Lecture 18. Terpenes and Cell Signaling, Part 2
CC Attribution - ShareAlike 3.0 Unported:
You are free to use, adapt and copy, distribute and transmit the work or content in adapted or unchanged form for any legal and non-commercial purpose as long as the work is attributed to the author in the manner specified by the author or licensor and the work or content is shared also in adapted form only under the conditions of this
Weiss, Gregory Alan
 Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. 
 I can't believe it's gone so quickly. So it's a little bittersweet. I love teaching this class, so it's hard for me to give this very last lecture. Okay, so today we're going to pick up where we left off last time. Small molecule control over signaling pathways. We already talked about this last lecture. Today, proposals are due. Make sure you don't leave the class without hitting them in. If you miss handing it in for whatever reason, then just bring it by my office as soon as possible. You have till noon as a grace period. This also applies if you need to get an abstract in or something like that. I'd like you to attach your abstract to the proposal. Your graded abstract. If you don't have your graded abstract with you today, run home, get it, bring it back either to me or to the TAs, Krithika or Miriam. Do that ASAP. There's a little bit of a grace period, but you don't want to extend it too far. I understand there's some issues with turnitin.com. Did anyone have issues with that? All right, let's try to get those in the next 24 hours uploaded. Sometimes it just jams with, this is kind of the end of the quarter, so there's a lot of assignments that are coming in. Hopefully, turnitin.com will get their act together. If it persists as a problem, then I'll make arrangements. For now, just keep trying and then get that turned in in 24 hours. I do have a quick question for you. For next year, did you like having this do today, or would you rather have it do Monday? Everyone in favor of today, raise your hand. One, two, three, four. Like Monday or Monday? No, no, no. Monday is in upcoming Monday, like a few days from now. Today, all the paper of today. Okay, and Monday? Wow, it is almost completely evenly divided. All right, well, in that case, we'll probably stick with today then. The reason is, this gives me more time to read the proposals. It actually takes a lot of time for me to read 120 10-page papers and provide comments on half of them. Because of that, I need the extra days. And also, I feel like you guys need a little bit of break to study for your other final exams. If you had this hanging over you, you would imagine just working on it, working on it, working on it until the very last minute. So, okay, well, I'm looking forward to reading them. So, you can now relax now that you're here. And just make sure that I get them at the end of today on this table over here. All right, I do have office hours today, immediately after class. I imagine that you don't have too many important things to talk to me about. I will have office hours next quarter if you want to stop by. You're always cordially welcome to my office. Okay, so you're always welcome to stop by. Usually office hours are the best way to get a hold of me or send me an email. You know, if you need advice about anything, about careers, about finding a job or whatever, don't hesitate to call on me. Okay, so you've been in my class. You worked hard for me this quarter. I'll work hard for you in the future. All right, here's what we talked about on Thursday. So Thursday we wrapped up our discussion of terpenes. And then we talked about cell signaling. And one thing I wanted to press upon you is that this concept of using small molecules to control cells and to coordinate responses in cells is really a universal phenomena. This is carried out by the most simple organisms on the planet and the most complicated organisms. It's carried out by humans. It's done by humans. It's done by insects. It is truly universal. It's one of those aspects of life that we find. Small molecules are also used extensively by chemical biologists as power tools for dissecting biological phenomena. And I showed you examples of this using both reverse and forward chemical genetics. The example of the forward chemical genetics were those massive screens to identify compounds that could interfere with mitosis. Okay, and we saw this during the cell division. The DNA, instead of lining up on the scrimmage line neatly in the center, was getting, was you know, pushed to the peripheries and it looked like the complete reverse of what should happen. So forward chemical genetics is a powerful way to identify unusual phenotypes in the cell. Then using reverse chemical genetics, you could use that small molecule as a powerful tool to figure out exactly what's happening. That compound monosterol emerged as a really interesting inhibitor of kinesin. And using that inhibitor, scientists were able to, chemical biologists more specifically, were able to define a particular kinesin variant that's critical to shoving apart the chromosomes and pushing them into place during cell division. And that's absolutely fascinating. Okay, so small molecules offer temporal control, meaning time control. They also offer localization. You can dose them in a particular spot. And we even talked about how they can be used to investigate phenotypes that would otherwise be lethal to the organism. So all of those aspects are what make small molecules such an integral part of chemical biology in the modern era. Okay, now because we often use these small molecules, kind of like toxicology, to investigate cell phenomena. We oftentimes find ourselves investigating cell signaling pathways. And my message to you there is don't panic. Okay, so if you open an issue of cell or something like that and you look at the last couple of pages, they always have signal transduction diagrams. And every month, every week, or every couple of weeks, they investigate a different signaling pathway. And these are daunting in their complexity. They look like the Milky Way in terms of the number of elements that are present. And they look completely nuts. But the message there is don't panic. Instead of looking at such crazy complexity, we as chemists can simplify things down to seven major pathways. And I contend that if you learn these seven pathways, you'll be equipped then to understand anything no matter how complex it is. All of that complexity can be reduced down to just seven elements, or seven different types. And if we learn those seven types, we'll be in good shape. Okay, we looked at the very first of these seven, which were the nuclear receptor-based pathways. And we looked at how steroids have potent activity through binding to these nuclear receptors. And then we saw that these have two different modes, that there were two modes of the nuclear receptor pathways, heterodimerization and homodimerization. Any questions? All right, well, let's return to our seven canonical pathways. I'll remind you again that we looked at the steroids binding to their nuclear receptors. And the key element here was the notion that these nuclear receptors bind directly to the steroids. And then these complexes make their way all the way to the nucleus where DNA is turned on or turned off and transcription takes place. That's a very powerful paradigm for affecting cell division, cell growth. And this is why these steroids like testosterone and estrogen have such potent cell-based effects. Today I want to pick up our discussion and discuss with you the other six pathways found in the cell. And again, I'm going to be emphasizing what's common and what's simple about these pathways. We could get crazy complex, but it's just not pedagogically useful. So I'll show you kind of the stripped down version. And if you start launching a research project on one of these pathways, you'll find quickly that things get a little more complex, but not much more complex. All right, so let's get started with the two component pathways. These are pathways in which a ligand binds to the outside of the cell up here and then dimerizes to receptors. And by the way, just as a quick preview, note that many of the upcoming pathways are going to feature ligands that don't even get inside the cell, like these guys over here. So here's a ligand, it's binding up here. Here's another ligand and purple binding up here. Another ligand binding up here. None of these ligands get through the plasma membrane. Instead, they're going to affect the confirmation of proteins that are found on the cell surface and that conformational change will transduce the signal from outside the cell to inside the cell. Okay, so that's going to be our paradigm for today. All right, let me skip on one moment. Let me see if I can find where we left off. Okay, we talked about quite a bit last time, didn't we? Okay, here we are, number two in our seven. In this case, I want to talk to you about controlling cell differentiation through a class of molecules called cytokines, also known as interleukins, variously called interleukins. This is one of the annoying aspects about biology is that they don't have a uniform nomenclature the way we chemists do. The periodic table is the same here as it is in Russia or Sudan or anywhere in the world, but biologists don't have a UPAC, so they have a little bit more difficulties getting a standardized nomenclature. Be that as it may, cytokines are an important class of molecules for causing cells to differentiate. The differentiation I want to talk to you about today is the differentiation into various blood cells. This includes red blood cells, erythrocytes. It also includes B cells and T cells, which are crucial to the immune response to responding to attack. It also includes a number of other different variants, like platelets, which we've already seen as being crucial to blood clotting, et cetera. All of these signaling ligands highlighted in bold and blue are examples of cytokines that all more or less have the same signaling pathway. Rather than us saying, all right, let's learn about IL-4. Now, forget everything I told you about IL-4. We're going to talk about IL-2 next. Rather than doing that, I could just tell you about one example, and then from that one example you can figure out everything else. Let's dive right in. I'm going to be talking to you about a favorite of ours, one that we've seen before. This is growth hormone. Human growth hormone causes acromegaly. This is characterized by abnormal growth features in the face. The face looks a little distorted, hands and feet. Just abnormally sized large hands and feet. I mean, really abnormal. So I'm realizing this slide is out of place. All right. We'll get back to growth hormone. Let's get back to, let's focus in the example I'm going to show you will be erythropoietin right here. And again, it's generalizable to a bunch of other molecules. Okay. So this is a very typical mode for these two component pathways, two meaning that they are going to cause dimerization on the cell surface. And so here's the molecule Epo up here, erythropoietin. Erythropoietin stimulates the formation of red blood vessels, of red blood cells. And the way it works is erythroepo up here dimerizes an Epo receptor on the cell surface and that allows an associated kinase, Jack 2 kinase, to phosphorylate each other. Okay. So this is an example of transphosphorylation. This guy phosphorylates this guy, which phosphorylates this guy. That in turn allows phosphorylation of stat 5 which dimerizes and then stat 5 actually gets into the nucleus and causes transcription down here. Now a closer look at the phosphorylation is in order because this is going to be a major mode for relay of the signal. Okay. So like in track and field when you have a relay there's a baton and you kind of pass the baton hand over hand. This is a similar type of phenomena. In this case the baton is going to be a phosphorylated amino acid side chain on the surface of the protein. And that phosphorylated amino acid side chain is going to create a new surface that then allows something to bind that otherwise would not be allowed to bind or it could change the confirmation of the protein after it's phosphorylated. Okay. So what that means is stat 5 has one confirmation up here. It gets phosphorylated and then it dimerizes. And that's a phosphorylation. It's not dimerized. It's a monomeric protein. Upon dimerization it has a different confirmation. One that can bind DNA. One that can turn on transcription. Oh, and one that can get through the nuclear pore to get into the nucleus. Okay, so this is also termed a jack stat pathway because jack kinase and stat 5 or stat proteins are playing a key role in this cell signaling. And note too that it's fairly direct. There's only a small number of proteins between signal up here and then effect down here in the nucleus. I'll show you examples that are nearly as direct. This is a good place to start though. All right, let's zoom in and take a look at exactly what's happening. So here is erythropoetin binding to its Epo receptor on the cell surface. These are also called Janus face kinases because this is Janus the doorway god that also has two faces, two homodimerized faces looking both ways. In any case, Epo is up here. So even though Epo is not a perfectly symmetrical protein, it results in a pseudo symmetrical dimer of these Epo receptors. So it's not, nothing is exactly symmetric, but it's pretty close to symmetric. Okay, and again, that's the characteristic of these two component signaling pathways. Now earlier I showed you that the stat 5 molecules upon dimerization combined to DNA. Here's the mode of binding to DNA where the DNA is coming out towards us. So imagine this long B strand or B helix DNA coming out towards us and extending way out in space. And you can see that the dimer has the DNA, kind of a vice grip. It's clamped over the DNA and it's looking actively for a correct DNA sequence that it can then grab onto and signal for transcription. And again, the transcription is going to involve the usual players. RNA polymerase and other transcription factors that jump on and get involved. Okay, so if all we have to do is dimerize something, bring, grab two things together on the cell surface, then we chemists can probably do something like that. And so one way to do this would be to identify a molecule that binds to one half of the dimer, like this guy over here that binds to gyro B. And then simply form a pseudo-symmetric dimer of it. It's pseudo-symmetric because notice the stuff in pink is not exactly symmetric, so this is not C2 symmetric. You can't just flip it over like that. But it doesn't really matter because as long as you get these guys close enough for the two jack kinases to autophosphorylate each other, then the signal transduction takes place and then the stat 5 gets phosphorylated and then it dimerizes and then it goes on to transcription. Okay, so again, all we have to do is identify a binder to one of these receptors and then by simply forming that monomer into a dimer, boom, we get something that could turn on signal transduction. And anyone have an idea why you might want to turn on EPO or why you might want to provide erythropoietin to patients, for example? What circumstances would you want red blood cells being produced? After surgery. Yeah, so after surgery, the patient is low on blood, lost a lot of blood, blood all over the place. So you routinely give EPO as a way of stimulating blood cell production, red blood cell production. Of course, this is wildly abused by athletes who also value red blood cells for their oxygen carrying capacity. And up until recently, there were no effective tests for erythropoietin abuse. They're now really good tests. And this is one of the reasons why you're seeing all these scandals come out as athletes are getting caught. In fact, there was one in the news in the last week