transcript advances in g protein-coupled receptor ......sean sanders: hello to our viewers and...
TRANSCRIPT
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Advances in G Protein‐Coupled Receptor Research [0:00:00] Sean Sanders: Hello to our viewers and welcome to today’s Science/AAAS live webinar
entitled “Advances in G Protein‐Coupled Receptor Research.” My name is Sean Sanders and I’m the commercial editor at Science Magazine.
Slide 1 Membrane bound G protein‐coupled receptors or GPCRs play an
integral role in sensing the external environment of the cell. A broad range of external stimuli signal through GPCRs including neurotransmitters, hormones, odor molecules, cations, and even photons. Their position at the apex of essential signal transduction pathways means that malfunctioning of these molecules frequently leads to disease, making them a perfect target for drug therapies. Further, naturally occurring and induced mutations in GPCRs can provide valuable information about these signaling pathways and their role in human pathologies.
For today’s webinar, I’m very pleased to have with us a truly
exceptional panel of scientists and thought leaders who we trust will give us valuable insight into GPCR proteins and their role in signaling and disease, as well as share their experience of the techniques and technologies available to study GPCRs.
In the studio today, we have sitting to my left Dr. Jeffrey Conn from
Vanderbilt University Medical Center in Nashville, Tennessee; next is Dr. Michel Bouvier from the Institute for Research in Immunology and Cancer at Montreal University; and at the far end of the table is Dr. Brian Kobilka from Stanford University School of Medicine in California.
Before we begin our first presentation, I wanted to remind you all that
if you wish to see an enlarged version of any of the slides presented today, you can simply click the enlarge slides button located just underneath the slide window of your web console. If you like, you can also download a PDF copy of all the slides using the download slides button. Please be aware that the file is quite large so it might take a few minutes to download.
To submit a question live to our panel or to a particular speaker, just
type it into the ask‐a‐question box on the bottom left of your viewing console and click submit. I’ll do my best to pose as many questions as
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possible to the panel. Keeping them short and to the point will give you the best chance of having them answered.
Finally, I’d like to thank Roche Diagnostics and their xCELLigence System
who are kind sponsors for today’s webinar. Okay, I think we’re ready to begin. Slide 2 So our first speaker today is Dr. Jeffrey Conn. Dr. Conn received his
Ph.D. in Pharmacology from Vanderbilt University in 1986 and pursued postdoctoral studies in the Department of Pharmacology at Yale University. He then joined the faculty of the Department of Pharmacology at Emory University and in 2000 assumed the position of Senior Director and Head of the Department of Neuroscience at Merck and Company’s site in West Point, Pennsylvania.
Dr. Conn returned to Vanderbilt University in 2003 to start a new
program in drug discovery, with a primary mission of facilitating translation of recent advances in basic science to novel therapeutics. He is currently the Lee E. Limbird Professor of Pharmacology and Director of the Vanderbilt Program in Drug Discovery. Dr. Conn is Editor‐in‐Chief of Molecular Pharmacology, Regional Editor for North America of Current Neuropharmacology, and also serves on the editorial board of six other international journals.
Dr. Conn has received numerous awards and honors and was named an
ISI Most‐Cited Scientists in Pharmacology & Toxicology. Dr. Conn’s current research is focused on the development of novel treatment strategies for schizophrenia, Parkinson’s disease, and other brain disorders.
Welcome, Dr. Conn. Dr. Jeffrey Conn: Thank you, Sean. Slide 3 So before I begin to discuss some of the more recent work that we have
pursued in our lab, I will focus on work that we have done in recent years and I will focus on just a brief overview of the GPCR field.
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So thinking about the GPCRs, the GPCRs are positioned in a setting where they have an extracellular domain and an intracellular domain. The extracellular domain interacts with extracellular stimuli such as hormones and neurotransmitters and then activates to proteins in the intracellular environment that have a wide variety of effects on cell function. So this is the largest class of receptors, cell‐surface receptors. There are three major subclasses within the GPCR super family. They play critical roles in virtually every major organ system and are activated by a wide range of ligands including ions, odorants, neurotransmitters, hormones as I mentioned, and photons of light, so quite a diverse array of ligands that activate these receptors, and coupled to a broad range of effector systems and second messenger pathways that we’ll hear more about.
[0:05:04] One of the important things about the G protein‐coupled receptors is
that they are involved in multiple human disease states and are the targets of approximately 50% of all modern drugs. To they are very critical for novel therapeutics and thinking about new therapeutic strategies for different human disorders.
While we understand the GPCRs and have ligands for many of these
receptors, there are still many receptors in this family for which we do not have ligands. Approximately 1000 genes exist for G protein‐coupled receptors, many of which we do not have ligands to either activate or inhibit.
Slide 4 So when we think about some of the major advances that have
occurred in GPCR research in recent years, we’ll hear about many of those today, there have been major advances in our understanding of this function of GPCRs and how that relates to the structure, so new insights into the structure of GPCRs and their associated functions of representing a major breakthrough and increasing understanding of hetero‐ and homodimerization and oligomerization, how these may impact GPCR function, increased understanding of complex signaling pathways and this includes large networks of signaling pathways as well as G protein‐independent signaling mechanisms. And then what I will talk to you about today are studies that we have performed and others in recent years, focused on novel approaches to regulate G protein‐coupled receptors with allosteric modulators that provide in many cases an unprecedented cell activity for specific GPCR subtypes.
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Slide 5 So the work that I will talk to you about began with an interest that we
had in developing new therapeutic strategies for treatment of schizophrenia, and we had identified a specific G protein‐coupled receptor termed mGluR5, metabotropic glutamate receptor 5, which is a receptor for the major excitatory transmitter in the brain, glutamate, and this is just a cartoon illustrating the position of mGluR5 at the glutamatergic synapse. And without going into all of the biology that led to the hypothesis that agonists of this receptor might have antipsychotic activity, I’ll just focus on the strategies that we have taken to try to develop these compounds.
So when we began these studies, mGluR5 was similar to many other
receptors in this class of GPCRs in that there were no known highly selective agonists or antagonists for this specific receptor subtype.
