Optimizing The Clinical Efficacy Of Opioids By TLR4 Blockade
Hang Yin
Chemistry And Biochemistryuniversity Of Colorado At Boulder
Grant 1R21DA026950-01 from National Institute On Drug Abuse IRG: ZDA1
Abstract: Opioid-induced glial activation, which compromises pain treatment and contributes to the development of drug addiction and abuse, is regulated via a signaling pathway downstream of toll-like receptor-4 (TLR4), a membrane spanning receptor that functions in complex with its accessory protein MD-2. As current opioid pharmacotherapeutics have failed to control pain while avoiding the negative consequences, there is an urgent need to understand opioid dysregulation via TLR4. The central hypothesis of the current proposal is that disruption of the TLR-4/MD-2 complex formation can inhibit opioid-induced glial activation, thereby enhancing analgesia and reducing opioid tolerance and dependence. The rationale underlying the proposed research is that the identified inhibitors, which selectively block the critical protein-protein interactions between TLR4 and MD-2, will provide a useful tool for investigating the role of the TLR4-mediated signaling pathway in glial activation. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The proposed high risk/high reward approach, if successful, is projected to yield significant novel outcomes. First, the results will shed light on the mechanism of the clinically relevant opioid-induced glial activation. Second, if successful, the peptide and peptidomimetic antagonists of the TLR4/MD-2 interactions identified in the proposed research can serve as prototypes for more drug-like small-molecule inhibitors. These inhibitors may eventually find application in the development of novel therapeutics to enhance the clinical efficacy of opioid analgesics and to treat opioid addiction and abuse, as well as other clinically relevant indications. The proposed studies are built on a strong collaborative team with expertise that optimizes its chance to effectively bridge between atomic detail of the TLR4/MD-2 interaction and its macroscopic effect, namely pain management and avoiding negative consequences of opoid use. In Aim 1, antagonists of TLR4 or MD-2 that block the TLR4/MD-2 complex formation will be developed using a cutting-edge computational technology. The working hypothesis here is that conformationally strained peptides derived from the binding region can compete with the full-length protein and thereby inhibit the TLR4/MD-2 interaction. These peptides can serve as starting points for the computational design of stronger inhibitors. In Aim 2, the second working hypothesis, that the inhibitors of the TLR4/MD-2 interactions can non-competitively prevent opioids from inducing TLR4-mediated glial activation, will be tested. Cellular assays and animal models will be used to evaluate the inhibition of glial activation by the TLR4 antagonists both in vitro and in vivo. The proposed research is significant because it is expected to establish the TLR4/MD-2 protein-protein complex as a novel therapeutic target for optimizing opioid analgesia while preventing and treating opioid abuse. Regarding its positive impact on scientific advancements, this work will (1) improve scientific understanding of drug dependence and pain suppression and (2) allow the development of a new generation of therapeutics. The proposed research aims to unravel the mechanism of opioid-induced glial activation that both hinders the ability of opioids to effectively control pain and also importantly contributes to the development of drug addiction and abuse. State-of-the-art technologies will be employed to define, design, create, and test new chemical entities predicted to prevent opioid induced glial activation, thereby optimizing opioid analgesia while preventing negative consequences of clinical opioid use
Project start date: 2009-07-01
Project end date: 2011-06-30
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Hang Yin
PROBING OPIOID-INDUCED GLIAL ACTIVATION WITH PEPTIDE ANTAGONISTS
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 1R03DA027977-01 from National Institute On Drug Abuse