where athletes that had competed eight years ago were caught because their samples were still refrigerated and you can actually detect the artificial EPO in those samples. So from eight years ago. And I think that's going to happen more and more. That we chemists are going to get better and better at analyzing samples and finding tiny little quantities of unnatural compounds and kneeling athletes who are abusing them. And I think this is a good trend because as a sports watcher, I want to watch real athletics and not chemical athletics. So a little off the topic. So you now know that you can readily make a chemical version of EPO using a strategy like this. So all right. Now another strategy would be a lot cruder. So the thing about this that's lovely is that this compound over here is specific just for gyrobeat. But a much more crude way to do this would be to simply get in and form a covalent dimer. This is a non-covalent dimer and it has some chance to be specific. But a much less specific way to do this would be to simply add a chemical cross-linker. And I'm showing you this because it illustrates a really important reaction and one that's used quite a bit in chemical biology and biochemistry labs. So in this reaction, the interferon beta receptor 2, there are going to be lysines. It's almost unavoidable to have lysines on the surface of proteins. Lysine is this amino acid side chain that's frequently charged, typically positively charged, and it's very solubilizing. So the outer surfaces of proteins are almost always studded with lysine residues. And so for this reason, you can react these lysine amine side chains, shown here in their neutral state, with a activated carboxylate. So this is like a carboxylate, except instead of an OH here, instead of an O minus, if it's carboxylate or OH, if it's carboxylic acid, instead this has an N-hydroxy-sacidimide ester. And this NHS ester is a really good leaving group. And so what that means then is that this could very readily form an amide bond and give you a covalent bond. This is completely analogous to the O-acyl-isoyuria activated carboxylic acid that we saw when we talked about amide bond formation by DCC many weeks ago. Four weeks ago or something like that, when we talked about how to make amide bonds, we talked about how you have to activate the carboxylic acid. In this case, we're starting with something that's already activated as an NHS ester. And what's extraordinary about this reactivity is that it reacts specifically with amines and not all of those OH groups that are present at 55 molar concentration in water. Water is a whole bunch of OH groups. And so this kind of chemical specificity or chemo specificity allows this to pick out these amine functionalities. And then if there are two amines from neighboring interferon beta receptors, these two amines can be dragged together to form a covalent bond where they're linked together covalently. And again, once you've covalently cross-linked things, you've brought these guys together and you can start signaling through the jack stat pathway. Now, I want to take a moment to show you how this is useful, how this reaction is useful. This thing, not so useful. Yeah, you can do this experiment. It will work. But honestly, you're not going to be giving your patients this compound as a pharmaceutical because it will react with every other lysine that it finds. It has zero specificity, zip. And who knows what kind of allergic reaction the patient will come down with if you inject that into them. But a much better way to do this, or a much more useful aspect of this reaction, is to use it to modify proteins to improve their solubility. So for example, interferon alpha 2B, this compound here, it's a protein therapeutic, is typically peggulated, meaning there is this polyethylene glycan, polyethylene glycol tail that's appended to the protein. And so the way this reaction works is you start with an NHS ester of pegg. Okay, so again, this is polyethylene glycol. This is a polymer that you've seen before, maybe not in this class, but certainly in sophomore organic chemistry. And because it has an activated carboxylate, it will come along and modify all these lysines and even these histidine residues. And what's remarkable about this is if you control the pH and the buffer conditions, you could set this up so that there is only one position in this complicated protein that actually gets modified. And when we look closely at this, what we find is not the lysines at pH 6.5 that get modified. At pH 6.5, all these lysines are protonated. And again, if they're protonated, if these guys are protonated, there's no way they can react with the activated carboxylate. Instead, at pH 6.5, the only thing that's available to react is one of these histidines. I believe it's this one. And so the pegg gets appended, gets conjugated specifically just to one of these histidines over here and nowhere else in this complicated protein. And that's really exquisite reactivity and selectivity. And so for this reason, this is used very extensively. And these types of things are used extensively in therapeutics, in protein therapeutics. Okay, why would you want to do this? Why would you, anyone want to put pegg on your protein? It's a little bit of a trick question, but come on, you guys. B. You seem to be thinking about something. You're debating something about like being, being crazy with solubility? Yes. All right, well done. So what B suggested is that this pegg over here is going to be highly water soluble, right? It's going to have a lot of hydrogen bond acceptors. And so you could take a protein that would be relatively insoluble and make it more soluble. In addition, you're increasing its molecular weight, and over here can be really big. It can be hundreds to thousands. And so you can take a relatively small protein that would get excreted very readily by the kidneys, increase its molecular weight, and then it gets recycled back and prevented from being excreted. So there's some huge advantages to solubilization and even doing things like decreasing the protein's antigenicity. Okay? So you're increasing how recognized it is by the immune system as being a foreign invader. Okay? All those are really important properties, and so this reaction is used very extensively. Okay? Any questions about this? All right. Notice that the HPLC over here indicates that you're getting one and only one protein out. If this was getting peggalite in a whole bunch of places, you would see lots and lots of peaks. Okay? So the dominant peak here is number two. And these guys are relatively small. All right. I'm going to move on. I want to talk to you next about growth hormone and growth hormone pathways. And earlier I showed you this idea of acromegaly, which is too much growth hormone leading to giganticism, you know, excessively large hands, feet, and chin. So this is our third example of a signaling pathway. This is a classic. This is used extensively by many different signaling ligands, including TGF, beta up here. And in general, so this is very common, very typical. This is a biggie. In general, all of these work by the same mechanism. The mechanism is dimerize the receptors up here and then autophosphorylate, meaning this kinase over here phosphorylates the one next to it and then this guy phosphorylates the one next to this, and then that leads to some sort of, and then that allows a kinase to phosphorylate a nearby other protein that then goes on and eventually get down to the DNA down here. Okay? But the important thing is dimerization, then autophosphorylation. And we're just going to see that again and again and again. Okay? So here, for example, is growth factor receptor up here. So sorry, this is, there was growth factor receptor in purple. These are also called receptor tyrosine kinases because they're acting as kinases to phosphorylate tyrosines on each other. And then you end up with a whole series of different, you end up with a whole series of different phosphorylated tyrosines. This thing basically becomes loaded up with all these phosphoryltyrosines. Okay. Everyone stay with me on the self-surface stuff. Dimerize autophosphorylate. All right. Here's where it gets complicated. Once you get the dimerization and the phosphorylation, a total of four pathways are then activated. This thing goes on and, for example, it engages the stat subpathway that we saw earlier when we talked about the two-component signaling pathways. It also gets into the map kinase subpathway, which I'll describe next to you. But it also starts to affect PI3 kinase and PLC gamma pathways as well. These are pathways associated with fatty acids, which we briefly alluded to earlier last, or later on Thursday of last week when we looked at fatty acid synthesis and signaling in the cell. And I'm going to show you, I'll be able to pick up and show you exactly what's going on when we talk about that kind of signaling. All right. Before we do, let's take a closer look. This protein at the top is a growth factor, human growth hormone, or growth factor, it's a ligand, and it's going to dimerize the receptor tyrosine kinase, the RTK at the cell surface. And good news, we've already seen this protein. This is a familiar protein to us. This is human growth hormone over here. And it's going to initiate this dimerization through having two binding sites. And earlier in this quarter, we talked about, in fact, this is one of our very earliest lectures, this might have been like the third week or something, we talked about one of these binding sites and how it had a hot spot of binding energy for binding to the receptor tyrosine kinase, human growth hormone binding protein on the cell surface. There is a second binding site that looks completely different than this one on the other side that allows the dimerization of the receptor tyrosine kinase to take place. Okay. So this is our model for receptor ligand interactions, which is why we've seen it before. But you don't necessarily have to have large proteins doing this dimerization. In fact, small molecules can be very potent at doing this. And so, for example, this PD compound over here has a great specificity for particular growth factor receptors on the cell surface. And these numbers over here are IC50s and nanomolar. And recall that smaller number indicates higher potency, right, because that means a lower KD, which means greater affinity. And notice that these compounds are selective for the FGF, the fetal growth factor receptors. FGF are 1, 2, and 3 versus VEGF. This is a vascular endothelial growth factor receptor, which stimulates angiogenesis, the growth of blood vessels that we talked about earlier when we talked about proteases and methionine aminopeptidase. Okay, so this sort of specificity for one class of these receptors over other receptors in the same class is prized. And medicinal chemists will spend a lot of time trying to optimize the structure of a compound like this one such that it will be specific for one of these receptors or maybe for a whole class of these receptors, but then avoid, say, EGFR, endothelial growth factor receptor, or something like SARC, which is really broad activity. And what this means then is then you can have ways of targeting tumors selectively. And what we're showing here are tumors treated, or tumors dissected out of an animal that have been treated with the compound on the left versus controls. And check out how much smaller these guys are. So that's a good sign. So if we had ways of targeting, blocking, inhibiting growth factor receptors, we'd have really potent anti-cancer compounds. And this is one of the frontiers in medicinal chemistry and chemical biology. Many of these get their specificity through binding not to the ligand binding site that was highlighted by this ligand over here where the ligand likes to bind. Rather, they seem to prefer to bind to the ATP binding site. And that actually seems to be a surprising area for gaining specificity. You wouldn't expect this, right? Because you'd expect every kinase that uses, all kinases use ATP. That's their source of phosphate. But you'd expect them all to bind ATP in the same sort of way. But what we're finding through efforts of medicinal chemistry is that actually there's enough variation in the ATP binding site that you can alter the structures of these compounds and gain specificity for, say, FGFR versus all of these other receptors down here. And so by what I mean by this, by bearing the structure would be, for example, changing the structure of this urea functionality. I'm showing you this with a T-butyl urea. You might want to try a phenyl urea. You might want to try, I don't know, a straight butyl urea, et cetera. As you can imagine, making lots and lots of little changes over here, over here, over here, and doing this to dial in the specificity for a particular receptor. This is a property that's also known as selectivity. Make sense? All right. Let's take a closer look then at phosphorylation. Phosphorylation is not just random. So these guys are going to be auto-phosphorylating each other. You know, it's like Punch and Judy or something. But they're going to do it in a very specific sequence of blows. In other words, they're going to have a very defined pathway of one phosphorylation allows the next phosphorylation, which allows the next phosphorylation. And that's illustrated by these numbers over here, which detail this guy gets phosphorylated first, this guy number two, second, three, four, and five, and so on and so forth. And so the longer this dimer hangs around, the more likely it is to actually allow signaling into those various pathways. And remember, there's four pathways that are going to be implicated by this one receptor up here. So the receptor tyrosine kinase goes to town, but it doesn't start going wild randomly. Instead it is very specific. And in that specificity, it's going to be turning on specific pathways. Let's take a look at one of these pathways. This is the map kinase pathway. This is one of the four that the protein has got a signal through. So after this phosphorylation takes place, a protein with an SH2 domain called SHIC1 can grab onto this phosphate and then get phosphorylated itself, allowing grab2 to bind. Grab2 also has an SH2 domain. SH2 domains bind to phosphorylated tyrosine. In fact, I think we saw them earlier in the quarter. But grab2 has a polyprolene helix that can be bound by an SH3 domain, allowing binding to RAS, allowing binding to RAF. And then in the end, MEK can phosphorylate ERC, which then gets into the nucleus. And remember earlier, I told you how there was alternative nomenclature. So MEK can also be called map kinase kinase. And then if phosphorylates map kinase, I've even seen map kinase kinase kinase. So there's a bunch of other kinases in this pathway that I'm sort of not showing you. And I'm also simplifying the nomenclature. I'm using the simplest nomenclature as well. Okay. The important part here is that there's kind of a handoff of signal that involves not just phosphorylation, but interaction through an SH2 domain. And we've seen that earlier in the quarter. Earlier in the quarter, I showed you how polyprolene sequences will bind into SH3 domains. In fact, here's the structure that I showed you. Here's an SH3 domain in green. Here's the polyprolene helix. And remember, this is the helix. This is the 310 helix. That if you look down this axis, do you see how it looks triangular? It looks triangular here. This is what the polyprolene looks like. So this is a familiar concept to us. This is something that we've seen before. We've also seen SH2 domains as well. So grab2 has an SH2 domain to bind to the phosphotyrosine on SH1, but it also has an SH3 domain that binds to sun of 7 less, sauce over here. And then sauce binds to RAS, which binds to RAP, et cetera. Okay. So everyone's still with me. All right. There's one other element that this pathway illustrates