Conference Overview


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Company representative for PharmaDiscovery Conference. Company presentation to disseminate knowledge acquired at conference; talks presented in context of basic pharmacological concepts & drug discovery approaches.

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  • Note: The slides presented here are not the originals.
  • This is a list of talks I attended as well as the topics which they covered. Reviews of screening technologies were usually covered within the context of a channel presentation. If you would like a copy of my notes for a particular talk, please see me.
  • What I decided to do with this talk was to present several talks which I thought either demonstrated how research is conducted in this field, important lessons learned that could be useful in our own research, or challenged conventional thought.
  • Trp family: TRPV1, also know as the capsacain or vanalloid receptor, was the first discovered.
  • Trypv1 is assumed to be involoved in pain: 1) it is expressed in sensory neurons and drgs 2) it is upregulated in pain states, 3) it is activated (excitatory) by noxious heat, and acid, inducing pain or hyperalgesia 4) the receptor is also modulated by pain inducers, ie.bradykinin 5) finally TRYPV1 KO data suggests a role in pain.
  • Mechanism of pain: A pain stimulus is detected by sensory neurons which transduct sensory information from the periphery into the spinal chord. This physiological pathway provides several ways to develop therapies for pain. One potential target could be the mechanosensory neuron, where signals are first generated, influencing AP propagation; another target could be transmitter release, cell body of a receiving neuron.
  • This is TRPV1’s respone to various stimuli: 1) Heat gated: When the temperature reaches 42C, an inward current (depolarization) is activated. 2) Below is the activation of TRPV1 by acid. 3) And to the right is the chili pepper experiment: Capsaicin was the ingredient found to activate TRPV1. Piperine, found in black pepper, you will note also activates the channel. The comparison of the capsaicin and piperine response demonstrates an important concept in pharmacology that was discussed last week. Note that although piperine has a much lower binding affinity, it’s response is much greater than capsacain which has a relatively much greater affinity for the receptor. In fact piperine is considered to be a full agonist whereas capsaicin is a partial agonist. This is an important demonstration that the reasons for responses of partial and full agonists (which by definition remember, are binding to the same receptor site) cannot be explained by binding affinity alone, and raise the issue of whether a more complex model of receptor binding, is necessary to explain the behavior of partial & full agonists (ie. conformational state equilibrium).
  • Here is in vivo data which validates the TRYPV1’s potential role in pain, particularly within the GI tract. Above we see staining of TRPV1 in human tissue of a patient experiencing oesophageal hypersensitivity. At the other end of the GI tract we also see TRPV1 upregulated in a patient experiencing rectal hypersensitivity. TRYPV1 knock outs confirm this observation: Here in the Visceral Pain Model, acid is instilled directly into the GI tract. Constrictions (sensory neuron discharge in response to distension in the gut), which are correlated with visceral pain response, are far less experienced in the KOs than controls.
  • Now TRPV1 has been validated as a potential target for pain, next is to determine if TRPV1 antagonists are effective in reducing pain. This was tested in the hotplate test, where the mouse paw is placed on a hotplate and the time to remove the paw form the hotplate is correlated with thermal sensitivity. Here it is indeed demonstrated that animals treated with a TRPV1 antagonist experience less pain, and the response is dose dependent. Mechanical hypersensitivity
  • This TRPV1 antagonist is also effective at reducing pain in the Seltzer neuropathic pain model.
  • Importantly a potential therapeutic has to demonstrate receptor selectivity, and normally receptor with close homology to the target receptor is chosen to verify the drug’s selectivity. Here TRYPV3, a close relative of TRYPV1, is not activated by the TRYPV1 antagonist, which suggests selectivity and that the drug is an excellent candidate to prepare for clinical trials.
  • So what turned out to be stopping development of this potential wonder drug, as you may know as capsazepine???
  • Because as it turns out HOMOLOGY CANNOT PREDICT SELECTIVITY: Although V1’s close relative was not blocked by capsazepine, here we see it’s inhibitory activity of TRP receptors not very related to TRYPV1, for example TRPM8 and TRYPM2 from different family.
  • In fact capsazepine blocks receptor outside of the TRP family, and at concentrations similar to its activity at the TRYPV1 receptor. Here we see capsazepine’s effect at the HCN1 channel.
  • Even more dramatic it turns out that most TRYPV1 antagonists are not selective.
  • So what important concepts in pharmacology does this illustrate? Certainly drug selectivity is one point, yet importantly as you may remember TRYPV1 antagonists were found to effective in the classic pain models. So what may the data be telling us.
  • If TRYPV1 AA has been found to be effective in pain models, but not selective IS TRYPV1 THE TARGET RECEPTOR FOR PAIN MODULATION???
  • In order to address the crucial point that other TRYP receptors may be active in modulating pain perception, other TRYP receptors that are activated by TRPV1 antagonists have to be tested. Yet how to demonstrate which TRYP receptors are active in pain?