Slide 6 However, when individuals began to focus on developing highly
selective tools, the first breakthroughs that occurred were developing selective antagonists, and what was unique about the antagonists of this receptor is that they did not act at the orthosteric glutamate binding site, but instead acted in the 7‐transmembrane‐spanning domain of the receptor to inhibit receptor function, so stabilizing a conformation of the receptor that prevented coupling to G proteins.
In our search for selective activators of the receptor, we began to
reason that if it’s possible to develop allosteric antagonists of the receptor, it may also be possible to develop compounds that interact at a site on the receptor removed from the glutamate binding site, the orthosteric agonist binding site that shift the receptor or stabilize the receptor in a conformation that favors coupling to G proteins and therefore act as allosteric activators.
Slide 7 So if we look at this slide, what we’re looking for is a compound that
has no effect on its own. So this is just a schematized representation on the right of this slide of the type of response we may look for in which we add a test compound at the beginning of the trace, then two minutes later we add an EC20 concentration of glutamate to slightly activate the receptor. And we’re looking for a compound that has no
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effect by itself so it’s not an agonist but will potentiate the response to the EC20 glutamate concentration. So once again in this schematized illustration, you’ll see what we are looking for in a potentiator that would enhance this response to the EC20 glutamate addition.
So in looking for these compounds, there were no such compounds
that were known. So really, the only way to get a foot in the door was to perform random screens of small molecule libraries, and we did that using a calcium fluorescence assay. These receptors lead to an increase in intracellular calcium. And on the left panel of this slide shows the assay that we used, and again, we’re adding our test compound at the beginning of the trace. Several minutes later, we add an EC20 concentration of glutamate. We’re looking for a compound that has no effect on its own but potentiates the response to glutamate, and you can see the response to one of the first allosteric potentiators that we identified shown here, which has exactly that profile.
[0:10:03] One of the biggest breakthroughs for us was discovery of the
compound shown, CDPPB, which is a robust allosteric potentiator of mGluR5, and you see on the right the concentration response curve for glutamate at activating the receptor in the absence of CDPPB and in the presence of increasing concentrations of CDPPB. And CDPPB induces a robust shift to the left of the glutamate concentration response curve and has no effect on the maximal response.
This was a breakthrough relative to previous allosteric potentiators we
had identified in that it was the first compound that had properties that would make it useful for studies in native systems. We’ve found that it’s highly selective for mGluR5 relative to other members of this receptor subclass. It potentiates mGluR5 in native systems when we drive glutamate synapses so it potentiates responses to endogenous glutamate that is released from presynaptic terminals, and most importantly, when we took this compound to animal models that predict antipsychotic activity, we’ve found that it did have antipsychotic‐like effects in these animal models. So a major breakthrough in achieving a high level of selectivity, taking a hypothesis that was somewhat speculative from a cell biology perspective, and now being able to test that hypothesis more rigorously in animal models.
Slide 8
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Another receptor that we’re interested in is especially of interest to many in the field in that the glutamate receptors, metabotropic glutamate receptors or class C GPCRs, whereas another receptor that we have studied in this vein is a class A GPCR, a muscarinic acetylcholine receptor. And what is of interest here is that the family A GPCRs have a somewhat different structure than the class C in that they do not have this large extracellular domain, but the natural ligand, acetylcholine, interacts with the receptor in the 7‐transmembrane‐spanning domain. So while previous studies had established that it’s possible to develop allosteric modulators of these receptors, we and others have had questions about whether the class A GPCRs would be as a minimal to robust allosteric potentiation that might be used for in vivo studies.
So again, we performed the same types of experiments in looking for
allosteric potentiators of these receptors, and this was also in the context of a schizophrenia program, looking specifically for M1 and M4 allosteric potentiators.
Slide 9 This shows one of the allosteric potentiators of M4 that we’ve recently
reported, a compound called VU10010, and this is a very robust, highly selective allosteric potentiator of M4. So shown in the left panel of this slide, you see a dose response curve for acetylcholine alone, and then in the presence of VU10010, we see a 50‐fold shift to the left of the acetylcholine concentration response curve, very robust potentiation, and most notably, very high selectivity for M4 relative to any other muscarinic receptor subtype with M1, 2, 3, and 5 shown on the right. So this is an unprecedented selectivity for a class of receptors for which that level of selectivity has been largely intractable based on traditional orthosteric ligands.
We understand a lot about this compound and compounds that have
come from this initial compound from studies at a molecular pharmacology level have found that this compound increases affinity of acetylcholine for the orthosteric site but also increases efficiency of coupling of these receptors to G proteins.
Also, we’ve found that it selectively potentiates M4 responses in native
systems and we have optimized compounds in this series and now performed in vivo studies where we find that they also have activity in some animal models that might predict antipsychotic‐like activity.
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Slide 10 Now, a question that has come up in this vein in developing allosteric
modulators is that they may interact with G protein‐coupled receptors in a promiscuous way in which selectivity across a broad range of receptors may be difficult to achieve. From what I just showed you, these compounds are highly selective for the individual GPCR in their sub‐family, but looking across a broader range of GPCRs, for the M4 potentiator, we have looked at a panel of GPCRs from the Millipore GPCR profile screen, and with the panel of the 15 closest relatives to the muscarinic receptors, we have found that only M4 is potentiated. So this is very encouraging and suggesting that there is a possibility of developing very highly selective compounds that interact with these allosteric sites to have their activity.
[0:15:00] Slide 11 So in thinking about this field, and I’ve just touched on a couple of
highlights of some of the studies that we have performed in recent years with allosteric potentiators, but it’s part of a much broader field and we’ve learned a lot about these allosteric site ligands. We now have allosteric site ligands for multiple GPCRs that include family A, B, and C across the field with different investigators. Different allosteric modulators can act at distinct sites on the same GPCR to have allosteric regulator activity. As I’ve mentioned, allosteric modulators can regulate both affinity of the orthosteric ligand and coupling to G proteins. And we’ve found that within a structural class, we can identify allosteric modulators to have a range of activities from antagonist to potentiator and include natural ligand or neutral ligands that interact with the allosteric site but have no detectible activity but block the effects of either allosteric potentiators or antagonists.
Also, novel modes of pharmacology have become possible such as
partial antagonists by targeting these allosteric sites, and something that’s very interesting and relates to the next presentation is that these allosteric modulators can differentially regulate coupling of a receptor to different signaling pathways.