that's also prototypical. This is one of those things that you see quite often. On the cell surface, the cell surface is not a sterile, neatly denuded environment. Rather, the cell surface is complicated. It has a lot of shrubbery on it. And in fact, amongst the shrubbery is long chains of this stuff called heparin sulfate. This can then attract FGF. It can track the ligands that these growth factor receptors like to bind and increase their, enhance their apparent affinity for binding to the receptor simply by a proximity effect. Okay. So the shrubbery up here, the heparin sulfate, grabs on to any stuff that happens to be passing by. And then because now it's localized up close to the receptor, the receptor can then more likely find, more easily find it. And that in turn enhances the signaling abilities. Sound good? Okay. So these are common modes. We're seeing the common mode that I want you to learn is this idea that heparin is out here to enhance binding. Dimerization takes place. Autophosphorylation takes place. There's binding through SH2 and SH3 domains. And then a relay of phosphorylation, phosphorylation, and then finally into the nucleus for transcription. Okay. It's like Yogi Berra said. Baseball, it's just running, throwing, catching and hitting. What could be more complicated than that? Okay. So there's a few elements here, but we're going to just, when you get into this area, you just see these same elements repeated over and over again. Learn these elements. You'll be able to understand everything else. Any questions so far? We good? All right. Let's talk a little bit about the other pathways that these receptor tyrosine kinases like to stimulate. One of these, another pathway, are these pathways that are fatty acid signaling pathways. These include phospholinosatol-3 kinase. This is phospholinosatol-3. There are kinases that phosphorylate this. So PI3K, phospholinosatol-3 kinase. I'll just call it PI3K. It's the third hydroxy of this glycan. So this glycan is appended to a fatty acid chain. And upon phosphorylation, this gives you something, it gives you a different structure. And we now have something that has three phosphate groups attached to this glycan. And this then can allow binding. This is called phospholinosatol-3 or PI3. PI3 can then be bound by these PI3 binding sites. The effect here, so this is a protein called protein kinase C or PKC. PKC is floating around the cell. It's happy as a bee. It's banging into flowers, looking for whatever it does all day. Until it finds PI3, once it finds PI3, it gets ensnared like a fish getting stuck onto a fish hook. At that point, PKC is stuck at the cell membrane. And this localizes PKC at the cell membrane where it can start doing all kinds of things. So again, this is that principle of localization. Localization is what allows these things to then get turned on and then in turn affect the pathways. It's kind of like that old adage in real estate. Location, location, location. And so in the same way that heparin is confining these ligands to particular spots in the cell, this PI3 is going to confine PKC to particular spots at the plasma membrane and that in turn is going to allow the pathway to get kicked off. So it's not simply activity. It's also localization, localization, localization that gets things into the right spot where they can do the most signaling. Okay, it makes sense? Okay, now this is a gristly simplified portrait. When you start looking at this in more detail, you start finding that there are other ways of shutting this off. So for example, you can free up the glycan from the rest of the acyl tail by a hydrolase, PLC gamma over here, and things get more complicated as well. Okay. In addition to the heparin sulfate that I've already told you about, there's also the various IP3s and PIP3s. There's also a curious role played by calcium and the release of calcium. So IP3, the release of the IP3 from the PIP3 has the effect of releasing calcium into the cell and this in turn unhinges a binding site to allow binding to stuff found here at the plasma membrane. Okay, so all of this stuff is working together. Okay, so you have a really complicated dance where one of the major goals of the dance is simply to get the players into the right space at the right time. Okay, and calcium is great because it diffuses very readily through the cell and it works really well for this. Okay, any questions about receptor tyrosine kinases, growth hormone, et cetera? Okay. This is 90% of cell signaling really is what I just told you. Okay? I'm now going to go on and I'll talk to you about the other, oh, sorry, I didn't mean to say 90%. 30%, 40% of cell signaling that you read about in science, nature, and cell deals with these pathways. I want to talk to you about another 30% or so. This is a really important one. G-protein couplin receptors are some of the most common proteins in the human proteome. These really are extremely abundant to us humans and in fact these are the proteins that make life worth living. These are the things that allow us to see like rhodopsin over here, allow us to smell like these olifactory things, allow us to taste. Here's some tasty ones, et cetera. All of these smell molecules, taste molecules work by binding to G-protein coupled receptors which look like this. This is a transmembrane protein where the membrane is going through here. The membrane cuts through this over here and the ligand is very deeply buried. It gets snuggled down deep into this GPCR. Inside the GPCR it changes the confirmation of the GPCR such that the stuff in here that's going to be signaling on the inside in the cytoplasm of the protein has a new confirmation. So these bind to a very wide range of ligands. In fact, many of these are promiscuous in their activity meaning they'll bind to a bunch of tasty things. Once that happens, that allows the conformational change that communicates from what's happening outside the cell to the inside of the cell. That's kind of at the very cell surface. Let's take a closer look. I have to tell you a little bit about how these work. I first want to tell you about dynamic range. I'm going to digress for a moment and then we're going to take a closer look at GPCR. If for example you had some receptor that simply was turned on and off depending on if you had binding by a ligand, you'd have a kind of a lousy sensor. These have to act as sensors. If you're going to be able to say sense light changing for moving towards light if you're some organism or being able to smell stuff so that it determines whether or not you eat this food that's going to kill you or not kill you or make it possible for you to live, there's an incredible premium on being able to sense tiny little quantities and at the same time being able to sense really big quantities. And that's a property called dynamic range. If the sensor simply operates on the principle of bound or unbound, your dynamic range is going to be very limited. Let me remind you that this principle of bound versus unbound is governed by an on rate and an off rate that together have a dissociation constant associated with it. The truth is this offers a limited dynamic range as epitomized by this percent activity plot that we showed earlier in the class. Recall all of these have this sigmoidal plot, the sigmoidal type of relationship between concentration of ligand and activity on the y-axis. The problem with this is that this type of receptor would be sensitive only in these three or four orders of magnitude in concentrations from 10 to the minus seven to 10 to the minus four. So like submillimolar to submicromolar. And that might be okay, but it's not so okay if you have to smell, I don't know, tiny quantities of some pheromone to know whether or not you're ever going to find a mate in your lifetime. That could be kind of important if you're an organism, right? And so this business about sensing only in one particular range is pretty much worthless for really good sensing and devising really good sensors. So instead, all of the GPCRs rely on a different principle. If I get to a different principle, does anyone have questions about this one? Does it make sense to you that this is kind of limited in its utility? Okay. Always get nervous when there's no questions. Take your word for it. Instead the G protein coupled receptors signal through a G protein. They all will bind directly to something called the G protein. And the G protein is a catalyst that catalyzes hydrolysis of GTP, this guy up here, magnesium GTP complex, to magnesium GDP. So it's simply a hydrolysis of the gamma phosphate of GTP to give us GDP. Okay. Straightforward reaction. A reaction we've seen before, an easy one, right? An easy reaction we can derive in our sleep. We can draw a mechanism in our sleep. This is an important part. By coupling binding to the activity of an enzyme, you can very sensitively tune the activity and you can tune this sensor. So it becomes a really exquisite sensor for low concentrations and high concentrations. And I realize it's not clear at this moment. I'm going to show you on the next slide. Before I do, though, everyone with me have this idea. GPCRs bind to G proteins. G proteins are catalysts. We're good with that one? Okay. So check this out. Here's what's really going on. So the G protein in its GDP bound state says signal off. So GPCRs up here, if the G protein, however, is bound to GDP, signal gets shut off. On the other hand, if it's bound to GTP, the signal gets turned on. And what's important is that there are two intermediary enzymes that are going to catalyze and help push this equilibrium between GDP bound and GTP bound. These two are a guany nucleotide exchange factor, a GEP, that helps push out GDP to allow binding by GTP. And then in the opposite direction, a GTPase activating protein or GAP that helps drive the phosphorylation of GTP to GDP. Okay. Now here's the way this works. Your cells can carefully control the ratio of GEP to GAP or GEP to GEP. And by doing that, they can make the signal, the sensor, more sensitive or less sensitive. Okay, and if you want it to be more sensitive, you basically set it up so it's always signal off, right? So then you'd have lots of gaps around. The gaps keep it in this off state. So if anything turns it on over here, then boom, everything goes on. On the other hand, if you want it to be less sensitive, you keep it in the signal on state, so it really takes quite a bit to get it shut off again. Makes sense? Okay, this is a really powerful principle. And it's one that I really have not seen in too many areas of electronics or chemistry. And it's one that I think really could be exploited for interesting uses. B? There is only water present in cells, so how can you actually turn it off? Okay, so you mean how does this happen? Actually I think it's only happened because there is only water inside the system. It doesn't have water. All of it is taking place in water. It's very close to 55 molar water. But the GTP over here bound by the G protein is getting hydrolyzed at a very slow rate. The catalyst here is not such a great catalyst. And GTP in water is completely stable. It's not going to get hydrolyzed. It needs an enzyme to hydrolyze it on the time scale that's relevant for biology. Okay, any question over here, Sergio? Sure. Okay, so this is happening right up close to the plasma membrane, right up close to the cell surface. Okay, so the GPCR is right there at the cell surface. The G protein is right below it, grabbing on, holding on for dear life. And then these gaps and gaps are forming another piece of the sandwich. So in the end you get these monster complexes. Okay, yeah, and over here. So instead of having that system, you just have ligand and receptor and then just the concentration of ligand. And that's where you could have the dynamic range. You want to get the same dynamic range. So if you adjust the concentration of ligand down here, you're only going over four or some magnitude of dynamic range. But if you have this gap and gap system, then you can tune this to kind of shove this over so that it's now responsive to ligand over here or ligand over here. This gives you a responsiveness over all of these, this dynamic range. Okay, and this is what you need. Let me explain. Okay, so this is the same set of receptors that's going to allow you to see. And there's times when you need to be able to see in dark caves to be able to survive. Maybe not you, but certainly your primeval ancestors, right? They would need to be able to survive in dark caves where humans can sense one photon. We can sense just a few photons. That kind of low light sensitivity, incredibly important. On the other hand, eventually you get out of the cave and you're wandering around in the bright sunlight. And if all you can see are, if you could see each photon individually, you're going to get totally overwhelmed and you're not going to be able to see anything, right? So you need a system that allows you to be able to sense things way down here, but then also be able to shut off so that it doesn't get overwhelmed way up here. Okay, and that's really the key to this activity. So affecting the gap and gap ratio controls that sensitivity. Okay. Question in the middle? Yeah? Is that why our eyes are pupils dialling? That's also part of it as well. Okay, but you know, the rhodopsin that's sensing photons, that has to have some way of being less sensitive at times and more sensitive at other times. But certainly, yes, you're right. Dilation of the pupils will have an effect as well. And over here? Is there any way that we can modify the ligand to be more sensitive? Okay, but if you modify the ligand and you make it more sensitive, let's say you're doing taste or something, you'd make it more sweet, right? But eventually, you know, you're over here. So you're going to be like hammering on it. So now it binds really well over here. Eventually it's just going to get overwhelmed. It gets saturated. Okay? Right? And so everything tastes sweet. That's not so pleasant either. Okay. So what happens after the G proteins? So we're up here. We have binding to the G protein coupled receptors. Here are the G proteins. These have three subunits, an alpha, beta, and gamma subunit. Alpha is the alpha that's going to be doing the GTPase activity that I talked about. This is then going to lead to denolate cyclase, this protein over here, which turns over ATP to give cyclic AMP. What's neat about this is that all of these G protein coupled receptors converge on this at denolate cyclase. All is a little bit too strong, but they're largely going to converge over here and a denolate cyclase acts as an integrator of signals such as you can get signals from a couple of different cell surface receptors turning on a denolate cyclase, which in turn the cyclic AMP turns on protein kinase A. Protein kinase A we've seen before. We've talked about this protein. This is the protein that does the waltz as it phosphorylates different proteins in the cell. We looked at a single molecule level earlier in this class and it's a good friend of everyone who's taking Chem128. PKA allows CREB, this transcription factor, to turn on genes, for example, to turn on genes to do something about cardiac myocyte hypertrophy. It turns out that the molecules that bind to these GPCRs are extremely diverse. In fact, we humans have learned to revel in this chemical diversity. For example, these are the molecules associated with the smell of peanuts. You know that nice, roasty smell of peanuts? It's actually not one molecule. Rather, it's a plethora, a little tiny combinatorial library of these molecules over here that are present at these different concentrations that have different odor thresholds and different odor activity values. The odorant receptors, which are G-protein coupled receptors, are going to bind to each of these and then, in turn, these are going to map to specific neurons. So lots and lots of different