  • Here is a list of potential TRYP candidates and their activators. Yet how can it be determined which TRYP receptors are active in the GI tract in an efficient manner?
  • To address this experimentally, a novel ex vivo model was developed where the GI tract was removed from the animal and hooked up to an apparatus where at one end agonists to the various TRYP receptors were introduced and at the other end changes in pressure, a measure of afferent activity, were recorded in response to receptor activity.
  • The results here demonstrate that agonists to Tryp A1 induce afferent activity, however agonists to Tryp M8 do not. And at present TRYPA1 is being pursued as a potential drug target for pain, and I am certain that others not discussed are being pursued as well.
  • So the conclusions of this work are: 1) Phylogeny does not predict selectivity. 2) Selective antagonists for the TRP family are difficult to develop- 3) but a condition as ubiquitous and important as pain warrants the effort.
  • Finally in closing I believe this talk illustrated an important issue in drug development that being for all the effort to develop a more “rational” approach (ie. crystallography, etc.) towards drug development, will less informed effort of screening with functional assays continue to be our most productive approach?
  • Family One a lot of the classical pharmacological agents fall into this category Type III: what characterizes this group is that have found AMs for almost every class within a family N terminal h Nice model for a allosteric modulators # Families Have identified allosteric modulators from every class within of family Unique Class 2 Receptors in One N terminal region Binds amino acids including glutamate, orthosteric binding domain Transmembrane analogy; Possible to have AM of this site
  • Self Explanatory. Transmembrane region which is considered to be the effector domain may offer us another potential target for allosteric modulation of the receptor. Now if TM configuration is what drives activation of the receptor, one could find molecules which bind directly to this region inducing the activated configuration of the receptor to form particularly at lower concentrations of orthosteric agonists, increasing the therapeutic index of the agonist.
  • Orthosteric agonist/glutamate binds to its site at the N terminal & induces the orthosteric region to change configuration (closed state), causing the adjacent TM regions to come closer and allow for interaction with G proteins which in turn activates the signaling pathway. Positive allosteric modulators: As discussed in the previous slide, this is the region targeted for activating the signaling pathway, could develop molecules which bind directly to this region and activate the pathway without the presence of ligand binding. Antagonists: Competitive antagonists block the activation of the receptor via binding to the orthosteric site where agonists bind. Importantly since it competes for the same site as receptor agonists, the block can be overcome by orthosteric agonist at high concentrations of an agonist. This highlights this disadvantages of orthosteric antagonists as potential therapeutics in that you would need to use a very high dose to overcome endogenous ligands in order for it to be effective and at such doses, run into toxicity and adverse effect (ie. reduces the therapeutic index). Negative Allosteric Modulators : Blocks receptor activity at site other than orthosteric site, therefore is noncompetitive blocker of the receptor. Allosteric Potentiators : Bind to the allosteric region, but do not active the Gprotein on its own; however it can change the probability of the orthosteric site (clamshell) to close, and essentially increase the potency of the orthosteric agonist. What was not clear was whether endogenously the potentiator would “do nothing functionally” so to speak, or is just undetectable in its endogenous tissue. For example can a high expression system “boost” the activity to detectable levels of activation? Assume that the major emphasis for targeting AM is not to increase the efficacy of poor agonists, but either to increase the potency (lower the EC50) of the agonist OR to increase the selectivity of response to the drug by binding to a less common receptor site. Both of these points are important for expanding the therapeutic window. ANTAGONISTS Can bind at orthosteric or allosteric site. Inactive receptor and agonist cannot activate. Implications for pharmacology: Competitive antagonist at orthosteric site, if agonist concentration is high can displace antagonist and achieve activation. Therefore if want to develop antagonist would need to administer very high concentrations, increase side effects and toxicity. So industry wide allosteric antagonists is the focus, since noncompetitive. ???Are allosteric sites endogenous as well???? Then I would think may need to worry about AM agonist unless again targeting different TM regions for AMA & AMAA.??? Agonist –Binds to another region close clam shell and TMs together receptor to activate. Potentiator- on its own does nothing as far as activation however increases the probability of clamshell to close, or probability for agonist bind and activate pathway.
  • Cross Coupling Gi Receptor to G Protein: The existence of allosteric modulators is yet another demonstration of the point made earlier concerning the limits of structural approach towards drug development. The existence of AMs was discovered through functional assays such as the FLIPR, and it is the only method at present which can detect allosteric modulators. Cells were exposed to [glut] which would give a submaximal response (ie. 10% max response), in order to determine whether a potentiator would indeed potentiate the response. if pretreat with potentiator, see potentiated response with low efficacy dose of glu. Potentiation is closed to glu max. So can set up dose response curves of pot., curve shifts, etc. So can do a lot of characterization all in the same system. AND I THINK IT IS IMPORTANT TO MENTION THAT WHILE ANALYZING THE DATA THROUGHOUT THIS PRESENTATION MAKE NOTE OF THE CONCENTRATIONS OF POT. & GLUT. SINCE [GLU] IS OFTEN SUBEC50.