Abstract: The pharmacological treatment of pain has long been limited by the negative side effects of opioids development of tolerance, dependence, and possibility of overdose. A literature has developed linking opiate side effects to their influence on glial cells of the central nervous system (CNS). Herein lies a proposal to investigate the role of an opiate-mediated glial activation, via the signaling pathway mediated by toll-like receptor 4 (TLR4). TLR4, an integral membrane receptor expressed in glia but no in neurons within the CNS, functions in complex with its accessory protein, Myeloid Differentiation protein-2 (MD-2). The TLR4/MD-2 complex is crucial to the TLR4-signaling transduction. The proposal´s central hypothesis is that inhibition of the TLR4/MD-2 association will impede the TLR4-signaling pathway, thereby preventing the negative side effects from opiate-induced glial activation. By selectively blocking the critical protein-protein interactions between TLR4 and MD-2, opiate tolerance and dependence is predicted to attenuate, thereby increasing the efficacy of current pain pharmacotherapies. This approach is innovative, as it is the first proposal aimed at the inhibition of glial- mediated opioid side effects. Further, the research is expected to yield significant outcomes (1) inhibitors of the TLR4/MD-2 interaction will be prototypes for the development of drugs to counteract opioid side effects from tolerance to addiction and overdose. The TLR4/MD-2 interaction is also implicated in other pathologies (e.g. sepsis), and will therefore serve potential targets for various diseases. (2) Importantly, antagonists of the TLR4/MD-2 interaction will elucidate the contribution of the TLR4 pathway to opiate-induced glial activation. The inhibitors will help determine the mechanism of action of the TLR4 pathway itself, shedding light on the molecular specificity with which TLR4 recognizes its ligands. The proposed studies are built on a strong collaborative team with a spectrum of expertise covering from protein design, biochemistry and biophysical assay development, x-ray structural analysis, and animal models for pain management. The proposed studies employ computationally designed peptides derived from the TLR4-bidning regions of MD-2, which is expected to compete with the full-length MD-2 protein and prevent the TLR4 signal transduction. These peptides will provide starting points for the small-molecule inhibitors. The significance of this work lies in its impacts on both clinical and scientific advancement. Dissecting the mechanism of opiate-induced glial activation will help us understand the development of opioid tolerance and addiction, as well as establish a novel angle from which to address drug dependence and abusing of these opiates. Regarding its impact on scientific advancement, the proposed studies will illuminate the molecular mechanism of TLR4 activation, which is relevant to a many interrelated signaling and immunomodulatory pathways, and crucial to the understanding of pain suppression. The proposed research aims to unravel the mechanism of opioid-induced glial activation that both hinders the ability of opioids to effectively control pain and also importantly contributes to the development of drug addiction and abuse. State-of-the-art technologies will be employed to define, design, create, and test new chemical entities predicted to prevent opioid induced glial activation, thereby optimizing opioid analgesia while preventing negative consequences of clinical opioid use
Keywords: Absence of pain sensation; Absence of sensibility to pain; Addiction, Drug; Addiction, Opiate; Address; Adverse effects; Analgesics, Opioid; Animal Model; Animal Models and Related Studies; Arts; Assay; Attenuated; Binding; Binding (Molecular Function); Bioassay; Biochemistry; Biologic Assays; Biological Assay; Biology; Cell Communication and Signaling; Cell Signaling; Cellular Assay; Central Nervous System; Chemical Dependence; Chemicals; Chemistry, Biological; Clinical; Collaborations; Complex; Dependence; Dependence, Drug; Dependence, Opiate; Development; Disease; Disorder; Drug Addiction; Drug Dependency; Drug Therapy; Drugs; Feels no pain; Generations; Glia; Glial Cells; Goals; In Vitro; Intracellular Communication and Signaling; Knowledge; Kolliker`s reticulum; Length; Letters; Ligands; Light; Link; Literature; Mediating; Medication; Membrane; Molecular; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Myelogenous; Myeloid; Nerve Cells; Nerve Unit; Nervous System Physiology; Nervous System, CNS; Neural Cell; Neuraxis; Neurobiology; Neurocyte; Neuroglia; Neuroglial Cells; Neurologic function; Neurological function; Neurons; No sensitivity to pain; Non-neuronal cell; Opiate Addiction; Opiates; Opioid; Opioid Analgesics; Opioid Receptor; Outcome; Overdose; Pain; Pain Control; Pain Therapy; Pain management; Painful; Pathology; Pathway interactions; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacotherapy; Photoradiation; Plant Embryos; Proteins; Receptor Protein; Receptors, Opiate; Research; Role; Seeds; Sepsis; Services; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Specificity; Structure; TLR4 receptor; Technology; Testing; Toll-4 receptor; Treatment Side Effects; Work; Zygotes, Plant; addiction; analgesia; assay development; biological signal transduction; bloodstream infection; clinical applicability; clinical application; clinical efficacy; clinical