And in each case in which we have been able to optimize compounds ‐‐
when I say “we” the larger field, not just our own studies ‐‐ to a point where we can go into in vivo studies, we see robust effects in in vivo animal models, and now, there are at least two allosteric modulators
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that are in the clinic and on the market for treatment of different disorders. So there have been major advances in this area.
Slide 12 So many people who are involved in this research. I won’t go into the
details, but this is just the individuals that are part of the Drug Discovery Program at Vanderbilt. Those highlighted in yellow have been especially involved in the work that I talked to you about today.
Sean Sanders: Great. Thank you very much, Dr. Conn. Slide 13 I think we’re going to move right on to our next speaker. Slide 14 Our second speaker today is Dr. Michel Bouvier. Following his Ph.D. in
Neurological Sciences at Montreal University in 1985, Dr Bouvier went on to complete a postdoctoral fellowship at Duke University in the United States. In 1989, he returned to Montreal as a scholar of the Medical Research Council of Canada and Assistant Professor of Biochemistry in the Faculty of Medicine of Montreal University. He was Chairman of the Biochemistry Department between 1997 and 2005 and was awarded the Hans‐Selye Chair in Cell and Molecular Biology in 1997 and the Canada Research Chair in Signal Transduction and Molecular Pharmacology in 2001.
He is now the director of the University Research Group on Drug
Discovery and professor of Biochemistry at the Institute for Research in Immunology and Cancer at Montreal University. Dr. Bouvier is the author of over 160 scientific papers and has delivered numerous invited conferences. He is a world renowned expert in the field of G protein‐coupled receptors and has made seminal contributions to our understanding of this major class of drug targets. He has served on many peer review committees and scientific advisory boards and his contributions to the field of molecular pharmacology have been recognized by many awards.
Welcome, Dr. Bouvier. Dr. Michel Bouvier: Thanks, Sean.
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Slide 15 As Dr. Conn mentioned in the first presentation, G protein‐coupled
receptors are classically viewed to transmit their activity through their interaction with heterotrimeric G protein, thus modulating the activity of a vast diversity of effectors into the cell, leading to the biological response.
Slide 16 In that context, the activity of these receptors were viewed as their
ability to either activate such as for partial agonists and full agonists or inhibit such as for partial or full inverse agonists the activity of their cognate G proteins.
Slide 17 However, in recent years, it became evident that the signaling capacity
of GPCR were more complex. For one thing, GPCR can form homo‐ or hetero‐oligomers, but also they can engage a large diversity of proteins that can either modulate the ability of the receptor to interact with its G proteins or serve as signaling molecules themselves, and in some case can mediate the activity of the receptor independently of the G protein. Also, it became obvious that many G protein‐coupled receptors can talk to more than one G protein and therefore can modulate the activity of different signaling pathways downstream.
In that context, then the question becomes are ligands able to
modulate the efficacy of all these signaling in the same direction? Could some ligands selectively activate some of these signaling pathways or could have different efficacy towards these various pathways?
[0:19:56] Slide 18 Recently or a few years ago, we’ve looked at that and acquired the
detail for the human beta‐2 adrenergic receptor, looking at the ability of various beta‐adrenergic ligands to modulate the ability of the receptor to either modulate the activity of adenylyl cyclase or the production of cyclic AMP through activation of Gs or the activation of the MAP kinase pathway, ERK1 and ERK2.
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As can be seen here on the left panel looking at adenylyl cyclase activity, some compounds such as isoproterenol and labetalol can be either partial or full agonists for the cyclic AMP production pathway, whereas other compounds such as bucindolol and carvedilol that are neutral antagonists and other compounds such as propranolol, metoprolol, bisoprolol, and atenolol are inverse agonists for adenylyl cyclase activity.
However, when looking at the right panel, it will become clear that
some of the compounds that are either neutral antagonists or inverse agonists for the cyclic AMP production are in fact partial agonists for the MAP kinase; therefore, they have a reverse efficacy toward the MAP kinase when compared to the adenylyl cyclase activity.
Slide 19 Also some compounds are inverse agonists for the MAP kinase and
we’re saying this for both the cyclase and the MAP kinase. Therefore, you have a vast diversity of possible signaling efficacy when encountering more than one pathway.
Slide 20 And you can represent that efficacy in graphical manner by doing a
Cartesian representation of the efficacy of these compounds by plotting their efficacy along for example two of the signaling pathways. On the X‐axis here is the adenylyl cyclase activity, and on the Y‐axis is the ERK1/2 or MAP kinase activity. And as you can see, the compounds can find themselves either being agonists for the two pathways. They would be in the upper right quadrant; inverse agonist for the two pathways, they would be in the lower left quadrant; or being an inverse agonist adenylyl cyclase and an agonist on the ERK1/2 pathway.
Slide 21 So without going into the details of the experiment that were done to
show that, but we’re also able to show that the mechanism by which the activation of these different pathways occurred through the receptors bound by different ligands differ. For instance, the compound isoproterenol which is a classical agonist for the beta‐2 adrenergic receptors, when we’re looking at the ability to activate the ERK1, does it partially through the activation of Gs, partially through the activation of Gi, and partially through the activation of the accessory protein beta‐arrestin.
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However, the compound ICI and propranolol, which are inverse
agonists within the adenylyl cyclase pathway but agonists for the MAP kinase pathway, do activate the ERK1/2 exclusively through a beta‐arrestin‐dependent mechanism and do not involve either Gs or Gi.
Slide 22 And in order to probe that directly, we used some assays that allowed
to monitor directly protein‐protein interaction in living cells in real time. This assay is based on the transfer of energy between the bioluminescent enzyme luciferase and the fluorescent protein GFP. In a nutshell, when the two proteins, as you can see in the left panel, do not interact and you add the substrate of luciferase, you have light emission that corresponds simply to the emission of light by luciferase.
However, if there is recruitment of beta‐arrestin to the receptor or
engagement of the beta‐arrestin by the receptor, then luciferase becomes very close to GFP, and part of the energy of luciferase is transferred to the GFP such that now, instead of having only the light emitted by the luciferase, you also have light emitted by the GFP, and you can calculate the transfer of energy by doing the ratio of light emitted by the GFP over the amount of light emitted by the luciferase. And by doing so, you can monitor the protein‐protein interaction.