  • So next verify that potentiator is not binding to orthosteric site. See that as increase concentration of orthosteric agonist or ligand, decreases radioligand binding, whereas the potentiator has little impact on binding. This demonstrates the important fact that the potentiator binds to a site other than orthosteric.
  • Here is a list of the characteristics of potentiators, which will then be followed by the supporting data on the next several slides. 4) Potentiation of response seen in recombinant systems can be replicated in native tissue (demonstrating the response is not just an artifact of high expression, and has physiological relevance). 5) If have high spare receptors, such as 97-95% spare receptors (and therefore only need to activate 5% receptors to get max. response), the potentiator functional EC50 are good estimate of KD (true affinity) of potentiator. This important fact implies that you do not need to develop a radioligand to determine a potentiator’s affinity for the receptor 6) Finally GTP binding increases without hydrolyzing galpha 15, and may explain how potentiators work.
  • Potentiators are agonist dependent: The dependency of a potentiator’s activity on an orthosteric agonist such as glutamate is illustrated here by the loss of potentiation in the presence of a glutamate site antagonist, which illustrates that the potentiation is glutamate dependent.
  • Self explanatory.
  • Here is an example of radioligand binding. ECG4 is a glutamate analog which binds to the orthosteric site. Checked its binding characteristics -/+ potentiator. See 2 fold change in affinity of EGC4 for ortho site.
  • This is the experiment to demonstrate that the EC50 value in FLIPR noneq., fast response conditions, where 95% of receptors are spare, potentiator EC50 is similar to its KD.
  • This is an example of the GTP binding method in a cell line expressing mGluR2. In the potentiator alone condition , do see GTP binding actually similar to glutamate alone when its at its maximum concentration. And as well can see binding is additive, and that an orthosteric AA does not blocked the potentiator’s binding. This not only supports that potentiator is binding to another site, but importantly provides some insight as to how the potentiator is increasing the agonist’s response. Namely that the potentiator can change the conformation of the receptor, but not activate the signalling pathway on its own.
  • Finally in vivo models validate the cellular findings that potentiators are selective and functional. As well mGluR2 & mGluR3 knock out mice demonstrate the potentiator’s specificity. ( But remember the earlier demonstration of
  • Self explanatory.
  • Self explanatory.
  • Self-explanatory. This study reinforces the advantages of functional assays to identify not only potential therapeutics, but classes of therapeutics, which would could not be discovered by any other means.
  • I was particularly interested in this talk because it was somewhat related to my reason for performing clinical research in brain injury and functional recovery. The administration of methylphenidate has been highly correlated with functional recovery outcomes; it was thought that its role in recovery was related to increased attentional ability during occupational therapy, similar to its role in improving child attention deficits. Yet a reviewing the clinical studies revealed that no measure of attention could be correlated with the noted recovery. It could be that the attention measures developed are not appropriate for such an impaired group. Yet as well it may be indicating that methylphendiate has a neuroregenerative role in TBI patients that is separate for its role in adolescent attention deficits. Here in this group they are thinking about channels in a novel way-- as having a neuroregenerative role, distinct from neurofunctional processing, and potential target for neuroprotection or regeneration. 2) Another relevant point of this talk was the demonstration of transitional states as targets for channel modulation, which could only be detected by functional assays. 3) There is no available slides for this talk, as with the majority of presentations of the conference. Therefore I am presenting my written summary of the talk. It is abbreviated so if you would like more details of the experiments, etc. please see me.
  • Self explanatory.
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  • ??? Wouldn’t you need to test point mutations at other sites not just the adjacent to verify this???
  • Self explanatory.
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  • Conference Overview

    1. 1. PHARMADISCOVERY CONFERENCE 2005 Washington D.C.