compounds, each one with their own characteristics smell, but the sort of symphony that we associate with the roasty smell of peanuts is a bunch of these present at different concentrations. We need receptors that are going to respond to each of these different molecules and do it in a way that then integrates the signal and gives us something that reminds us of roasty peanuts. Here's the way this works. These are the smell receptors, the olfactory receptors in the nose. Here is the odorant over here. Odoring can bind to several different receptors. So here's the odorant. It's going to bind weakly to this guy, strongly to this guy, not at all to this guy and maybe to this guy over here. All of the olfactory cells that have this receptor on their surface all signal back through the same neuron. Okay, so if we map the cells over here in this olfactory region, they're all going to go back to one and only one neuron, all the ones that have the purple GPCR on their surface. All the ones that have the green GPCR go back to a different neuron. This is an astonishing result that came up, was discovered in the last two decades or so. But in short, what this means is that everything that binds to this receptor signals in the same way. You get the same neurons firing. In the end, using that gap to geth ratio that I discussed with you, this can allow signaling that tells the neurons whether or not you have a strong binder or a weak binder. Over here, you have a strong binder. You get more signal out. Over here, you have a weaker binder, so you have a weaker signal coming back out. And again, you can control the dynamic range such that you don't get totally overwhelmed if you go into a peanut factory so that you're smelling peanuts for the next week or something like that. All right, let's talk about vision. I mentioned this earlier. This is one of those amazing chemical reactions that all of us are reliant upon. We humans are especially reliant upon vision as our primary sensing organ. And what's remarkable about this is the goal here is to capture a photon. Your expectation is you can't bind to a photon. How do you capture a photon? So instead, the GPCR, associated with vision, called Rodopsin, covalently links to a cofactor called retinol. So this is the structure of retinol. It forms a shift base with a lysine in the GPCR. This is normally found in a cis-olefin form. Okay, so this 11-12 carbon-carbon double bond, cis. Upon being irradiated by a photon, that's H nu, the cis-olefin isomerizes to give a trans-olefin. And the net effect here is a flipping of the retinol. Okay, so we go from cis to trans, and this whole cyclohexane has flipped from being over here to being now extended. That has the fact of changing the confirmation of the GPCR and allowing you to see. This is how you are seeing me right now. This is how it really works. Okay, now in recent years, chemists have started to get creative, and we've been devising retinols that are responsive to different wavelengths. And some of these might be really interesting for allowing us to see at different wavelengths. For example, to be able to see in the infrared, to see in dark, and so on and so forth. You can imagine a lot of interest in this by people who like to see in the dark. All right, let's take a closer look at the structure of rhodopsin. So again, it's a GPCR. Notice that its structure looks very similar to the GPCR I showed on the first slide. All of these GPCRs have a common structural motif consisting of seven alpha helices that circumvent the membrane, that are transmembrane. Here in purple is the retinol, again forming a shift base at one end to a lysine residue on this alpha helix. And the protein undergoes a dramatic confirmation change in response to that cis trans isomerization. Cis trans isomerization is huge. Up here, this little change up here, photon comes flashing in over here, boom. You get isomerization and boom, the whole protein changes confirmation, changing, affecting its binding to the G protein and turns signaling through that G protein cascade that we talked about earlier and turn leading to stimulation of neurons, which again is what you're doing when you see. Okay, that's kind of extraordinary. All right, any questions about GPCRs? Moving on. Oh, Carl. Yeah, so it's constantly flipping back and forth between cis and trans, and that's what you're interpreting as vision. All right, ion signaling, so this is number five in our discussion. So these arrows are going the wrong way. Potassium is constantly being pumped out of the cell, calcium is being controlled carefully, chloride is being pumped in and out of the cell, potassium is constantly being pumped in and out of the cell. The cell maintains an equilibrium of specific concentrations of its ions. Those ions provide a way of signaling very quickly in the cell. In fact, most of the cells' fastest responses, such as vision and stuff like that, are probably going through ion channels. So the way this works is that these ion channels can open up pores that allow ions to flow in and this leads to an electrical signal that can propagate along a neuron, for example. So it's carefully controlled through transporters and it provides really fast response. This is the kind of response where if I suddenly make a loud noise, then you jump or something like that, that's the ion response. I'm hearing that, ion channel response, seeing ion channel response. So here's what these channels look like. This is a calcium-activated potassium channel that allows potassium to flow in. Calcium binds down here and changes the conformation and then allows potassium to flow out of the cell. You can imagine that because these channels allow really fast response, such as a muscle response, toxins would very potently target these types of channels. And caribatoxin is one in a large number of different toxins that target these types of channels. Many of these toxins cause paralysis. These are things that are used by scorpions and cobras, et cetera. All right, that's all I have to say about ion channels. They're fascinating. Ions flow in, they flow out. I want to move on. Death receptors. So this is a class of compounds that binds to the cell surface and instead of causing dimerization, they cause trimerization. So you get this active trimer. In fact, dimerization is associated with shutting off the pathway. And so, for example, this compound over here allows, encourages formation of an inactive dimer that shuts off the pathway. But I'm getting a little ahead of myself. Here's what it looks like. Lygand is up here. Lygand is a trimer. Yand binding, the receptor forms a trimer. And then you get a series of kinases, I-Capa, you get a series of kinases passing off the phosphorylated response. And then in the end, NF-Capa B gets into the cell and causes transcription and inflammation. This is a highly regulated process that's used extensively in the immune system. This includes things like TNF. This includes interferon, et cetera. These are really important pathways for turning on inflammation, turning on the immune system. Okay. Last pathway, number seven, defusible gas molecules. It turns out that we humans are susceptible to signaling by tiny little gas molecules, things like oxygen. In fact, all life is going to be very dependent upon being able to sense oxygen. You can imagine that if you cannot sense low oxygen conditions, you're going to die, right? Because you need to be able to learn to move towards areas where you can breathe, right? That instinct to breathe when you're underwater is fundamental to life. And it's really fundamental to all organisms that depend on oxygen. And so the way this works is oxygen sensing by the cell works by taking advantage of the chemical properties of oxygen as an oxidant. So there's a concentration of Hif1 alpha in the cell. This Hif1 alpha is constantly being synthesized and constantly being degraded. And this Hif1 alpha upon encountering oxygen oxidizes, gets oxidized to introduce an OH group that in turn allows it to be degraded. And that in turn will prevent these transcription pathways from taking place. So when Hif1 alpha is present, nuclear, this gets in, causes transcription, which in turn turns on anaerobic respiration, right? That tells the cell, we don't have a lot of oxygen present. We better take into gear the anaerobic respiration aspects of metabolism. On the other hand, if oxygen is present, that oxidation introduces a hydroxyl that in turn leads to degradation of Hif1 alpha. So the key concept here is that you have this concentration of a Hif1 alpha that's constantly being formed, constantly being degraded, and that gives you a very rapid sense of how much oxygen is present. It's kind of like this Hif1 alpha is like a thermostat for oxygen levels. It's constantly monitoring how much oxygen the cells have. If the cell doesn't have enough oxygen, it's going to die if it doesn't switch on these anaerobic respiration pathways. Nitric oxide is also another very common cell signaling molecule. And this one works by binding to iron-porphorin cofactors of guanylate cyclase and in turn causing blood vessels to relax. So this is important for the regulation of blood pressure. And a number of compounds were invented to affect this pathway. And what was astonishing is actually they weren't compounds like Viagra were found to be less effective as blood pressure regulators and more interesting for their side effects. And here are their structures over here. These are structures of Cialis and Viagra compounds in the little blue pill. Here's the way this works. Okay, so this is nitric oxide over here. It's going to bind to a guanylate cyclase and in turn that's going to affect GTP being converted to cyclic GMP. These compounds inhibit guanylate cyclase and in turn that causes blood vessel relaxation. This is one pathway that's very dependent upon calcium concentration, et cetera. Oh, sorry. Actually, sorry. The compounds are going to be targeting PDE5, this compound over here, which is affected by cyclic GMP levels. Okay. Any questions about any of the seven signal transduction pathways? All right. In that case, that brings us to my wrap-up and conclusion. What I've been talking to you about all quarter is a new way of thinking about biology, thinking about it at the level of atoms and bonds where we can actually make changes, where we can think creatively. And I hope, for example, the proposal assignment has shown you what it's like to be at the frontiers of this really exciting field. So these are processes that you've probably seen in other classes, certainly DNA, DNA base pairing, et cetera, but I hope by thinking about these in terms of chemicals, in terms of being compounds that have atoms and bonds, we've introduced into your thinking new ways of thinking about their reactivity. For example, forming cross-links, depurination, et cetera. Chemical biology lets us start to address who are we. It really gets us to answer really big questions in biology. And all of the structures that I'm showing up here are things that we've seen this quarter, either we've discussed in lecture or they're in the textbook. These include things like a conantoxin that targets ion channels and causes paralysis. So these are organisms on the planet, and we've seen lots and lots of their bioactivities in this class. It also lets us address where do we come from? When we talked about prebiotic synthesis, it was very abstract. We're using it as an example of error pushing, but chemical biology gives us a foundation to start to address really philosophical questions, things that humans have been grappling with for a very long time. And finally, we get to address them really at the level of atoms and bonds. And then chemical biology gives us the control over our destiny. It gives us the tools that we need to devise compounds that allow us to overcome our being human, that allow us to address things like disease, starvation, nutritional deficit, et cetera. And we've been talking about this all quarter. Here for example is the PEG intron molecule that I showed earlier in today's lecture. This is the PEG, this big blob next to it, and then that's the protein that I showed earlier. These are really important molecules. These are molecules that affect the world and that allow us to address diseases. And in the end, chemical biology ends up changing society. The compounds we talk about have a big impact on our lives. They have an impact that goes beyond just medical health. They have impacts on, for example, affecting athletes, for example. So I want to encourage you to join us. Join us at this frontier of chemical biology. I hope to see you in the laboratories here at UC Irvine. And I'm really looking forward to reading your proposals. So I want to thank Natalie for being our projectionist, for doing all the videotaping all the time. And I have to thank Prithika up here and Miriam down here for being really outstanding TA's. And I want to thank all of you for a really fun quarter. I've really enjoyed teaching you, and if you're joining us on YouTube, I hope to see some comments from you on the class and whether you enjoyed it as well. But I've really enjoyed teaching this class, and so I thank you for a really great quarter. Thanks a lot, everyone. Thank you. 개편지 writings, anne legalencia, tactical policy, maarie lacので하는 사랑 어머hhwhistleffenf
 I can't believe it's gone so quickly.  So it's a little bittersweet.  I love teaching this class, so it's hard for me to give this very last lecture.  Okay, so today we're going to pick up where we left off last time.  Small molecule control over signaling pathways.  We already talked about this last lecture.  Today, proposals are due.  Make sure you don't leave the class without hitting them in.  If you miss handing it in for whatever reason, then just bring it by my office as soon as  possible.  You have till noon as a grace period.  This also applies if you need to get an abstract in or something like that.  I'd like you to attach your abstract to the proposal.  Your graded abstract.  If you don't have your graded abstract with you today, run home, get it, bring it back  either to me or to the TAs, Krithika or Miriam.  Do that ASAP.  There's a little bit of a grace period, but you don't want to extend it too far.  I understand there's some issues with turnitin.com.  Did anyone have issues with that?  All right, let's try to get those in the next 24 hours uploaded.  Sometimes it just jams with, this is kind of the end of the quarter, so there's a lot  of assignments that are coming in.  Hopefully, turnitin.com will get their act together.  If it persists as a problem, then I'll make arrangements.  For now, just keep trying and then get that turned in in 24 hours.  I do have a quick question for you.  For next year, did you like having this do today, or would you rather have it do Monday?  Everyone in favor of today, raise your hand.  One, two, three, four.  Like Monday or Monday?  No, no, no.  Monday is in upcoming Monday, like a few days from now.  Today, all the paper of today.  Okay, and Monday?  Wow, it is almost completely evenly divided.  All right, well, in that case, we'll probably stick with today then.  The reason is, this gives me more time to read the proposals.  It actually takes a lot of time for me to read 120 10-page papers and provide comments  on half of them.  Because of that, I need the extra days.  And also, I feel like you guys need a little bit of break to study for your other final  exams.  If you had this hanging over you, you would imagine just working on it, working on it,  working on it until the very last minute.  So, okay, well, I'm looking forward to reading them.  So, you can now relax now that you're here.  And just make sure that I get them at the end of today on this table over here.  All right, I do have office hours today, immediately after class.  I imagine that you don't have too many important things to talk to me about.  