And as you can see in the right panel, the compound ICI and
propranolol, which are inverse agonists for the production of cyclic AMP but agonists for the MAP kinase, were able to recruit beta‐arrestin, therefore promoting recruitment of beta‐arrestin, and therefore activating this pathway.
When you look at the ability of propranolol to block isoproterenol‐
stimulated recruitment of beta‐arrestin on the extreme right of the panel, you can see that propranolol acts a partial agonist. In other words, it blocks the extent where propranolol can itself promote beta‐arrestin recruitment.
Slide 23 So it means that when we are considering more than one pathway, we
can start finding that there is a lot of possibility of representation of signaling efficacy. In fact, the number of potential efficacy profile for a given compound will be 2n number of output or signaling cascade that
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are being considered. That means that we need to be able to look at more than one pathway at the same time.
Slide 24 And this is a representation of how this may be happening at the
molecular level. At the top of the slide here are some of the possible signaling mechanisms and just a fraction of these possible signaling mechanisms that can be engaged by the G protein‐coupled receptors ‐‐ the G protein dependent signaling leading to desensitization, endocytosis of the receptor, but also G protein independent signaling.
[0:24:55] So the lower part of the slide shows that some ligands, by stabilizing a
specific conformation, may allow the engagement of a subset of these signaling effectors, leading to only part of the signaling repertoire of the receptor. In order to be able to detect these possible signaling pathways, we need to be able to measure more than one signaling activity in the cells. In most cases, it would be nice to have it simultaneously.
Slide 25 And so we started developing in the laboratory a number of biosensors
that allow to measure the activity of some of these signaling pathways in real time in living cells and hopefully in parallel, looking at more than one pathway. And I won’t give you an extensive list but just a few examples of this.
And here is a recent BRET‐based biosensor that we’ve developed to
monitor the accumulation of cyclic AMP in the cell. This is based on the structure of EPAC, which is a RhoGEF, and the idea here is that the proximity between luciferase and GFP that are fused to the EPAC will be modified upon binding of the cyclic AMP to the EPAC molecule, allowing a change in the BRET signal that is being measured in the cell. And you can see that by using this in living cell, we can monitor either a Gs‐coupled receptor, the V2 vasopressin receptor on the left that will lead to a decreased in BRET upon the increase of cyclic AMP production or a Gi‐coupled receptor, the delta opioid receptor that will lead to an increase in BRET upon the activation of the Gi.
Slide 26
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Also, we are using currently in the laboratory a biosensor for calcium flux into the cells which is obelin. Obelin is a luminescent protein that is completely silent in the absence of calcium but become highly luminescent in the presence of calcium. This protein that is originally I think from Obelia longissima can be expressed in cells and then we can look at the ability of a receptor. The example given here is the GABAb receptor, promoting the increase in calcium that is reflected by an increase in the luminescence of the obelin, and in that case, we documented that this was phospholipase C dependent as it could be blocked by a phospholipase C inhibitor.
Slide 27 We can also try to monitor the conformational changes or the change
in the engagement of the various effectors that the receptor can engage with upon stimulation with different ligands that have different patterns. Here is an example only that is given where we’re looking at three compounds ‐‐ isoproterenol, bucindolol, and propranolol and their ability to activate or inhibit adenylyl cyclase or the ERK1/2. And you can see these three compounds have different signaling efficacies depending on which signaling pathway you considered.
And then you can look at their ability to either induce the engagement
of alpha‐I by the receptor by monitoring the BRET on the left panel between the receptor and G‐alpha‐I‐1. And you can see here that the signature of the BRET signal obtained by isoproterenol is clearly different from that bucindolol and propranolol, indicating therefore that there’s a change in conformation which is distinct between these compounds, the change in conformation of the receptor G protein complex.
And to the right‐hand side, we can see here the change in BRET
between the G‐alpha subunit and the G‐gamma subunit which is seen here as an indication of deactivation of Gi, and you can see that isoproterenol here can engage Gi, whereas bucindolol and propranolol cannot engage Gi in this pathway.
Slide 28 Also, we are now considering using another technology that is based on
the measurement of impedance of cell that has been proposed to be able to distinguish between different signaling pathways. Here in that slide you can see the pattern observed with this measurement of impedance looking at Gq‐coupled receptor, top; Gi‐coupled receptor,
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bottom; or dopamine receptor which is a Gs receptor on the right. And you can see the traces obtained for these different receptors are different and are believed to really indicate which type of G protein there is that they’re engaged with. We are now asking ourselves if the granularity of this assay is sufficient to get more information out of it and to really obtain more information about which ligand can selectively engage a subset of signaling pathways and dissect more precisely the information that is given by these trace of impedance measurement.
Slide 29 So in closing, I’d like to thank the people who did the work and that are
either in my laboratory or in the laboratory of collaborators. Thank you very much.
Sean Sanders: Thank you, Dr. Bouvier. Slide 30 Well, we’ve had a number of questions come in, but I think in the
interest of time, we’re going to move on to our final talk and we’ll get to those at the end in our Q&A session.
Slide 31 So our final speaker today is Dr. Brian Kobilka. Dr. Kobilka completed his
undergraduate training in Biology and Chemistry at the University of Minnesota and his medical degree at Yale University School of Medicine.
[0:30:00] Following a research fellowship at Duke University Medical Center in
North Carolina, Dr Kobilka joined the faculty of Duke in 1987. He later took up an Assistant Professorship at Stanford University School of Medicine where he moved up the ranks to his current position as Professor in the Departments of Medicine and Molecular and Cellular Physiology. Dr Kobilka has published numerous papers in eminent peer‐reviewed journals as well as receiving many awards and honors for his research. He currently sits on the editorial boards of the Journal of Biological Chemistry and Molecular Pharmacology. His research is focused on understanding the structural basis for the functional properties of G protein‐coupled receptors.
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Dr. Kobilka. Dr. Brian Kobilka: Thank you, Sean. Slide 32 So my presentation today will focus largely on the beta‐2 adrenergic
receptor and our efforts to understand ‐‐ to use this as a model system to understand the structural basis for G protein‐coupled receptor signaling. And I’ll show on this slide just some of the properties that the beta‐2 receptor exhibits in a cell to show you more or less its versatility as a signaling molecule.