    2. 2. <ul><li>Talks Attended List </li></ul><ul><li>Presentations </li></ul><ul><ul><li>TRP Family </li></ul></ul><ul><ul><li>Allosteric Modulation of mGluR </li></ul></ul><ul><ul><li>Allosteric Modulation of AMPA Receptors </li></ul></ul><ul><li>In Vivo trends/Potential uses for chromovert in POC? </li></ul>PHARMADISCOVERY CONFERENCE 2005 Preview
    3. 3. <ul><li>Screening Platforms </li></ul><ul><li>High-Throughput Ion Channel Screening </li></ul><ul><li>PatchedExpress </li></ul><ul><li>IonWorks </li></ul><ul><li>Atomic Absorption </li></ul><ul><li>Technology Assessment </li></ul><ul><li>Maximizing the tools of discovery to improve productivity </li></ul><ul><li>Drug Design </li></ul><ul><li>Ion Channel Screening: The impact of new technologies & targeted </li></ul><ul><li>Libraries (Soft Focus Libraries) </li></ul><ul><li>Biotechnology Research & Microgravity </li></ul><ul><li>New Rhodopsin Crystal and the Prospects for GPCR Crytstallization </li></ul>PHARMADISCOVERY CONFERENCE 2005 Talks Attended
    4. 4. <ul><li>Modulation of Receptor-Ligand Interactions </li></ul><ul><li>Receptor Mediated Channels </li></ul><ul><li>Discovery of Novel Modulators of the Class C GPCR Metabotropic Glutamate Receptors </li></ul><ul><li>GPCRs vs. Flavor Enhancers </li></ul><ul><li>Ligand-Gated Channels </li></ul><ul><li>Molecular Determinants Influencing Allosteric Modulation of Glutamate AMPA Receptors </li></ul><ul><li>TRP Family Members as Pharmaceutical Targets </li></ul><ul><li>Voltage-Gated Channels </li></ul><ul><li>Drug Discovery for T-type Calcium Channels </li></ul><ul><li>Atomic Absorption Rb Efflux Assay & its Application in Screening of K+ Channel Modulator </li></ul><ul><li>Animal Modeling to Establish Physiological Efficacy </li></ul><ul><li>CNS Models of Neuroprotection </li></ul><ul><li>CNS Rodent Models of Depression </li></ul><ul><li>Imaging Techniques to Study Pharmacodynamics Effects in CNS Diseases </li></ul><ul><li>CNS Models of Cognition </li></ul>
    5. 5. THEMES COVERED in PRESENTATION <ul><li>Demonstrated how research is conducted in this field. </li></ul><ul><ul><li>“ Working” concepts utilized </li></ul></ul><ul><ul><li>Experimental design </li></ul></ul><ul><ul><li>Data Analysis: Inferential </li></ul></ul><ul><li>Provided interesting lessons learned. </li></ul><ul><li>Challenged conventional theories in pharmacology. </li></ul>
    6. 6. 1) TRP Family Members as Pharmacological Targets Andy Randall Director Neurophysiology, Neurology & GI CEDD GlaxoSmithKline 2) Discovery of Novel Modulators of Class C GPCR Metabotropic Glutamate Receptors Michael Johnson, Principle Research Scientist, Neuroscience, Eli Lilly Corp. 3) Molecular Determinants Influencing Allosteric Modulation of Glutamate AMPA Receptors Eric S. Nisenbaum Neuroscience Discovery Research, Eli Lilly Corp. Presentations Distilled & Rearranged to Reflect Stated Themes
    7. 7. TRP Family Members as Pharmacological Targets Andy Randall Director Neurophysiology, Neurology & GI CEDD GlaxoSmithKline
    9. 9. <ul><li>Predominantly expressed in sensory neurons/ dorsal root ganglion (DRGs) </li></ul><ul><li>Excitatory Receptor (Depolarizing) </li></ul><ul><li>Up regulated in animal & human pain states </li></ul><ul><li>Polymodal Activation </li></ul><ul><ul><li>Noxious heat & acid </li></ul></ul><ul><li>Agonists produce pain or hyperalgesia </li></ul><ul><li>Activated/modulated by other pain-inducing stimuli </li></ul><ul><ul><li>Bradykinin </li></ul></ul><ul><li>Knock out data suggest hyperalgesia role </li></ul>ASSUMED TRPV1 ROLE IN PAIN
    10. 10. PAIN MODEL <ul><li>Sensory neurons transduct sensory information from periphery into spinal chord. </li></ul><ul><li>Transmitter release in response to firing. </li></ul><ul><li>Strategies for therapeutic development </li></ul><ul><ul><li>Target mechanoreceptor: AP propagation. </li></ul></ul><ul><ul><li>Transmitter release from neuronal cell body. </li></ul></ul>
    12. 12. Correlation of hTRPV1 & In Vivo Pain Models <ul><li>Human GI tract </li></ul><ul><li>VR1 is Up-Regulated in inflammatory states </li></ul>2) TRPV1 Null Mice Altered mesenteric Activity
    13. 13. PRECLINICAL EFFICACY of TRPV1 ANTAGONIST 1) Hotplate Test Reverses inflammatory thermal hyperalgesia 2) Reverses inflammatory mechanical hypersensitivity
    14. 14. PRECLINICAL EFFICACY of TRPV1 ANTAGONIST Effective in Seltzer neuropathic pain model!
    15. 15. TRPV1 ANTAGONIST DOES NOT BLOCK CLOSEST RELATIVE TRYPV3 has similar homology to TRPV1 Control TRPV1 AA
    16. 16. PAIN WUNDER DRUG Why not developed as pain therapeutic???