I will have office hours next quarter if you want to stop by.  You're always cordially welcome to my office.  Okay, so you're always welcome to stop by.  Usually office hours are the best way to get a hold of me or send me an email.  You know, if you need advice about anything, about careers, about finding a job or whatever,  don't hesitate to call on me.  Okay, so you've been in my class.  You worked hard for me this quarter.  I'll work hard for you in the future.  All right, here's what we talked about on Thursday.  So Thursday we wrapped up our discussion of terpenes.  And then we talked about cell signaling.  And one thing I wanted to press upon you is that this concept of using small molecules  to control cells and to coordinate responses in cells is really a universal phenomena.  This is carried out by the most simple organisms on the planet and the most complicated organisms.  It's carried out by humans.  It's done by humans.  It's done by insects.  It is truly universal.  It's one of those aspects of life that we find.  Small molecules are also used extensively by chemical biologists as power tools for dissecting  biological phenomena.  And I showed you examples of this using both reverse and forward chemical genetics.  The example of the forward chemical genetics were those massive screens to identify compounds  that could interfere with mitosis.  Okay, and we saw this during the cell division.  The DNA, instead of lining up on the scrimmage line neatly in the center, was getting, was  you know, pushed to the peripheries and it looked like the complete reverse of what should  happen.  So forward chemical genetics is a powerful way to identify unusual phenotypes in the  cell.  Then using reverse chemical genetics, you could use that small molecule as a powerful  tool to figure out exactly what's happening.  That compound monosterol emerged as a really interesting inhibitor of kinesin.  And using that inhibitor, scientists were able to, chemical biologists more specifically,  were able to define a particular kinesin variant that's critical to shoving apart the chromosomes  and pushing them into place during cell division.  And that's absolutely fascinating.  Okay, so small molecules offer temporal control, meaning time control.  They also offer localization.  You can dose them in a particular spot.  And we even talked about how they can be used to investigate phenotypes that would otherwise  be lethal to the organism.  So all of those aspects are what make small molecules such an integral part of chemical  biology in the modern era.  Okay, now because we often use these small molecules, kind of like toxicology, to investigate  cell phenomena.  We oftentimes find ourselves investigating cell signaling pathways.  And my message to you there is don't panic.  Okay, so if you open an issue of cell or something like that and you look at the last couple  of pages, they always have signal transduction diagrams.  And every month, every week, or every couple of weeks, they investigate a different signaling  pathway.  And these are daunting in their complexity.  They look like the Milky Way in terms of the number of elements that are present.  And they look completely nuts.  But the message there is don't panic.  Instead of looking at such crazy complexity, we as chemists can simplify things down to  seven major pathways.  And I contend that if you learn these seven pathways, you'll be equipped then to understand  anything no matter how complex it is.  All of that complexity can be reduced down to just seven elements, or seven different  types.  And if we learn those seven types, we'll be in good shape.  Okay, we looked at the very first of these seven, which were the nuclear receptor-based  pathways.  And we looked at how steroids have potent activity through binding to these nuclear receptors.  And then we saw that these have two different modes, that there were two modes of the nuclear  receptor pathways, heterodimerization and homodimerization.  Any questions?  All right, well, let's return to our seven canonical pathways.  I'll remind you again that we looked at the steroids binding to their nuclear receptors.  And the key element here was the notion that these nuclear receptors bind directly to the  steroids.  And then these complexes make their way all the way to the nucleus where DNA is turned  on or turned off and transcription takes place.  That's a very powerful paradigm for affecting cell division, cell growth.  And this is why these steroids like testosterone and estrogen have such potent cell-based effects.  Today I want to pick up our discussion and discuss with you the other six pathways found  in the cell.  And again, I'm going to be emphasizing what's common and what's simple about these pathways.  We could get crazy complex, but it's just not pedagogically useful.  So I'll show you kind of the stripped down version.  And if you start launching a research project on one of these pathways, you'll find quickly  that things get a little more complex, but not much more complex.  All right, so let's get started with the two component pathways.  These are pathways in which a ligand binds to the outside of the cell up here and then  dimerizes to receptors.  And by the way, just as a quick preview, note that many of the upcoming pathways are going  to feature ligands that don't even get inside the cell, like these guys over here.  So here's a ligand, it's binding up here.  Here's another ligand and purple binding up here.  Another ligand binding up here.  None of these ligands get through the plasma membrane.  Instead, they're going to affect the confirmation of proteins that are found on the cell surface  and that conformational change will transduce the signal from outside the cell to inside  the cell.  Okay, so that's going to be our paradigm for today.  All right, let me skip on one moment.  Let me see if I can find where we left off.  Okay, we talked about quite a bit last time, didn't we?  Okay, here we are, number two in our seven.  In this case, I want to talk to you about controlling cell differentiation through a  class of molecules called cytokines, also known as interleukins, variously called interleukins.  This is one of the annoying aspects about biology is that they don't have a uniform  nomenclature the way we chemists do.  The periodic table is the same here as it is in Russia or Sudan or anywhere in the world,  but biologists don't have a UPAC, so they have a little bit more difficulties getting  a standardized nomenclature.  Be that as it may, cytokines are an important class of molecules for causing cells to differentiate.  The differentiation I want to talk to you about today is the differentiation into various  blood cells.  This includes red blood cells, erythrocytes.  It also includes B cells and T cells, which are crucial to the immune response to responding  to attack.  It also includes a number of other different variants, like platelets, which we've already  seen as being crucial to blood clotting, et cetera.  All of these signaling ligands highlighted in bold and blue are examples of cytokines  that all more or less have the same signaling pathway.  Rather than us saying, all right, let's learn about IL-4.  Now, forget everything I told you about IL-4.  We're going to talk about IL-2 next.  Rather than doing that, I could just tell you about one example, and then from that one  example you can figure out everything else.  Let's dive right in.  I'm going to be talking to you about a favorite of ours, one that we've seen before.  This is growth hormone.  Human growth hormone causes acromegaly.  This is characterized by abnormal growth features in the face.  The face looks a little distorted, hands and feet.  Just abnormally sized large hands and feet.  I mean, really abnormal.  So I'm realizing this slide is out of place.  All right.  We'll get back to growth hormone.  Let's get back to, let's focus in the example I'm going to show you will be erythropoietin  right here.  And again, it's generalizable to a bunch of other molecules.  Okay.  So this is a very typical mode for these two component pathways, two meaning that they  are going to cause dimerization on the cell surface.  And so here's the molecule Epo up here, erythropoietin.  Erythropoietin stimulates the formation of red blood vessels, of red blood cells.  And the way it works is erythroepo up here dimerizes an Epo receptor on the cell surface  and that allows an associated kinase, Jack 2 kinase, to phosphorylate each other.  Okay.  So this is an example of transphosphorylation.  This guy phosphorylates this guy, which phosphorylates this guy.  That in turn allows phosphorylation of stat 5 which dimerizes and then stat 5 actually  gets into the nucleus and causes transcription down here.  Now a closer look at the phosphorylation is in order because this is going to be a major  mode for relay of the signal.  Okay.  So like in track and field when you have a relay there's a baton and you kind of pass  the baton hand over hand.  This is a similar type of phenomena.  In this case the baton is going to be a phosphorylated amino acid side chain on the surface of the  protein.  And that phosphorylated amino acid side chain is going to create a new surface that then  allows something to bind that otherwise would not be allowed to bind or it could change  the confirmation of the protein after it's phosphorylated.  Okay.  So what that means is stat 5 has one confirmation up here.  It gets phosphorylated and then it dimerizes.  And that's a phosphorylation.  It's not dimerized.  It's a monomeric protein.  Upon dimerization it has a different confirmation.  One that can bind DNA.  One that can turn on transcription.  Oh, and one that can get through the nuclear pore to get into the nucleus.  Okay, so this is also termed a jack stat pathway because jack kinase and stat 5 or stat proteins  are playing a key role in this cell signaling.  And note too that it's fairly direct.  There's only a small number of proteins between signal up here and then effect down here in  the nucleus.  I'll show you examples that are nearly as direct.  This is a good place to start though.  All right, let's zoom in and take a look at exactly what's happening.  So here is erythropoetin binding to its Epo receptor on the cell surface.  These are also called Janus face kinases because this is Janus the doorway god that also has  two faces, two homodimerized faces looking both ways.  In any case, Epo is up here.  So even though Epo is not a perfectly symmetrical protein, it results in a pseudo symmetrical  dimer of these Epo receptors.  So it's not, nothing is exactly symmetric, but it's pretty close to symmetric.  Okay, and again, that's the characteristic of these two component signaling pathways.  Now earlier I showed you that the stat 5 molecules upon dimerization combined to DNA.  Here's the mode of binding to DNA where the DNA is coming out towards us.  So imagine this long B strand or B helix DNA coming out towards us and extending way out  in space.  And you can see that the dimer has the DNA, kind of a vice grip.  It's clamped over the DNA and it's looking actively for a correct DNA sequence that it  can then grab onto and signal for transcription.  And again, the transcription is going to involve the usual players.  RNA polymerase and other transcription factors that jump on and get involved.  Okay, so if all we have to do is dimerize something, bring, grab two things together  on the cell surface, then we chemists can probably do something like that.  And so one way to do this would be to identify a molecule that binds to one half of the dimer,  like this guy over here that binds to gyro B. And then simply form a pseudo-symmetric  dimer of it.  It's pseudo-symmetric because notice the stuff in pink is not exactly symmetric, so this  is not C2 symmetric.  You can't just flip it over like that.  But it doesn't really matter because as long as you get these guys close enough for the  two jack kinases to autophosphorylate each other, then the signal transduction takes  place and then the stat 5 gets phosphorylated and then it dimerizes and then it goes on  to transcription.  Okay, so again, all we have to do is identify a binder to one of these receptors and then  by simply forming that monomer into a dimer, boom, we get something that could turn on  signal transduction.  And anyone have an idea why you might want to turn on EPO or why you might want to provide  erythropoietin to patients, for example?  What circumstances would you want red blood cells being produced?  After surgery.  Yeah, so after surgery, the patient is low on blood, lost a lot of blood, blood all over  the place.  So you routinely give EPO as a way of stimulating blood cell production, red blood cell production.  Of course, this is wildly abused by athletes who also value red blood cells for their oxygen  carrying capacity.  And up until recently, there were no effective tests for erythropoietin abuse.  They're now really good tests.  And this is one of the reasons why you're seeing all these scandals come out as athletes  are getting caught.  In fact, there was one in the news in the last week where athletes that had competed  eight years ago were caught because their samples were still refrigerated and you can  actually detect the artificial EPO in those samples.  So from eight years ago.  And I think that's going to happen more and more.  That we chemists are going to get better and better at analyzing samples and finding tiny  little quantities of unnatural compounds and kneeling athletes who are abusing them.  And I think this is a good trend because as a sports watcher, I want to watch real athletics  and not chemical athletics.  So a little off the topic.  So you now know that you can readily make a chemical version of EPO using a strategy  like this.  So all right.  Now another strategy would be a lot cruder.  So the thing about this that's lovely is that this compound over here is specific just for  gyrobeat.  But a much more crude way to do this would be to simply get in and form a covalent dimer.  This is a non-covalent dimer and it has some chance to be specific.  But a much less specific way to do this would be to simply add a chemical cross-linker.  And I'm showing you this because it illustrates a really important reaction and one that's  used quite a bit in chemical biology and biochemistry labs.  So in this reaction, the interferon beta receptor 2, there are going to be lysines.  It's almost unavoidable to have lysines on the surface of proteins.  Lysine is this amino acid side chain that's frequently charged, typically positively charged,  and it's very solubilizing.  So the outer surfaces of proteins are almost always studded with lysine residues.  And so for this reason, you can react these lysine amine side chains, shown here in their  neutral state, with a activated carboxylate.  So this is like a carboxylate, except instead of an OH here, instead of an O minus, if it's  carboxylate or OH, if it's carboxylic acid, instead this has an N-hydroxy-sacidimide ester.  And this NHS ester is a really good leaving group.  And so what that means then is that this could very readily form an amide bond and give you  a covalent bond.  This is completely analogous to the O-acyl-isoyuria activated carboxylic acid that we saw when  we talked about amide bond formation by DCC many weeks ago.  Four weeks ago or something like that, when we talked about how to make amide bonds, we  talked about how you have to activate the carboxylic acid.  In this case, we're starting with something that's already activated as an NHS ester.  And what's extraordinary about this reactivity is that it reacts specifically with amines  and not all of those OH groups that are present at 55 molar concentration in water.  Water is a whole bunch of OH groups.  And so this kind of chemical specificity or chemo specificity allows this to pick out  these amine functionalities.  