First of all, the beta‐2 receptor mediates cardiovascular and smooth
muscle responses to adrenaline and noradrenaline. It signals through both Gs and Gi and arrestin as Michel just discussed. It also has been shown to reside in specific plasma membrane micro domains with other signaling molecules. It undergoes agonist‐induced internalization and trafficking to different cellular compartments. It exists in both homo‐ and hetero‐oligomeric forms and exhibits a moderate amount of basal activity. As Michel mentioned, a number of receptors exhibit basal activity let’s say in contrast to rhodopsin where it’s literally silent in the absence of activation, and it’s basal activity can be modulated by inverse agonists. And finally, it’s a very good receptor to study because as you’ve seen in the previous talk, there are a large number of ligands available with interesting functional behavior.
Slide 33 Our approaches to study the structure of the beta receptor are
illustrated here. Chimeric receptors and site‐directed mutagenesis have provided a tremendous amount of information through many labs including ours, but many other labs have contributed to identifying domains involved in ligand binding and G protein coupling specificity.
We’ve recently been able to solve the structure of the beta‐2 receptor
and this structure is a three‐dimensional structure of an inactive state of the beta‐2 receptor. And we’ve used fluorescence spectroscopy to characterize conformational changes involved in agonist binding and activation and to get a better understanding of the dynamic aspects of the receptor.
Slide 34
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So I’d briefly like to outline some of the changes that were required in
order for us to be able to get a crystal structure. The beta‐2 receptor like many membrane proteins becomes a rather flexible dynamic molecule when it’s removed from the plasma membrane, and in order to get high quality crystals, one must have very uniform proteins so you have to minimize heterogeneity.
Slide 35‐36 And the structure modifications that we made included removing
glycosylation sites, truncating some of the unstructured regions which are shown as grey circles.
Slide 37 And finally, the receptor within the transmembranes is quite dynamic
and we have evidence that that two domains boxed here behave as independent folding domains are a particularly dynamic interface.
Slide 38‐39 And to stabilize that interface, we’ve used both a high affinity
antagonist or an inverse agonist, carazolol, and two different approaches ‐‐ one using an antibody fragment to stabilize the three‐dimensional epitope composed of transmembranes 5 and 6 and a fusion protein.
Slide 40 And those two approaches are illustrated here. Slide 41 The antibody as you can see is binding to an epitope at the end of
transmembrane 5 and 6, and lysozyme is fused to the end of transmembrane 5 and 6, and both of these approaches resulted in obtaining diffraction‐quality crystals.
Slide 42 The two different properties of the receptor in complex with the
antibody and lysozyme are shown here. It’s obviously important that whatever we do to get crystal structures have serious negative impact
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on the function of the protein. Both the antibody receptor complex and T4 lysozyme exhibit wild‐type antagonist binding properties. The only slight modification in properties would be that the receptor lysozyme fusion has a slight increase in agonist affinity, but both structures are able to undergo agonist‐induced conformational changes. And I think that the fact that we were able to get structures in two different ways provides some confidence that the structures we get are probably accurate.
[0:34:56] Slide 43 So I’d like to briefly go through some interesting comparisons between
the beta‐2 receptor and rhodopsin, and probably, there are a couple particularly interesting differences.
Slide 44 And while the overall typology of the proteins are extremely similar,
there are some particularly interesting differences, one of which is shown here, in that the extracellular loop 2 of the rhodopsin forms a lid over the retinal binding pocket. And as you can see here in the beta‐2, the extracellular loop 2 is displaced away from the binding pocket, providing open access for diffusion into and out of the binding pocket.
Slide 45 Another interesting difference is shown in the next slide where we’re
looking at the cytoplasmic domains. In rhodopsin and many family A G protein‐coupled receptors, there’s a highly conserved sequence, DRY or ERY, at the cytoplasmic end of transmembrane 3 that forms a stabilizing interaction with a glutamate residue at the end of transmembrane 6. So this a conserved sequence and it stabilizes the rhodopsin in inactive state.
Slide 46‐47 What we’ve found to be of interest is that both the lysozyme fusion
structure and the receptor Fab structure, this ionic lock is broken. So the stabilizing interaction is not observed in our crystal structures.
Slide 48
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A possible explanation is that this might be a crystallography artifact. This is unlikely since both crystal structures have the same ‐‐ both show the same loss of the ionic lock. It’s also possible that it’s a carazolol‐specific conformation or that it might be one of several basal conformations that the beta receptor exists in.
Slide 49 We’ve also been able to observe some interesting behavior or
properties of the ligand binding site that might have implications on our ability to identify selective drugs for specific receptors. And I show here in white are residues that surround the carazolol binding pocket and carazolol is shown in yellow. And these residues are all within fractions of the carazolol molecule.
Slide 50‐51 If we now try to look at what residues differ between beta‐1 and beta‐2
adrenergic receptors, you can see that there’s only one difference, and what this illustrates is the extreme degree of difficulty in generating ligands that are specific for one subtype. And now, if we extend the differences to the whole molecule, you can see these are all the amino acid differences between beta‐1 and beta‐2, and in order to achieve sound selectivity, you’d have to extend the ligand to the outside of the binding pocket and possibly sample some of the differences in the pore of the binding pocket.
Slide 52‐54 Now, the structure is an inactive structure, but it does provide some
clues as to the possible pathway of activation. And shown here is an interesting collection of water molecules that are resolved in both the beta‐2 receptor and rhodopsin, and this collection of water molecules involves interactions with highly conserved residues. So these residues form a network of hydrogen bonding interactions between the water and these conserved residues, and this is likely to be a signal transduction pathway for conformational changes extending from the agonist binding site of the G protein coupling domain.
Slide 55 Now, briefly what we would like to next learn about is what is the
structure of an active receptor and how do we get these active structures, and this is going to be a very challenging problem as I outlined here briefly. The challenges involve getting a very high affinity
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agonist and possibly the necessity for obtaining a structure of a receptor and a G protein complex. So I think this is, in terms of structural biology, the next frontier for us.