    17. 17. Capsazepine Is Not the Marrying Type
    18. 18. Capsazepine Is Not the Marrying Type
    19. 19. It’s an Epidemic… Various TRPV1 AAs Inhibit TRPM8
    20. 20. THERAPEUTIC ISSUES RAISED BY FINDING THAT TRPV1 AA IS NOT SPECIFIC <ul><li>Selectivity/Therapeutic Index </li></ul><ul><li>2) </li></ul>
    21. 21. THERAPEUTIC ISSUES RAISED BY FINDING THAT TRPV1 AA IS NOT SPECIFIC <ul><li>Selectivity/Therapeutic Index </li></ul><ul><li>Is TRPV1 the actual pain target? </li></ul><ul><ul><li>Efficacy of antagonist is validated in preclinical pain models. </li></ul></ul><ul><ul><li>Is its therapeutic actions due to another receptor? </li></ul></ul>
    23. 24. Rat Colon-Nerve Preparation In Vitro Method to Address Presence of TRP Members
    24. 25. TRP CHANNELS & COLONIC AFFERENT ACTIVITY TRPA1 is Another Potential Target for GI Pain
    25. 26. CONCLUSIONS <ul><li>Cannot always predict drug activity from phylogeny </li></ul><ul><li>Difficult to develop selective antagonists for TRP family </li></ul><ul><li>Yet such ubiquitous conditions as pain & </li></ul><ul><ul><li>the validation of TRP targets in pain models warrant the effort </li></ul></ul>
    26. 27. MAINPOINTS <ul><li>Demonstrates: </li></ul><ul><ul><li>selectivity is important & difficult issue to address at present. </li></ul></ul><ul><ul><li>the rational approach of drug design (ie. designing drugs on </li></ul></ul><ul><ul><li>the basis of structure or genetic sequence) may not expedite </li></ul></ul><ul><ul><li>the discovery of hit molecules. </li></ul></ul><ul><ul><li>screening molecules via functional assays may continue our most </li></ul></ul><ul><li>Panel of receptor family cell lines: </li></ul><ul><ul><li>could be useful for validating the selectivity of target molecule. </li></ul></ul><ul><ul><li>important issue in therapeutic development. </li></ul></ul>
    27. 28. Discovery of Novel Modulators of Class C GPCR Metabotropic Glutamate Receptors Michael Johnson Principle Research Scientist, Neuroscience Eli Lilly Corp.
    28. 29. GPCR Families Allosteric Modulators Identified in 26% Allosteric Modulators Identified in 100%* *Nice model for studying allosteric modulation Type 1 Type 2 Type 3* Rhodopsin LH-hCG Thrombin Angiotensin Adenosine ( ά 2) Adrenergic ( β 2) Serotonin (5HT4S) ά -latrotoxin PTH Secretin VIO PACAP Glucagon CRF mGluR4 mGluR2 mGluR1 Ca ++ VR 1 G 0 VN 2 G 0 VN 1 GABA B (R1)
    29. 30. Overall Structure of Type III GPCRs <ul><li>Amino Terminal Region </li></ul><ul><li>Orthosteric binding site </li></ul><ul><li>Expressed as soluble fragment </li></ul><ul><li>mGlu1 co-crystallized (open & closed) with glutamate </li></ul><ul><li>Cysteine Rich Region (exc. GABA) </li></ul><ul><li>Links orthosteric & effector regions </li></ul><ul><li>Transmembrane Region (TM) </li></ul><ul><li>7 TMs via AA sequence </li></ul><ul><li>Effector domain </li></ul><ul><li>I2 loop ~ Selectivity </li></ul>
    30. 31. Allosteric Domains as Potential Drug Targets Increase Selectivity of Response & Therapeutic Window
    31. 32. mGluR2 Cell Line Potentiation of Glutamate Response Discovered via Functional Assay
    32. 33. Potentiator 4-MPPTS Does Not Interact with the Glutamate Binding Site
    33. 34. Pharmacological Characteristics of Potentiators <ul><li>Functional response is dependent on orthosteric agonist </li></ul><ul><li>Agonist efficacy (response) curves shift to the left & may increase maximal efficacy </li></ul><ul><li>Agonist binding affinity is increased* </li></ul><ul><li>Responses in native tissue can mimic those seen in recombinant expression systems** </li></ul><ul><li>Potentiator’s EC50*** is similar to its KD (binding affinity constant) </li></ul><ul><li>In high expression systems, GTP binding can be stimulated alone </li></ul><ul><li>* Binding affinity is correlated with efficacy—not casual. </li></ul><ul><li>** Not just artifact of high expression. </li></ul><ul><li>*** Clarification: Actual reference to agonist’s ∆ EC50 due to presence of potentiator binding??? </li></ul>
    34. 35. Potentiation is Eliminated in the Presence of Orthosteric Antagonist Potentiation Requires Presence of Glutamate
    35. 36. Potentiators Shift Agonist Dose Response Curve to Left
    36. 37. Potentiators Increase Agonist Affinity <ul><li>2X decrease in Kd for ortho site agonist </li></ul><ul><li>Results are similar for other agonists </li></ul><ul><li>Competition exps. demonstrate Ki vs. ortho site ligand > 100uM </li></ul>
    37. 38. Potentiator Affinity is Similar to EC 50 <ul><li>Ligand EC50 decreases with usual Emax </li></ul><ul><li>Potentiator (?)* EC50 similar to its Kd (140 nM) </li></ul>