And then if there are two amines from neighboring interferon beta receptors, these two amines  can be dragged together to form a covalent bond where they're linked together covalently.  And again, once you've covalently cross-linked things, you've brought these guys together  and you can start signaling through the jack stat pathway.  Now, I want to take a moment to show you how this is useful, how this reaction is useful.  This thing, not so useful.  Yeah, you can do this experiment.  It will work.  But honestly, you're not going to be giving your patients this compound as a pharmaceutical  because it will react with every other lysine that it finds.  It has zero specificity, zip.  And who knows what kind of allergic reaction the patient will come down with if you inject  that into them.  But a much better way to do this, or a much more useful aspect of this reaction, is to  use it to modify proteins to improve their solubility.  So for example, interferon alpha 2B, this compound here, it's a protein therapeutic,  is typically peggulated, meaning there is this polyethylene glycan, polyethylene glycol  tail that's appended to the protein.  And so the way this reaction works is you start with an NHS ester of pegg.  Okay, so again, this is polyethylene glycol.  This is a polymer that you've seen before, maybe not in this class, but certainly in  sophomore organic chemistry.  And because it has an activated carboxylate, it will come along and modify all these lysines  and even these histidine residues.  And what's remarkable about this is if you control the pH and the buffer conditions,  you could set this up so that there is only one position in this complicated protein that  actually gets modified.  And when we look closely at this, what we find is not the lysines at pH 6.5 that get  modified.  At pH 6.5, all these lysines are protonated.  And again, if they're protonated, if these guys are protonated, there's no way they can  react with the activated carboxylate.  Instead, at pH 6.5, the only thing that's available to react is one of these histidines.  I believe it's this one.  And so the pegg gets appended, gets conjugated specifically just to one of these histidines  over here and nowhere else in this complicated protein.  And that's really exquisite reactivity and selectivity.  And so for this reason, this is used very extensively.  And these types of things are used extensively in therapeutics, in protein therapeutics.  Okay, why would you want to do this?  Why would you, anyone want to put pegg on your protein?  It's a little bit of a trick question, but come on, you guys.  B.  You seem to be thinking about something.  You're debating something about like being, being crazy with solubility?  Yes.  All right, well done.  So what B suggested is that this pegg over here is going to be highly water soluble, right?  It's going to have a lot of hydrogen bond acceptors.  And so you could take a protein that would be relatively insoluble and make it more soluble.  In addition, you're increasing its molecular weight, and over here can be really big.  It can be hundreds to thousands.  And so you can take a relatively small protein that would get excreted very readily by the  kidneys, increase its molecular weight, and then it gets recycled back and prevented from  being excreted.  So there's some huge advantages to solubilization and even doing things like decreasing the  protein's antigenicity.  Okay?  So you're increasing how recognized it is by the immune system as being a foreign invader.  Okay?  All those are really important properties, and so this reaction is used very extensively.  Okay?  Any questions about this?  All right.  Notice that the HPLC over here indicates that you're getting one and only one protein  out.  If this was getting peggalite in a whole bunch of places, you would see lots and lots of  peaks.  Okay?  So the dominant peak here is number two.  And these guys are relatively small.  All right.  I'm going to move on.  I want to talk to you next about growth hormone and growth hormone pathways.  And earlier I showed you this idea of acromegaly, which is too much growth hormone leading  to giganticism, you know, excessively large hands, feet, and chin.  So this is our third example of a signaling pathway.  This is a classic.  This is used extensively by many different signaling ligands, including TGF, beta up here.  And in general, so this is very common, very typical.  This is a biggie.  In general, all of these work by the same mechanism.  The mechanism is dimerize the receptors up here and then autophosphorylate, meaning  this kinase over here phosphorylates the one next to it and then this guy phosphorylates  the one next to this, and then that leads to some sort of, and then that allows a kinase  to phosphorylate a nearby other protein that then goes on and eventually get down to the  DNA down here.  Okay?  But the important thing is dimerization, then autophosphorylation.  And we're just going to see that again and again and again.  Okay?  So here, for example, is growth factor receptor up here.  So sorry, this is, there was growth factor receptor in purple.  These are also called receptor tyrosine kinases because they're acting as kinases to phosphorylate  tyrosines on each other.  And then you end up with a whole series of different, you end up with a whole series  of different phosphorylated tyrosines.  This thing basically becomes loaded up with all these phosphoryltyrosines.  Okay.  Everyone stay with me on the self-surface stuff.  Dimerize autophosphorylate.  All right.  Here's where it gets complicated.  Once you get the dimerization and the phosphorylation, a total of four pathways are then activated.  This thing goes on and, for example, it engages the stat subpathway that we saw earlier when  we talked about the two-component signaling pathways.  It also gets into the map kinase subpathway, which I'll describe next to you.  But it also starts to affect PI3 kinase and PLC gamma pathways as well.  These are pathways associated with fatty acids, which we briefly alluded to earlier last,  or later on Thursday of last week when we looked at fatty acid synthesis and signaling  in the cell.  And I'm going to show you, I'll be able to pick up and show you exactly what's going  on when we talk about that kind of signaling.  All right.  Before we do, let's take a closer look.  This protein at the top is a growth factor, human growth hormone, or growth factor, it's  a ligand, and it's going to dimerize the receptor tyrosine kinase, the RTK at the cell surface.  And good news, we've already seen this protein.  This is a familiar protein to us.  This is human growth hormone over here.  And it's going to initiate this dimerization through having two binding sites.  And earlier in this quarter, we talked about, in fact, this is one of our very earliest  lectures, this might have been like the third week or something, we talked about one of  these binding sites and how it had a hot spot of binding energy for binding to the receptor  tyrosine kinase, human growth hormone binding protein on the cell surface.  There is a second binding site that looks completely different than this one on the  other side that allows the dimerization of the receptor tyrosine kinase to take place.  Okay.  So this is our model for receptor ligand interactions, which is why we've seen it before.  But you don't necessarily have to have large proteins doing this dimerization.  In fact, small molecules can be very potent at doing this.  And so, for example, this PD compound over here has a great specificity for particular  growth factor receptors on the cell surface.  And these numbers over here are IC50s and nanomolar.  And recall that smaller number indicates higher potency, right, because that means a lower  KD, which means greater affinity.  And notice that these compounds are selective for the FGF, the fetal growth factor receptors.  FGF are 1, 2, and 3 versus VEGF.  This is a vascular endothelial growth factor receptor, which stimulates angiogenesis, the  growth of blood vessels that we talked about earlier when we talked about proteases and  methionine aminopeptidase.  Okay, so this sort of specificity for one class of these receptors over other receptors  in the same class is prized.  And medicinal chemists will spend a lot of time trying to optimize the structure of a  compound like this one such that it will be specific for one of these receptors or maybe  for a whole class of these receptors, but then avoid, say, EGFR, endothelial growth factor  receptor, or something like SARC, which is really broad activity.  And what this means then is then you can have ways of targeting tumors selectively.  And what we're showing here are tumors treated, or tumors dissected out of an animal that  have been treated with the compound on the left versus controls.  And check out how much smaller these guys are.  So that's a good sign.  So if we had ways of targeting, blocking, inhibiting growth factor receptors, we'd have  really potent anti-cancer compounds.  And this is one of the frontiers in medicinal chemistry and chemical biology.  Many of these get their specificity through binding not to the ligand binding site that  was highlighted by this ligand over here where the ligand likes to bind.  Rather, they seem to prefer to bind to the ATP binding site.  And that actually seems to be a surprising area for gaining specificity.  You wouldn't expect this, right?  Because you'd expect every kinase that uses, all kinases use ATP.  That's their source of phosphate.  But you'd expect them all to bind ATP in the same sort of way.  But what we're finding through efforts of medicinal chemistry is that actually there's  enough variation in the ATP binding site that you can alter the structures of these compounds  and gain specificity for, say, FGFR versus all of these other receptors down here.  And so by what I mean by this, by bearing the structure would be, for example, changing  the structure of this urea functionality.  I'm showing you this with a T-butyl urea.  You might want to try a phenyl urea.  You might want to try, I don't know, a straight butyl urea, et cetera.  As you can imagine, making lots and lots of little changes over here, over here, over  here, and doing this to dial in the specificity for a particular receptor.  This is a property that's also known as selectivity.  Make sense?  All right.  Let's take a closer look then at phosphorylation.  Phosphorylation is not just random.  So these guys are going to be auto-phosphorylating each other.  You know, it's like Punch and Judy or something.  But they're going to do it in a very specific sequence of blows.  In other words, they're going to have a very defined pathway of one phosphorylation allows  the next phosphorylation, which allows the next phosphorylation.  And that's illustrated by these numbers over here, which detail this guy gets phosphorylated  first, this guy number two, second, three, four, and five, and so on and so forth.  And so the longer this dimer hangs around, the more likely it is to actually allow signaling  into those various pathways.  And remember, there's four pathways that are going to be implicated by this one receptor  up here.  So the receptor tyrosine kinase goes to town, but it doesn't start going wild randomly.  Instead it is very specific.  And in that specificity, it's going to be turning on specific pathways.  Let's take a look at one of these pathways.  This is the map kinase pathway.  This is one of the four that the protein has got a signal through.  So after this phosphorylation takes place, a protein with an SH2 domain called SHIC1  can grab onto this phosphate and then get phosphorylated itself, allowing grab2 to bind.  Grab2 also has an SH2 domain.  SH2 domains bind to phosphorylated tyrosine.  In fact, I think we saw them earlier in the quarter.  But grab2 has a polyprolene helix that can be bound by an SH3 domain, allowing binding  to RAS, allowing binding to RAF.  And then in the end, MEK can phosphorylate ERC, which then gets into the nucleus.  And remember earlier, I told you how there was alternative nomenclature.  So MEK can also be called map kinase kinase.  And then if phosphorylates map kinase, I've even seen map kinase kinase kinase.  So there's a bunch of other kinases in this pathway that I'm sort of not showing you.  And I'm also simplifying the nomenclature.  I'm using the simplest nomenclature as well.  Okay.  The important part here is that there's kind of a handoff of signal that involves not just  phosphorylation, but interaction through an SH2 domain.  And we've seen that earlier in the quarter.  Earlier in the quarter, I showed you how polyprolene sequences will bind into SH3 domains.  In fact, here's the structure that I showed you.  Here's an SH3 domain in green.  Here's the polyprolene helix.  And remember, this is the helix.  This is the 310 helix.  That if you look down this axis, do you see how it looks triangular?  It looks triangular here.  This is what the polyprolene looks like.  So this is a familiar concept to us.  This is something that we've seen before.  We've also seen SH2 domains as well.  So grab2 has an SH2 domain to bind to the phosphotyrosine on SH1, but it also has an SH3 domain that  binds to sun of 7 less, sauce over here.  And then sauce binds to RAS, which binds to RAP, et cetera.  Okay.  So everyone's still with me.  All right.  There's one other element that this pathway illustrates that's also prototypical.  This is one of those things that you see quite often.  On the cell surface, the cell surface is not a sterile, neatly denuded environment.  Rather, the cell surface is complicated.  It has a lot of shrubbery on it.  And in fact, amongst the shrubbery is long chains of this stuff called heparin sulfate.  This can then attract FGF.  It can track the ligands that these growth factor receptors like to bind and increase  their, enhance their apparent affinity for binding to the receptor simply by a proximity  effect.  Okay.  So the shrubbery up here, the heparin sulfate, grabs on to any stuff that happens to be passing  by.  And then because now it's localized up close to the receptor, the receptor can then more  likely find, more easily find it.  And that in turn enhances the signaling abilities.  Sound good?  Okay.  So these are common modes.  We're seeing the common mode that I want you to learn is this idea that heparin is out  here to enhance binding.  Dimerization takes place.  Autophosphorylation takes place.  There's binding through SH2 and SH3 domains.  And then a relay of phosphorylation, phosphorylation, and then finally into the nucleus for transcription.  Okay.  It's like Yogi Berra said.  Baseball, it's just running, throwing, catching and hitting.  What could be more complicated than that?  Okay.  So there's a few elements here, but we're going to just, when you get into this area,  you just see these same elements repeated over and over again.  Learn these elements.  You'll be able to understand everything else.  Any questions so far?  We good?  All right.  Let's talk a little bit about the other pathways that these receptor tyrosine kinases  like to stimulate.  One of these, another pathway, are these pathways that are fatty acid signaling pathways.  These include phospholinosatol-3 kinase.  This is phospholinosatol-3.  There are kinases that phosphorylate this.  So PI3K, phospholinosatol-3 kinase.  I'll just call it PI3K.  It's the third hydroxy of this glycan.  So this glycan is appended to a fatty acid chain.  And upon phosphorylation, this gives you something, it gives you a different structure.  And we now have something that has three phosphate groups attached to this glycan.  And this then can allow binding.  This is called phospholinosatol-3 or PI3.  PI3 can then be bound by these PI3 binding sites.  The effect here, so this is a protein called protein kinase C or PKC.  PKC is floating around the cell.  It's happy as a bee.  It's banging into flowers, looking for whatever it does all day.  