However, we have been able to learn something about the process of
activation using fluorescence studies, and in the last few slides, I’m just going to outline some of the approaches that we’ve used to study the process of agonist activation of the receptor.
Slide 56 And a particular method that we’ve employed recently is the use of
tryptophan quenching of bimane fluorescence. Slide 57 The experiments shown here is that we initially engineer a receptor so
that we’re able to specifically attach bimane, which is a small fluorescent molecule.
Slide 58 We then attach through site‐directed mutagenesis a tryptophan, which
if it’s within 15 Ångströms of the bimane will quench bimane fluorescence.
Slide 59 And then we’re able to observe conformational changes by changes in
bimane fluorescence if ligand‐induced conformational changes affect the distance between the tryptophan and the bimane.
[0:40:12] Slide 60 This just summarizes some of the movements that we’ve observed in
the beta receptor. This is the cytoplasmic end. The solid arrows indicate the positions where agonists induce an increase in distance, and the dotted lines indicate positions where agonists induce a decrease in distance. And what we’ve found is evidence that agonists and partial agonists induce distinct states and that agonists and partial agonists, in the presence of both agonists, partial agonists and in the absence of drugs, the receptor exists in more than one state. So the receptors, the
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dynamic protein, it’s not in the single conformational state even in the presence of these compounds, and this is perhaps one of the reasons why it’s so difficult to crystallize these proteins.
Slide 61 So here’s a brief summary of what I hoped to present and I’ll just move
on to the acknowledgements. Slide 62 And particularly, I’d like to acknowledge Dan Rosenbaum and Soren
Rasmussen who were the postdoctoral fellows involved in obtaining the tissue crystal structures that I discussed. Thank you.
Sean Sanders: Great. Thank you very much, Dr. Kobilka, and thank you all for your
excellent presentations. Slide 63 So it’s time to now move on to our Q&A portion of the webinar. But
just before we do, I want to mention that Sciences Online Signal Transduction Knowledge Environment (STKE) is now called Science Signaling, and for the first time, we are accepting original research. So if you would like to know more about this information about submitting your papers, you can go to the website which will come up in your slide viewer, www.sciencesignaling.org.
So we’re going to move on to our first question. I’m going to pose this
to Dr. Conn about a question that came in via email from one of our registrants asking, how is high specificity achieved in such a large family and such a large super family of proteins?
Dr. Jeffrey Conn: I think when we think about specificity, we can be thinking about two
different things ‐‐ the specificity of signaling for a G protein‐coupled receptor, how does a G protein‐coupled receptor specifically activate different signaling cascades, and also then the specificity of small molecule reagents like the ones that I mentioned.
In terms of specificity of signaling, I think a lot of evidence recently
suggests that the same receptor in different cellular environments can induce very different and specific effects. So in recording for instance in the nervous system where I work, recording from different neuronal populations the activity of a single GPCR, you may see that same GPCR
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regulate different effector systems, different ion channels, and different cell types even though all of those are present in each, and the thought is that that is due to different signaling complexes, so different protein‐protein interactions and scaffolding proteins within the cell that are very cell type specific.
So then a ligand inducing a conformation, stabilizing a conformation
can selectively as we’ve discussed stabilize a conformation that couples to one of those, and then there’s the question of the specificity of the ligands. And some of the allosteric molecules that we’re studying, the very unique specificity that we’re finding, is really not well understood why that is the case at this point. I think we’re just starting to understand something about the binding pockets for those ligands, how they specifically react with different receptors but really do not fully understand why that selectivity is possible.
Sean Sanders: Okay. Any other comments from the panel? Dr. Michel Bouvier: I think it was addressed very nicely. An additional level of selectivity can
also in some cases be achieved by sequestrating some of the component of this into microdomains which may be lipidic microdomains or some environment of the membrane that can also allow the selectivity of signaling in some cell type or in some conditions versus otherwise.
Sean Sanders: Okay, excellent. So I’m going to jump to Dr. Kobilka with the question
considering the large number and variety of GPCRs, are there any common structural similarities in drug binding sites for different GPCRs?
Dr. Brian Kobilka: Well, I believe that many of the family A members, particularly those
for small molecules such as dopamine, serotonin, histamine, all share overlapping binding pockets, and even for some of the family A members where larger peptides are involved in binding where they bind primarily in the extracellular loops, there are other parts of the peptides that in fact might share common space with small molecule receptors. But nevertheless, there are receptors such as the family C receptor where small molecules are binding to an extracellular domain.
[0:45:05] So there is both shared binding sites and very different binding sites,
but they all seem to ultimately change the arrangement of the
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transmembrane domains and lead to similar kind of conformational change on the inside recognized by G proteins.
Sean Sanders: Okay, great. Perhaps this is something that Dr. Bouvier can answer. Do
you feel that there are multiple activation site states of GPCRs such that the different states can activate different G proteins, leading to differential activation?
Dr. Michel Bouvier: Yeah. There’s increasing evidence as I mentioned a little bit during my
talk that different ligands can have different functional responses. We believe that this is due to different conformations of the receptor that may be stabilized differentially by the different ligands, allowing the receptor to add up a conformation that will engage only a subset of the signaling partners, the subset of signaling partners being either different G proteins or in some cases G protein‐independent signaling.
One other difficulty right now is finding the link between the elegant
structural work that had been accomplished by Brian and these functional responses. We don’t know how the different conformations that are being observed in fluorescence studies and other type of studies impinge on the selection of specific factors in the cell.
The other challenge also will be to identify how important these
different signaling pathways engaged by different ligands will lead to physiologically relevant different biological response so that we can exploit these different efficacies of the compound to really have better handle on the efficacy of potential therapeutic agents.
Sean Sanders: Okay, great. So a question from our online audience that I believe this
came in during your talk, Dr. Conn, asking, which family B receptors have you been successful with and have you found both positive and negative allosteric modulators for these?
Dr. Jeffrey Conn: So we have not in our lab studied the family B receptors so our studies
have focused on family A and family C. There’s nothing that would say to me that the family B receptors could not be as successful and there are examples in the literature. So when you look at some of the CRF receptor ligands, those are likely to be acting by allosteric mechanisms. And some of the compounds that were even discovered before individuals were specifically looking for allosteric modulators, but they were just looking at functional assays, some of the ligands that were discovered without really thinking about the mechanism in earlier days are now being reevaluated in terms of their allosteric mechanisms of action, and some of those do include family B GPCRs where this
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mechanism does appear to be viable, somewhat behind family A and family C in terms of different labs specifically going after those compounds.