    38. 39. GTP Binding Assay <ul><li>Binding occurs with potentiator in absence of agonist </li></ul><ul><li>Binding is additive with orthosteric agonist </li></ul><ul><li>Yet not blocked by orthosteric site antagonist </li></ul>* * *
    39. 40. In Vivo Validation of Potentiator’s Effect & Specificity GTP γ S35 Autoradiography <ul><li>mGlu2 agonist effect is potentiated by potentiator 3-MPPTS </li></ul><ul><li>Potentiation is specific to regions high in mGluR2 distribution </li></ul><ul><li>mGluR2 potentiator does not have effect in mGluR2 K/O mice </li></ul>
    40. 41. <ul><li>Further in vivo validation was performed in animal behavioral models </li></ul><ul><ul><ul><li>Potentiated rat fear potentiated startle anxiety response </li></ul></ul></ul><ul><ul><ul><li>PCP motor evoked motor response </li></ul></ul></ul><ul><li>Characterization studies with chimeras and point mutations </li></ul>Further Preclinical Validation & Molecular Characterization of Potentiator
    41. 42. <ul><li>Can allosterically modulate orthosteric agonists </li></ul><ul><ul><ul><li>Functional & Biochemical Characterization </li></ul></ul></ul><ul><ul><ul><li>Specificity Established via chimeras/knock outs (but remember the TRP family) </li></ul></ul></ul><ul><ul><ul><li>Preclinical Validation </li></ul></ul></ul><ul><li>Clinical Benefit </li></ul><ul><ul><ul><li>May increase potency &/or therapeutic index </li></ul></ul></ul><ul><li>Clinical Risk </li></ul><ul><ul><ul><li>May create more variance in quantal effect </li></ul></ul></ul><ul><ul><ul><ul><li>“ Silent” genetic mutations within patient population </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Limit population demonstrating efficacy </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><li>“ Active” genetic mutations </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Bind unexpectedly to other receptors (“think” TRP family) </li></ul></ul></ul></ul></ul>CONCLUSIONS
    42. 43. MAINPOINTS <ul><li>Unique case where can use FLIPR without worrying about Kd (ie. assess spare receptors). </li></ul><ul><li>High expression recombinant systems can identify modulating systems which exist, but are: </li></ul><ul><ul><ul><ul><ul><li>undetectable in endogenous systems </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>unidentifiable via crystallography or binding studies.* </li></ul></ul></ul></ul></ul><ul><li>* Reinforced by AMPA study; transitional states are important for modulation </li></ul>
    43. 44. Molecular Determinants & Functional Consequences of Allosteric Modulation of Glutamate AMPA Receptors Eric S. Nisenbaum Neuroscience Discovery Research Eli Lilly Corp.
    44. 45. <ul><li>4 Subunits (mgluR1-4) of receptor </li></ul><ul><li>3 TM spanning regions, 4 form pores of channel </li></ul><ul><li>Crystallography of EC binding domain verifies clamshell configuration </li></ul><ul><li>Tetromers assembled from 4 different SUs form: </li></ul><ul><ul><li>homomeric receptors (identical receptors) </li></ul></ul><ul><ul><li>heteromeric receptors (nonidentical receptors) </li></ul></ul>AMPA RECEPTOR STRUCTURE
    45. 46. <ul><li>Each SU has splice variant region called flip flop region </li></ul><ul><ul><li>in folded protein is positioned at the hinge of clamshell configuration </li></ul></ul><ul><li>Flip & flop are isomers of the receptor: </li></ul><ul><ul><li>gives rise to different levels of desensitization </li></ul></ul><ul><ul><li>different susceptibilities to allosteric modulation </li></ul></ul>FLIP FLOP REGION is IMPORTANT
    46. 47. <ul><li>Agonist Binding Site </li></ul><ul><ul><li>Dimer of dimers </li></ul></ul><ul><ul><li>Unbound state channel is closed </li></ul></ul><ul><ul><li>Bound state </li></ul></ul><ul><ul><ul><li>agonist binds to closed state then channel opens </li></ul></ul></ul><ul><ul><ul><li>if bound a long time channel configures to agonist bound densensitized state. </li></ul></ul></ul>MODULATION of AMPA RECEPTORS
    47. 48. <ul><li>Allosteric Modulation </li></ul><ul><ul><li>Influence deactivation rate by: </li></ul></ul><ul><ul><ul><li>slow rate of deactivation via slowing transition to that state. </li></ul></ul></ul><ul><ul><ul><li>cyclothiazide will activate densensitized receptor. </li></ul></ul></ul><ul><ul><li>Most compounds effect both processes, but preferential for one or other </li></ul></ul>MODULATION of AMPA RECEPTORS
    48. 49. <ul><li>Substrates for fast excitatory neurotransmission in brain </li></ul><ul><li>Bind to open channel: </li></ul><ul><ul><li>EPSP occurring in postsynaptic dendrite </li></ul></ul><ul><li>If depolarization great enough: </li></ul><ul><ul><li>can remove Mg++ occlusion of NMDA receptor </li></ul></ul><ul><ul><li>increase Ca influx through channels as well as through voltage gated Ca channels. </li></ul></ul>HOW TARGET ALLOSTERIC MODULATORS?
