones. We put this together. You don't need to have click chemistry This is just killed. But of course you can just make an MIT because you make it in vitro anyway. And then you edit and here's the painful guanine and everything that is on this pain so group gets transferred to sustain in this net deck. But only two that says they know rails because this is more or less unreactive also with Google file, and is a spatial electrophile in that way, and we have made one that maximum activates for 380 nanometers with UVA light, which is maybe not so good for in vivo work. But it is very nicely compatible with fluorophores. So as that, and we also made some that is activated with blue light for 60 nanometers maximally. And the only change, let's go back Look carefully here was the removal of one carbon rule here. So this one carbonyl here makes all the difference in terms of the action spectrum. And here's a model of what this looks like. Before rotated, I just want to explain this array to seven helix transmembrane domains. In the meantime, by the way, there is a cryo em structure of, I think Angular five, and a few other emulators. And then he has an adapter. So we'll switch the rich domain, this is just a lever. This is a very stiff adapter mechanical adapter. And here is the clamshell, the two clamshell, sort of back to back when I wrote it, this maybe it is located, you will see better the two clamshells and as the clamshell closes, this lever arm here pulls on the seven helix transmembrane portion, which then changes the interface and kicks out literally the heterotrimeric G protein that is attached here which then travels and does stop stressing signaling. And let's go back here. And in orange I have modeled in the snap text you get the sense how small or larger snap tag is. And in green we have modeled in the tether and the photoswitching here in red This is actually glutamate which was for the crystal structure. So get a good sense for how big this is. This fear the five nanometers fear here we put in the sort of measure and calculate the local concentration, effective concentration of this of this glutamate, which is very high, we are always in this sort of high millimolar range and mid mid to high millimolar range. And this construct was expressed that woody isikoff and pet john Flannery with a viral transfection method with an AV virus in the retina of blind mice. And then Mike Berry, the greatest student
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