Sean Sanders: Dr. Bouvier, Dr. Kobilka, have you studied any family B proteins? Dr. Michel Bouvier: We have studied a little bit some of the family B protein but not in the
realm of identifying allosteric regulators. In some of them, one other particularity if we think about the CGRP receptor is that they involve not only a seven‐transmembrane‐domain protein but also an accessory protein known as RAMP which is required for the pharmacological selectivity and also the trafficking of the receptor, which may offer other sites of action actually for allosteric regulators. So a molecule that would bind to the RAMP protein in the seven‐domain portion could be an allosteric regulator. To my knowledge, there’s no such compound identified yet, but this is certainly a possibility.
Sean Sanders: Okay. Dr. Jeffrey Conn: And having mentioned CGRP, that is an example where a recent paper
came out showing allosteric antagonists of the CGRP receptors and the exact mechanism, whether it involves RAMPs is not really understood but certainly allosteric mechanism of action.
Sean Sanders: Good. So I’m going to jump to the question. I’ve got a couple of queries
from our online audience and I know we talked a little bit about it earlier before the broadcast. This question is about more recent research that shows the existence of nuclear GPCRs in several cell types, and the viewer asked how do you think these GPCRs are different from the GPCRs in the plasma membrane? So Dr. Kobilka, would you like to jump in?
Dr. Brian Kobilka: I’m afraid I really can’t answer that question. I don’t know enough
about them. Sean Sanders: Okay. Dr. Bouvier? Dr. Michel Bouvier: Well, those are intriguing observations, finding that there is GPCRs that
may be nuclear. How exactly they function, I don’t think we know really right now. Some of the receptors which were described being at the nuclear membrane are also found at the plasma membrane. One other complexity there of course is that the membrane of the nucleus is continuous with the endoplasmic reticulum so it’s not always clear if it is a trafficking phenomenon that is being seen or whether or not there
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would be a real functional response. Some people have described nice functional response so most likely there will be some effect of GPCRs, but I don’t think at this point we know what distinguishes a receptor that can indeed be translocated to the nuclear membrane whereas those that do not.
[0:50:30] Sean Sanders: Okay. Anything to add, Dr. Conn? Dr. Jeffrey Conn: Really not a lot to add. It’s not an area that I am that familiar with, but
there had been recent studies. Michel mentioned some of the GPCRs that are on the plasma membrane, also the nuclear membrane. At least one metabotropic glutamate receptor falls into that category and it has been postulated that it interacts with intracellular glutamate under different conditions. I think it’s too early to really know for certain to fully understand that, but there are postulated roles that are starting to come out.
Sean Sanders: Do you think talking hypothetically that it’s possible that these nuclear
GPCRs could also be targets for drugs? Dr. Jeffrey Conn: I think it’s certainly possible. You have the additional barrier of getting
compounds into the cell which that’s common and for many enzyme targets and other targets. Certainly, there are many intracellular targets. If it is a nuclear receptor that also exists on the cell membrane, having developing compounds that would selectively interact with a nuclear receptor may be a real challenge. But in other cases, if it is predominantly a nuclear receptor, there should be nothing to make it particularly difficult to develop compounds that will have that activity.
Sean Sanders: Okay. Another question here about studies on the effects of membrane
composition on GPCR conformation and signaling. I’m going to throw that open if anybody would like to jump in. Dr. Kobilka?
Dr. Brian Kobilka: Yeah. I think it’s an extremely interesting point and there’s very little
that has been done. The only thing I’m aware of are some studies done in rhodopsin I think out of I believe Michael Brown’s lab where they’ve looked at the effect of alkyl chain length and head groups on signaling of rhodopsin molecules and they clearly show important differences, but there’s really been nothing done on other GPCRs. We’ve been interested in pursuing this, but it’s a very challenging problem.
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Sean Sanders: Okay. A question to the panel asking to highlight some recent methods for deorphanizing GPCRs, whether there’s a number of orphan receptors still out there. Any comments so we could start with you? Dr. Bouvier?
Dr. Michel Bouvier: Yes. Well, it’s still a daunting task. The numbers have been falling down.
I mean, there was a period where they were deorphanizing a lot of receptors rapidly. I think the one that were ‐‐ the easiest ones to deorphanize have been deorphanized. I think there’s still a number of them to be achieved. There’s no easy way. There’s many different ways that people are trying. People are trying using synthetic ligand, just screening randomly, trying to look for a generic activity and high throughput screening, and with this tool then go back into physiological system trying to find out what could be the endogenous ligand. People are still trying to look at extract from tissue to identify the ligands that can be binding and I also know of some people that are trying to use bioinformatic tools to predict what kind of ligands could be binding to the receptors. But each of these methods are challenging. They are difficult. There is no rapid solution to deorphanization that I can think of.
Dr. Jeffrey Conn: I would not add anything to that except that a real challenge is if you
are randomly screening libraries, having a readout that you have confidence couples to the receptor, the orphan receptor, and that has always been a challenge. People have used promiscuous G proteins and that has been effective. Now, there are other methods that are coming available. I think each of them has the same limitations. You can’t be absolutely certain that the receptor that you’re studying couples to that effector system even if it is designed to be a very generic readout of GPCR function until you have at least a foot in the door in some ligand that you know activates the receptor. Then once you find the agonist, knowing what the natural agonist is is another major task that does not necessarily come easily.
Sean Sanders: Okay. So I’m going to stay with you, Dr. Conn, and ask you a question
about the role of GPCRs in neurodegenerative disorders and in mental illness, if you could comment briefly.
[0:54:55] Dr. Jeffrey Conn: Sure. So when we look at the roles of GPCRs in CNS disorders, I think
we can think about two different aspects to that. One is the etiology, so a primary role of a GPCR in the etiology of a disorder, and the second is the role of a GPCR in regulating transmission through circuits that are
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important for the disorder in a way that might have a therapeutic benefit. And I think those are two quite different questions. In thinking about roles in primary etiology of a disorder, there are different genetic association studies that are providing hints to roles of GPCRs, but really, a primary causative role of a mutation say in a GPCR in neurodegenerative disorders is really something that I cannot point to specific examples.