    49. 50. <ul><li>Effect of Modulator (ie. Anaracitan) </li></ul><ul><ul><li>enhancement of ion flux through AMPA receptor. </li></ul></ul><ul><ul><li>facilitates potentiation of synaptic response. </li></ul></ul><ul><li>Importantly could facilitate: </li></ul><ul><ul><li>synaptic plasticity (ie. LTP). </li></ul></ul><ul><ul><li>Other postsynaptic changes (ie. recruitment of neurotrophic factors ). </li></ul></ul>HOW TARGET ALLOSTERIC MODULATORS?
    50. 51. <ul><li>Common </li></ul><ul><ul><li>Cognitive deficits & depression </li></ul></ul><ul><li>Novel </li></ul><ul><ul><li>Neurodegenerative Diseases </li></ul></ul><ul><li>Classes of Modulators: </li></ul><ul><ul><li>Cyclothiazide (benzothiadiazide group) </li></ul></ul><ul><ul><li>Puro inhibitines Anaracitan </li></ul></ul><ul><ul><li>Cortex compounds </li></ul></ul><ul><ul><li>Bioproposulfanomides </li></ul></ul><ul><ul><li>LY54330 </li></ul></ul>THERAPEUTIC TARGETS for ALLOSTERIC MODULATORS
    51. 52. <ul><li>Flipr assays: Potentiate Ca flux </li></ul><ul><ul><li>Q version of the receptor so will flux Calcium </li></ul></ul><ul><li>Homomeric mglur 1-4 receptors in flip or flop isoform </li></ul><ul><li>Modulator characteristics: </li></ul><ul><ul><li>preferential for gluR2 </li></ul></ul><ul><ul><li>splice variant preference for flip over flop isoform of receptor </li></ul></ul>WHAT ACCOUNTS for PREFERENTIAL SENSITIVITY? EXPERIMENTAL STRATEGY
    52. 53. <ul><li>Flip vs. Flop phenotypic response differences: </li></ul><ul><ul><li>Potentiate densensitized response in both flip & flop, although potency is much greater in flip. </li></ul></ul><ul><ul><li>Differences in kinetics: Flip isoform:time dependent enhancement </li></ul></ul><ul><li>Flop isoform:rapid increase to steady state </li></ul><ul><li>Determine molecular determinants of isoform response differences: </li></ul><ul><ul><li>What point mutations alter phenotypic response of isoform? </li></ul></ul>WHAT ACCOUNTS for PREFERENTIAL SENSITIVITY? EXPERIMENTAL STRATEGY
    53. 54. <ul><li>Robust potentiation of desensitized response </li></ul><ul><li>Preferential modulator of flip isoforms </li></ul><ul><li>Sequence homology between Flip & Flop </li></ul><ul><ul><li>Domain is 38 aa segment with EC S2 loop </li></ul></ul><ul><ul><li>Only @ 3 AA differ between 2 isoforms </li></ul></ul><ul><li>Which point mutations determine where sensitivity is being conferred? </li></ul><ul><ul><li>Subdivide into region 1,2,3 </li></ul></ul><ul><ul><li>Determine mutation altered phenotypic response </li></ul></ul><ul><li>Outcome </li></ul><ul><ul><li>Serine – Asparagine exchange account for cylclothiazide sensitivity . </li></ul></ul>WHAT ACCOUNTS for PREFERENTIAL SENSITIVITY? CHIMERIC APPROACH EXAMPLE: CYCLOTHIAZIDE
    54. 55. <ul><li>Flip-Flop Cassette Mutations: </li></ul><ul><ul><li>Divide AA in cassette into 2 regions </li></ul></ul><ul><ul><li>Make point mutations </li></ul></ul><ul><ul><li>Measure resulting response & compare to phenotype response </li></ul></ul><ul><li>Results: </li></ul><ul><ul><li>Mutate region 3 of gluR 2 Flip isoform to Flop sequence </li></ul></ul><ul><ul><ul><li>No resultant change in flip response </li></ul></ul></ul><ul><ul><li>Mutate other 4 AAs </li></ul></ul><ul><ul><ul><li>∆ in response from characteristic flip to flop response </li></ul></ul></ul><ul><ul><ul><li>decrease potency, kinetics typical of flop isoform </li></ul></ul></ul><ul><ul><li>Further subdivided region </li></ul></ul><ul><ul><ul><li>Exchanges: Serine/asparagines >> Valine/leucine </li></ul></ul></ul>WHAT ACCOUNTS for PREFERENTIAL SENSITIVITY? CHIMERIC APPROACH LILY COMPOUND
    55. 56. WHAT ACCOUNTS for PREFERENTIAL SENSITIVITY? CHIMERIC APPROACH LILY COMPOUND <ul><li>Conclusions: </li></ul><ul><ul><li>Serine/asparagine: </li></ul></ul><ul><ul><ul><li>Critical for conferring flop sensitivity although not completely! </li></ul></ul></ul><ul><ul><li>Valine/leucine: </li></ul></ul><ul><ul><ul><li>Must have synergistic effect to account for difference in response. </li></ul></ul></ul><ul><ul><li>Crystallography confirms results. </li></ul></ul>