Until it finds PI3, once it finds PI3, it gets ensnared like a fish getting stuck onto  a fish hook.  At that point, PKC is stuck at the cell membrane.  And this localizes PKC at the cell membrane where it can start doing all kinds of things.  So again, this is that principle of localization.  Localization is what allows these things to then get turned on and then in turn affect  the pathways.  It's kind of like that old adage in real estate.  Location, location, location.  And so in the same way that heparin is confining these ligands to particular spots in the cell,  this PI3 is going to confine PKC to particular spots at the plasma membrane and that in turn  is going to allow the pathway to get kicked off.  So it's not simply activity.  It's also localization, localization, localization that gets things into the right spot where  they can do the most signaling.  Okay, it makes sense?  Okay, now this is a gristly simplified portrait.  When you start looking at this in more detail, you start finding that there are other ways  of shutting this off.  So for example, you can free up the glycan from the rest of the acyl tail by a hydrolase,  PLC gamma over here, and things get more complicated as well.  Okay.  In addition to the heparin sulfate that I've already told you about, there's also the various  IP3s and PIP3s.  There's also a curious role played by calcium and the release of calcium.  So IP3, the release of the IP3 from the PIP3 has the effect of releasing calcium into the  cell and this in turn unhinges a binding site to allow binding to stuff found here at the  plasma membrane.  Okay, so all of this stuff is working together.  Okay, so you have a really complicated dance where one of the major goals of the dance is  simply to get the players into the right space at the right time.  Okay, and calcium is great because it diffuses very readily through the cell and it works  really well for this.  Okay, any questions about receptor tyrosine kinases, growth hormone, et cetera?  Okay.  This is 90% of cell signaling really is what I just told you.  Okay?  I'm now going to go on and I'll talk to you about the other, oh, sorry, I didn't mean  to say 90%.  30%, 40% of cell signaling that you read about in science, nature, and cell deals with these  pathways.  I want to talk to you about another 30% or so.  This is a really important one.  G-protein couplin receptors are some of the most common proteins in the human proteome.  These really are extremely abundant to us humans and in fact these are the proteins  that make life worth living.  These are the things that allow us to see like rhodopsin over here, allow us to smell  like these olifactory things, allow us to taste.  Here's some tasty ones, et cetera.  All of these smell molecules, taste molecules work by binding to G-protein coupled receptors  which look like this.  This is a transmembrane protein where the membrane is going through here.  The membrane cuts through this over here and the ligand is very deeply buried.  It gets snuggled down deep into this GPCR.  Inside the GPCR it changes the confirmation of the GPCR such that the stuff in here that's  going to be signaling on the inside in the cytoplasm of the protein has a new confirmation.  So these bind to a very wide range of ligands.  In fact, many of these are promiscuous in their activity meaning they'll bind to a bunch  of tasty things.  Once that happens, that allows the conformational change that communicates from what's happening  outside the cell to the inside of the cell.  That's kind of at the very cell surface.  Let's take a closer look.  I have to tell you a little bit about how these work.  I first want to tell you about dynamic range.  I'm going to digress for a moment and then we're going to take a closer look at GPCR.  If for example you had some receptor that simply was turned on and off depending on if you  had binding by a ligand, you'd have a kind of a lousy sensor.  These have to act as sensors.  If you're going to be able to say sense light changing for moving towards light if you're  some organism or being able to smell stuff so that it determines whether or not you eat  this food that's going to kill you or not kill you or make it possible for you to live,  there's an incredible premium on being able to sense tiny little quantities and at the  same time being able to sense really big quantities.  And that's a property called dynamic range.  If the sensor simply operates on the principle of bound or unbound, your dynamic range is  going to be very limited.  Let me remind you that this principle of bound versus unbound is governed by an on rate and  an off rate that together have a dissociation constant associated with it.  The truth is this offers a limited dynamic range as epitomized by this percent activity  plot that we showed earlier in the class.  Recall all of these have this sigmoidal plot, the sigmoidal type of relationship between  concentration of ligand and activity on the y-axis.  The problem with this is that this type of receptor would be sensitive only in these  three or four orders of magnitude in concentrations from 10 to the minus seven to 10 to the minus  four.  So like submillimolar to submicromolar.  And that might be okay, but it's not so okay if you have to smell, I don't know, tiny quantities  of some pheromone to know whether or not you're ever going to find a mate in your lifetime.  That could be kind of important if you're an organism, right?  And so this business about sensing only in one particular range is pretty much worthless  for really good sensing and devising really good sensors.  So instead, all of the GPCRs rely on a different principle.  If I get to a different principle, does anyone have questions about this one?  Does it make sense to you that this is kind of limited in its utility?  Okay.  Always get nervous when there's no questions.  Take your word for it.  Instead the G protein coupled receptors signal through a G protein.  They all will bind directly to something called the G protein.  And the G protein is a catalyst that catalyzes hydrolysis of GTP, this guy up here, magnesium  GTP complex, to magnesium GDP.  So it's simply a hydrolysis of the gamma phosphate of GTP to give us GDP.  Okay.  Straightforward reaction.  A reaction we've seen before, an easy one, right?  An easy reaction we can derive in our sleep.  We can draw a mechanism in our sleep.  This is an important part.  By coupling binding to the activity of an enzyme, you can very sensitively tune the activity  and you can tune this sensor.  So it becomes a really exquisite sensor for low concentrations and high concentrations.  And I realize it's not clear at this moment.  I'm going to show you on the next slide.  Before I do, though, everyone with me have this idea.  GPCRs bind to G proteins.  G proteins are catalysts.  We're good with that one?  Okay.  So check this out.  Here's what's really going on.  So the G protein in its GDP bound state says signal off.  So GPCRs up here, if the G protein, however, is bound to GDP, signal gets shut off.  On the other hand, if it's bound to GTP, the signal gets turned on.  And what's important is that there are two intermediary enzymes that are going to catalyze  and help push this equilibrium between GDP bound and GTP bound.  These two are a guany nucleotide exchange factor, a GEP, that helps push out GDP to  allow binding by GTP.  And then in the opposite direction, a GTPase activating protein or GAP that helps drive  the phosphorylation of GTP to GDP.  Okay.  Now here's the way this works.  Your cells can carefully control the ratio of GEP to GAP or GEP to GEP.  And by doing that, they can make the signal, the sensor, more sensitive or less sensitive.  Okay, and if you want it to be more sensitive, you basically set it up so it's always signal  off, right?  So then you'd have lots of gaps around.  The gaps keep it in this off state.  So if anything turns it on over here, then boom, everything goes on.  On the other hand, if you want it to be less sensitive, you keep it in the signal on state,  so it really takes quite a bit to get it shut off again.  Makes sense?  Okay, this is a really powerful principle.  And it's one that I really have not seen in too many areas of electronics or chemistry.  And it's one that I think really could be exploited for interesting uses.  B?  There is only water present in cells, so how can you actually turn it off?  Okay, so you mean how does this happen?  Actually I think it's only happened because there is only water inside the system.  It doesn't have water.  All of it is taking place in water.  It's very close to 55 molar water.  But the GTP over here bound by the G protein is getting hydrolyzed at a very slow rate.  The catalyst here is not such a great catalyst.  And GTP in water is completely stable.  It's not going to get hydrolyzed.  It needs an enzyme to hydrolyze it on the time scale that's relevant for biology.  Okay, any question over here, Sergio?  Sure.  Okay, so this is happening right up close to the plasma membrane, right up close to  the cell surface.  Okay, so the GPCR is right there at the cell surface.  The G protein is right below it, grabbing on, holding on for dear life.  And then these gaps and gaps are forming another piece of the sandwich.  So in the end you get these monster complexes.  Okay, yeah, and over here.  So instead of having that system, you just have ligand and receptor and then just the  concentration of ligand.  And that's where you could have the dynamic range.  You want to get the same dynamic range.  So if you adjust the concentration of ligand down here, you're only going over four or  some magnitude of dynamic range.  But if you have this gap and gap system, then you can tune this to kind of shove this over  so that it's now responsive to ligand over here or ligand over here.  This gives you a responsiveness over all of these, this dynamic range.  Okay, and this is what you need.  Let me explain.  Okay, so this is the same set of receptors that's going to allow you to see.  And there's times when you need to be able to see in dark caves to be able to survive.  Maybe not you, but certainly your primeval ancestors, right?  They would need to be able to survive in dark caves where humans can sense one photon.  We can sense just a few photons.  That kind of low light sensitivity, incredibly important.  On the other hand, eventually you get out of the cave and you're wandering around in  the bright sunlight.  And if all you can see are, if you could see each photon individually, you're going to  get totally overwhelmed and you're not going to be able to see anything, right?  So you need a system that allows you to be able to sense things way down here, but then  also be able to shut off so that it doesn't get overwhelmed way up here.  Okay, and that's really the key to this activity.  So affecting the gap and gap ratio controls that sensitivity.  Okay.  Question in the middle?  Yeah?  Is that why our eyes are pupils dialling?  That's also part of it as well.  Okay, but you know, the rhodopsin that's sensing photons, that has to have some way of being  less sensitive at times and more sensitive at other times.  But certainly, yes, you're right.  Dilation of the pupils will have an effect as well.  And over here?  Is there any way that we can modify the ligand to be more sensitive?  Okay, but if you modify the ligand and you make it more sensitive, let's say you're  doing taste or something, you'd make it more sweet, right?  But eventually, you know, you're over here.  So you're going to be like hammering on it.  So now it binds really well over here.  Eventually it's just going to get overwhelmed.  It gets saturated.  Okay?  Right?  And so everything tastes sweet.  That's not so pleasant either.  Okay.  So what happens after the G proteins?  So we're up here.  We have binding to the G protein coupled receptors.  Here are the G proteins.  These have three subunits, an alpha, beta, and gamma subunit.  Alpha is the alpha that's going to be doing the GTPase activity that I talked about.  This is then going to lead to denolate cyclase, this protein over here, which turns over ATP  to give cyclic AMP.  What's neat about this is that all of these G protein coupled receptors converge on this  at denolate cyclase.  All is a little bit too strong, but they're largely going to converge over here and a  denolate cyclase acts as an integrator of signals such as you can get signals from a  couple of different cell surface receptors turning on a denolate cyclase, which in turn  the cyclic AMP turns on protein kinase A. Protein kinase A we've seen before.  We've talked about this protein.  This is the protein that does the waltz as it phosphorylates different proteins in the  cell.  We looked at a single molecule level earlier in this class and it's a good friend of everyone  who's taking Chem128.  PKA allows CREB, this transcription factor, to turn on genes, for example, to turn on  genes to do something about cardiac myocyte hypertrophy.  It turns out that the molecules that bind to these GPCRs are extremely diverse.  In fact, we humans have learned to revel in this chemical diversity.  For example, these are the molecules associated with the smell of peanuts.  You know that nice, roasty smell of peanuts?  It's actually not one molecule.  Rather, it's a plethora, a little tiny combinatorial library of these molecules over here that  are present at these different concentrations that have different odor thresholds and different  odor activity values.  The odorant receptors, which are G-protein coupled receptors, are going to bind to each  of these and then, in turn, these are going to map to specific neurons.  So lots and lots of different compounds, each one with their own characteristics smell,  but the sort of symphony that we associate with the roasty smell of peanuts is a bunch  of these present at different concentrations.  We need receptors that are going to respond to each of these different molecules and do  it in a way that then integrates the signal and gives us something that reminds us of  roasty peanuts.  Here's the way this works.  These are the smell receptors, the olfactory receptors in the nose.  Here is the odorant over here.  Odoring can bind to several different receptors.  So here's the odorant.  It's going to bind weakly to this guy, strongly to this guy, not at all to this guy and maybe  to this guy over here.  All of the olfactory cells that have this receptor on their surface all signal back through the  same neuron.  Okay, so if we map the cells over here in this olfactory region, they're all going to  go back to one and only one neuron, all the ones that have the purple GPCR on their surface.  All the ones that have the green GPCR go back to a different neuron.  This is an astonishing result that came up, was discovered in the last two decades or  so.  But in short, what this means is that everything that binds to this receptor signals in the  same way.  You get the same neurons firing.  In the end, using that gap to geth ratio that I discussed with you, this can allow signaling  that tells the neurons whether or not you have a strong binder or a weak binder.  Over here, you have a strong binder.  You get more signal out.  Over here, you have a weaker binder, so you have a weaker signal coming back out.  And again, you can control the dynamic range such that you don't get totally overwhelmed  if you go into a peanut factory so that you're smelling peanuts for the next week or something  like that.  All right, let's talk about vision.  I mentioned this earlier.  This is one of those amazing chemical reactions that all of us are reliant upon.  We humans are especially reliant upon vision as our primary sensing organ.  And what's remarkable about this is the goal here is to capture a photon.  Your expectation is you can't bind to a photon.  How do you capture a photon?  So instead, the GPCR, associated with vision, called Rodopsin, covalently links to a cofactor  called retinol.  