If we look at Alzheimer’s disease as an example, there is a very long
field of study that preceded some of the more modern approaches. So thinking about Alzheimer’s disease with acetylcholine and muscarinic receptor signaling so there’s a very early degeneration of cholinergic neurons. There’s a thought that muscarinic receptors may play an important role, and a major target for Alzheimer’s disease in the 1990s were the muscarinic receptors which really waned in more recent years and that is now having a resurgence with some of these new more selective ligands. And as that occurs, more evidence for a role of these receptors in processing of some of the toxic proteins, the amyloid beta precursor protein, and that could play both in etiologic role potentially and also a role for therapeutics.
But when we look at a variety of CNS disorders, we can see roles of
GPCRs that have therapeutic value, and there are many good examples. So one of the most obvious would be schizophrenia where dopamine receptor antagonists have antipsychotic efficacy. So dopamine and dopamine receptors are in some way involved. Whether that is causative for the disorder or not, it provides a therapeutic potential. The same would be true for the loss of dopamine in Parkinson’s disease and therefore agonists of dopamine receptors having therapeutic effects. And we could point to several examples such as that where GPCRs are involved in the circuits that are impacted in both psychiatric and neurological disorders in important ways that lead to therapeutic approaches.
Sean Sanders: Okay. I’m going to come back to Dr. Bouvier ‐‐ a question that came in
during your talk. You mentioned that a single receptor can bind more than one G protein. Is there a possibility, the viewer asks, to have two different ligands that activate two different G proteins via a single receptor or a single ligand activating two different G proteins via a single GPCR?
Dr. Michel Bouvier: The answer is yes to both questions. There is for example, for the beta‐
2 adrenergic receptor, some ligand will lead to the activation of both Gs and Gi which may appear paradoxical because one stimulates adenylyl
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cyclase and then one inhibit adenylyl cyclase, but there is a kinetic random event here that is taking place, and each of these G proteins can have more effect than just regulating adenylyl cyclase. That’s one example where some ligands can lead to the activation of only Gs and others to both Gs and Gi.
The other example is the ligand that can promote two G proteins isn’t
the same answer really because isoproterenol and even norepinephrine and epinephrine, the natural ligands for the beta‐2 adrenergic receptor, can lead to the activation of both Gs and Gi, and there’s ample examples of that that are known for many GPCRs of different classes.
Sean Sanders: Okay. So we’re almost out of time but I’m going to ask one more
question that I’ll throw open to the panel. This was a question asking are there high throughput screening technologies available to discover new drugs targeting GPCRs? Maybe Dr. Conn, you would like to start.
Dr. Jeffrey Conn: A large number of different high throughput screening technologies
that are available and we wouldn’t have time to go through all of them, but starting with radioligand binding‐based assays that start with the knowledge of a specific binding site on the GPCR that you’re interested in developing molecules to functional readouts. Some of the more common functional readouts would be those such as what I showed in my talk with calcium fluorescence where you’re looking at a Gq‐coupled receptor that activates calcium mobilization from a cell and then in high throughput format, you can measure that quite readily. You can also do that with Gi‐coupled receptors and using promiscuous G proteins.
[1:00:00] There are a number of other technologies that are also available,
including technologies that’s specifically get beta‐arrestin‐mediated signaling, so high information content screening where you look at trafficking of GPCRs in response to novel ligands. New technologies that have recently been reported that can measure coupling to an ion channel in high throughput format using fluorescence. Plate readers but then also increasingly technology is moving towards an ability to do at least moderate throughput assays with rapid parallel patch clamp recording. So it ranges from ultra‐high throughput to more moderate throughput, but a wide range of technologies that are available depending on the specific needs.
Sean Sanders: Great. Dr. Bouvier, you have anything to add?
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Dr. Michel Bouvier: Well, I think that was very well covered, and as Dr. Conn mentioned,
depending on the type of questions that you want to ask, the screening that you will be able to pick will be determined by this. But also, we have to keep in mind that the type of molecules that we will identify will be impacted by the acid that we’ve selected, and one clear example of that comes from the fact that there is this ligand biased signaling and that, you know, some ligands can have different efficacies toward different pathways. When you select a given pathway, let’s say calcium, using a promiscuous G protein that couples everyone, you don’t know if a ligand which is an agonist on this assay will also be an agonist on the most pertinent physiological readout that could be something else. And it could be an inverse agonist or it could be a neutral antagonist.
So you have to keep that in mind when you’re selecting your assay that
if you are really looking for an agonist, you better choose an assay which is as close as possible to your assay, the physiologically relevant assay. And then you can also, if you just want to have something that binds, then whatever assay will give you information about what is binding to the receptor most likely.
Sean Sanders: Okay, thank you. Anything to add, Dr. Kobilka? Dr. Brian Kobilka: Well, just that one of the problems with high throughput screening is
you’re developing an assay which is very sensitive but not necessarily one that will pick up the physiologic signaling pathway which could be a minor pathway for that particular receptor and that particular cell in the body, and I think that’s more or less echoing with what Michel said. So it’s a very big challenge for drug discovery.
Sean Sanders: Right. Well, it seems like there’s a lot of challenges out there to keep
everyone busy for a good few years. Slide 64 So it just remains for me to thank all of our wonderful speakers very
much for being with us today and for sharing their knowledge and expertise: Dr. Jeffrey Conn from Vanderbilt University, Dr. Michel Bouvier from Montreal University, and Dr. Brian Kobilka from Stanford University School of Medicine.
Thank you to our online audience for your great questions. I wish we
had time to answer all of them, but unfortunately, we’re out of time. Please go to the URL at the bottom of your slide viewer now if you
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would like to learn more about products related to today’s discussion and look out for more webinars from Science in the near future available at www.sciencemag.org/webinar. We encourage you to share your thoughts about the webinar with us by sending an email to the address now up in your slide viewer, [email protected].
Again, thank you to our participants and to Roche Diagnostics and their
xCELLigence System for their generous sponsorship of this educational seminar.
Goodbye. [1:03:30] End of Audio