    56. 57. <ul><li>Method </li></ul><ul><ul><li>Treat animals 24 hours after inducing lesion for 15 days </li></ul></ul><ul><ul><li>On Day 15 administered apomorphine to induce rotational behavior </li></ul></ul><ul><li>Results </li></ul><ul><ul><li>See robust contralateral turn. </li></ul></ul><ul><ul><li>Animals treated with Lily compound see attenuation in contralateral turning </li></ul></ul><ul><ul><li>Histopathology confirms behavioral data: </li></ul></ul><ul><ul><ul><li>increased TH staining that corroborate with recovery. </li></ul></ul></ul><ul><ul><li>Delayed dosing 3-6 days: </li></ul></ul><ul><ul><ul><li>can still see statistically significant recovery/protection. </li></ul></ul></ul><ul><ul><ul><li>TH staining within the stratum corroborates behavioral data. </li></ul></ul></ul><ul><li>Similar results with intrastriatal approach & mouse MPTP models: </li></ul><ul><ul><li>Sparing of DA neurons within Substantia Nigra </li></ul></ul><ul><ul><li>Stat. sig. increase in BDNF containing neurons in lesioned treated w/ AMPA potentiator. </li></ul></ul><ul><li>Data suggests AMPA modulators provide neuroprotection/regeneration. </li></ul>AMPA MODULATOR as NOVEL THERAPEUTIC PARKINSON’S DISEASE
    57. 58. <ul><li>Relevance of transitional states cannot be detected via crystallography. </li></ul><ul><ul><li>Interestingly only functional assay determine the existence & relevance of valine-leucine; structural study could not determine. </li></ul></ul><ul><ul><li>As with earlier example of potentiators, only functional assay can detect molecules which modulate functional response via transitional states. </li></ul></ul><ul><ul><li>Illustrates limitations of “static” structural studies. </li></ul></ul><ul><li>Now using this type of information to determine: </li></ul><ul><ul><li>Physiological states corresponding with splice variant selectivity. </li></ul></ul><ul><ul><li>Which disease states would benefit from the administration of AMPA modulators. </li></ul></ul><ul><li>Illustrates a novel use of channel targets as not only neurofunctional modulators, but as well neuroregenerative activators. </li></ul>Conclusions
    58. 59. Mainpoint of In Vivo Talks Other Potential Uses for Chromovert <ul><li>There appears to be a movement away from behavioral models towards more quantifiable readouts such as autoradiography. </li></ul><ul><li>A potential use of chromovert could be for tagged-GOI knock ins (?) Could then be used for primary culture assays and possibly ex vivo studies if a transfection method could be developed for primaries & tissue (don’t know if feasible & stability of mRNAs). </li></ul><ul><li>Another related use for knock ins would be an alternative to in situ hybridization (FISH), & enzymatic readout knock ins (ie. lacZ), depending on feasibility of transfection & microscopy method. Possibly more reliable than IHC, where establishing conditions, including fixative & sectioning method, is tedious, lengthy work, & often requires expertise for a successful outcome. Antibodies are often not good, & suddenly become unavailable as was the case with SHH Abs. Potentially could be a more user friendly, accurate & reliable method. </li></ul><ul><li>Finally there was a strong interest in crystallography, & possibly the field would be interested in a secreted protein version of their POI, particularly if quantity is important to crystallography. I do not know if this an acceptable method for protein crystallization; however the CBSE @ NASA has focus on crystallization & is seeking projects; importantly the director, Lawrence J. DeLucas worked with IFF on the first commercial product to be developed by the research program. </li></ul>