So this is the structure of retinol.  It forms a shift base with a lysine in the GPCR.  This is normally found in a cis-olefin form.  Okay, so this 11-12 carbon-carbon double bond, cis.  Upon being irradiated by a photon, that's H nu, the cis-olefin isomerizes to give a  trans-olefin.  And the net effect here is a flipping of the retinol.  Okay, so we go from cis to trans, and this whole cyclohexane has flipped from being over  here to being now extended.  That has the fact of changing the confirmation of the GPCR and allowing you to see.  This is how you are seeing me right now.  This is how it really works.  Okay, now in recent years, chemists have started to get creative, and we've been devising retinols  that are responsive to different wavelengths.  And some of these might be really interesting for allowing us to see at different wavelengths.  For example, to be able to see in the infrared, to see in dark, and so on and so forth.  You can imagine a lot of interest in this by people who like to see in the dark.  All right, let's take a closer look at the structure of rhodopsin.  So again, it's a GPCR.  Notice that its structure looks very similar to the GPCR I showed on the first slide.  All of these GPCRs have a common structural motif consisting of seven alpha helices that  circumvent the membrane, that are transmembrane.  Here in purple is the retinol, again forming a shift base at one end to a lysine residue  on this alpha helix.  And the protein undergoes a dramatic confirmation change in response to that cis trans isomerization.  Cis trans isomerization is huge.  Up here, this little change up here, photon comes flashing in over here, boom.  You get isomerization and boom, the whole protein changes confirmation, changing, affecting  its binding to the G protein and turns signaling through that G protein cascade that we talked  about earlier and turn leading to stimulation of neurons, which again is what you're doing  when you see.  Okay, that's kind of extraordinary.  All right, any questions about GPCRs?  Moving on.  Oh, Carl.  Yeah, so it's constantly flipping back and forth between cis and trans, and that's what  you're interpreting as vision.  All right, ion signaling, so this is number five in our discussion.  So these arrows are going the wrong way.  Potassium is constantly being pumped out of the cell, calcium is being controlled carefully,  chloride is being pumped in and out of the cell, potassium is constantly being pumped  in and out of the cell.  The cell maintains an equilibrium of specific concentrations of its ions.  Those ions provide a way of signaling very quickly in the cell.  In fact, most of the cells' fastest responses, such as vision and stuff like that, are probably  going through ion channels.  So the way this works is that these ion channels can open up pores that allow ions to flow in  and this leads to an electrical signal that can propagate along a neuron, for example.  So it's carefully controlled through transporters and it provides really fast response.  This is the kind of response where if I suddenly make a loud noise, then you jump or something  like that, that's the ion response.  I'm hearing that, ion channel response, seeing ion channel response.  So here's what these channels look like.  This is a calcium-activated potassium channel that allows potassium to flow in.  Calcium binds down here and changes the conformation and then allows potassium to flow out of the  cell.  You can imagine that because these channels allow really fast response, such as a muscle  response, toxins would very potently target these types of channels.  And caribatoxin is one in a large number of different toxins that target these types of  channels.  Many of these toxins cause paralysis.  These are things that are used by scorpions and cobras, et cetera.  All right, that's all I have to say about ion channels.  They're fascinating.  Ions flow in, they flow out.  I want to move on.  Death receptors.  So this is a class of compounds that binds to the cell surface and instead of causing  dimerization, they cause trimerization.  So you get this active trimer.  In fact, dimerization is associated with shutting off the pathway.  And so, for example, this compound over here allows, encourages formation of an inactive  dimer that shuts off the pathway.  But I'm getting a little ahead of myself.  Here's what it looks like.  Lygand is up here.  Lygand is a trimer.  Yand binding, the receptor forms a trimer.  And then you get a series of kinases, I-Capa, you get a series of kinases passing off the  phosphorylated response.  And then in the end, NF-Capa B gets into the cell and causes transcription and inflammation.  This is a highly regulated process that's used extensively in the immune system.  This includes things like TNF.  This includes interferon, et cetera.  These are really important pathways for turning on inflammation, turning on the immune system.  Okay.  Last pathway, number seven, defusible gas molecules.  It turns out that we humans are susceptible to signaling by tiny little gas molecules,  things like oxygen.  In fact, all life is going to be very dependent upon being able to sense oxygen.  You can imagine that if you cannot sense low oxygen conditions, you're going to die, right?  Because you need to be able to learn to move towards areas where you can breathe, right?  That instinct to breathe when you're underwater is fundamental to life.  And it's really fundamental to all organisms that depend on oxygen.  And so the way this works is oxygen sensing by the cell works by taking advantage of the  chemical properties of oxygen as an oxidant.  So there's a concentration of Hif1 alpha in the cell.  This Hif1 alpha is constantly being synthesized and constantly being degraded.  And this Hif1 alpha upon encountering oxygen oxidizes, gets oxidized to introduce an OH  group that in turn allows it to be degraded.  And that in turn will prevent these transcription pathways from taking place.  So when Hif1 alpha is present, nuclear, this gets in, causes transcription, which in turn  turns on anaerobic respiration, right?  That tells the cell, we don't have a lot of oxygen present.  We better take into gear the anaerobic respiration aspects of metabolism.  On the other hand, if oxygen is present, that oxidation introduces a hydroxyl that in turn  leads to degradation of Hif1 alpha.  So the key concept here is that you have this concentration of a Hif1 alpha that's constantly  being formed, constantly being degraded, and that gives you a very rapid sense of how much  oxygen is present.  It's kind of like this Hif1 alpha is like a thermostat for oxygen levels.  It's constantly monitoring how much oxygen the cells have.  If the cell doesn't have enough oxygen, it's going to die if it doesn't switch on these  anaerobic respiration pathways.  Nitric oxide is also another very common cell signaling molecule.  And this one works by binding to iron-porphorin cofactors of guanylate cyclase and in turn  causing blood vessels to relax.  So this is important for the regulation of blood pressure.  And a number of compounds were invented to affect this pathway.  And what was astonishing is actually they weren't compounds like Viagra were found to  be less effective as blood pressure regulators and more interesting for their side effects.  And here are their structures over here.  These are structures of Cialis and Viagra compounds in the little blue pill.  Here's the way this works.  Okay, so this is nitric oxide over here.  It's going to bind to a guanylate cyclase and in turn that's going to affect GTP being  converted to cyclic GMP.  These compounds inhibit guanylate cyclase and in turn that causes blood vessel relaxation.  This is one pathway that's very dependent upon calcium concentration, et cetera.  Oh, sorry.  Actually, sorry.  The compounds are going to be targeting PDE5, this compound over here, which is affected  by cyclic GMP levels.  Okay.  Any questions about any of the seven signal transduction pathways?  All right.  In that case, that brings us to my wrap-up and conclusion.  What I've been talking to you about all quarter is a new way of thinking about biology, thinking  about it at the level of atoms and bonds where we can actually make changes, where we can  think creatively.  And I hope, for example, the proposal assignment has shown you what it's like to be at the  frontiers of this really exciting field.  So these are processes that you've probably seen in other classes, certainly DNA, DNA  base pairing, et cetera, but I hope by thinking about these in terms of chemicals, in terms  of being compounds that have atoms and bonds, we've introduced into your thinking new ways  of thinking about their reactivity.  For example, forming cross-links, depurination, et cetera.  Chemical biology lets us start to address who are we.  It really gets us to answer really big questions in biology.  And all of the structures that I'm showing up here are things that we've seen this quarter,  either we've discussed in lecture or they're in the textbook.  These include things like a conantoxin that targets ion channels and causes paralysis.  So these are organisms on the planet, and we've seen lots and lots of their bioactivities  in this class.  It also lets us address where do we come from?  When we talked about prebiotic synthesis, it was very abstract.  We're using it as an example of error pushing, but chemical biology gives us a foundation  to start to address really philosophical questions, things that humans have been grappling with  for a very long time.  And finally, we get to address them really at the level of atoms and bonds.  And then chemical biology gives us the control over our destiny.  It gives us the tools that we need to devise compounds that allow us to overcome our being  human, that allow us to address things like disease, starvation, nutritional deficit,  et cetera.  And we've been talking about this all quarter.  Here for example is the PEG intron molecule that I showed earlier in today's lecture.  This is the PEG, this big blob next to it, and then that's the protein that I showed  earlier.  These are really important molecules.  These are molecules that affect the world and that allow us to address diseases.  And in the end, chemical biology ends up changing society.  The compounds we talk about have a big impact on our lives.  They have an impact that goes beyond just medical health.  They have impacts on, for example, affecting athletes, for example.  So I want to encourage you to join us.  Join us at this frontier of chemical biology.  I hope to see you in the laboratories here at UC Irvine.  And I'm really looking forward to reading your proposals.  So I want to thank Natalie for being our projectionist, for doing all the videotaping  all the time.  And I have to thank Prithika up here and Miriam down here for being really outstanding TA's.  And I want to thank all of you for a really fun quarter.  I've really enjoyed teaching you, and if you're joining us on YouTube, I hope to see some  comments from you on the class and whether you enjoyed it as well.  But I've really enjoyed teaching this class, and so I thank you for a really great quarter.  Thanks a lot, everyone.  Thank you.  개편지 writings, anne legalencia, tactical policy, maarie lacので하는  사랑 어머hhwhistleffenf
Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_0_119.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_120_323.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_324_467.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_468_779.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_780_2099.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_2100_5051.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_5052_5351.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_5352_8063.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_8064_12863.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_12864_14207.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_14208_14255.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_14256_14351.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_14352_15455.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_15456_16991.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_16992_17315.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_17316_17531.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_17532_17903.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_17904_19775.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_19776_20963.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_20964_22463.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_22464_22727.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_22728_27983.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_27984_29111.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_29112_32687.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_32688_36263.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_36264_38543.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_38544_39107.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_39108_42599.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_42600_43055.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_43056_43487.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_43488_48503.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_48504_51575.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_51576_52943.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_52944_53999.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_54000_54563.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_54564_56135.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_56136_56507.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_56508_57719.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_57720_57935.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_57936_59867.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_59868_64355.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_64356_65147.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_65148_65603.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_65604_65771.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_65772_65831.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_65832_69755.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_69756_70259.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_70260_71963.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_71964_72599.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_72600_72659.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_72660_76619.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_76620_77483.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_77484_81047.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_81048_83375.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_83376_84047.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_84048_89075.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_89076_91907.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_91908_94739.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_94740_94991.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_94992_103439.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_103440_104135.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_104136_104831.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_104832_106619.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_106620_107051.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_107052_107099.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_107100_107219.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_107220_108167.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_108168_108239.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_108240_108287.png Introduction_to_Chemical_Biology_128__Lecture_18__Terpenes_and_Cell_Signaling__Part_2_108288_108430.png