relevance; clinically relevant; conformation; conformational state; design; designing; disease/disorder; drug development; drug/agent; gene product; high reward; high risk; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; insight; membrane structure; model organism; molecular recognition; nerve cement; nervous system function; neurobiological; neuronal; neuropathic pain; new therapeutics; next generation therapeutics; novel; novel therapeutics; opiate abuse; opioid abuse; opioid addiction; opioid dependence; painful neuropathy; pathway; prevent; preventing; protein protein interaction; prototype; public health relevance; receptor; receptor function; scaffold; scaffolding; seed; side effect; small molecule; social role; therapy adverse effect; toll-like receptor 4; treatment adverse effect
Relevance: The proposed research aims to unravel the mechanism of opioid-induced glial activation that both hinders the ability of opioids to effectively control pain and also importantly contributes to the development of drug addiction and abuse. State-of-the-art technologies will be employed to define, design, create, and test new chemical entities predicted to prevent opioid induced glial activation, thereby optimizing opioid analgesia while preventing negative consequences of clinical opioid use
Project start date: 2009-09-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2009
Budget end date: 31-AUG-2010
PFA/PA: RFA-DA-09-021
1R03DA027977-01 (2009): $37875
Transforming Clinical Pain Control By Targeting A Novel Non-Neuronal Receptor
Hang Yin
Chemistry And Biochemistryuniversity Of Colorado At Boulder
Grant 1R03DA025740-01 from National Institute On Drug Abuse IRG: ZRG1
Abstract: Opioid-induced glial activation, which compromises pain treatment and contributes to the development of drug addiction and abuse, is regulated via a signaling pathway downstream of toll-like receptor-4 (TLR4), a membrane spanning receptor that functions in complex with its accessory protein MD-2. As current opioid pharmacotherapeutics have failed to control pain while avoiding the negative consequences, there is an urgent need to understand opioid dysregulation via TLR4. The central hypothesis of the current proposal is that disruption of the TLR-4/MD-2 complex formation can inhibit opioid-induced glial activation, thereby enhancing analgesia and reducing opioid tolerance and dependence. The rationale underlying the proposed research is that the identified TLR4 inhibitors, which selectively block the critical protein-protein interactions between TLR4 and MD-2, will provide a useful tool for investigating the role of the TLR4-mediated signaling pathway in glial activation. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The proposed high risk/high reward approach, if successful, is projected to yield significant novel outcomes. First, the results will shed light on the mechanism of the clinically relevant opioid-induced glial activation. Second, if successful, the peptide and peptidomimetic antagonists of the TLR4/MD-2 interactions identified in the proposed research can serve as prototypes for more drug-like small-molecule inhibitors. These inhibitors may eventually find application in the development of novel therapeutics to enhance the clinical efficacy of opioid analgesics and to treat opioid addiction and abuse, as well as other clinically relevant indications. The proposed studies are built on the complementary strength of the PI in the design, synthesis, and evaluation of novel protein-protein interaction inhibitors, and of the Co-PI, who has extensive expertise in glial neurobiology and will provide support in animal testing of the identified inhibitors. In Aim 1, antagonists of TLR4 that block the TLR4/MD-2 complex formation will be developed. The working hypothesis here is that conformationally strained peptides derived from the TLR4-binding region of MD-2 can compete with the full-length MD-2 protein and thereby inhibit the TLR4/MD-2 interaction. In Aim 2, the second working hypothesis, that the inhibitors of the TLR4/MD-2 interactions can non-competitively antagonize opioids to block TLR4-mediated glial activation, will be tested. Cellular assays and animal models will be used to evaluate the inhibition of glial activation by the TLR4 antagonists both in vitro and in vivo. The proposed research is significant because it is expected to establish the TLR4/MD-2 protein-protein complex as a novel therapeutic target for preventing and treating opioid abuse. Regarding its positive impact on scientific advancements, this work will (1) improve scientific understanding of drug dependence and pain suppression and (2) allow the development of a new generation of therapeutics. We aim to unravel the mechanism of opioid-induced glial activation that importantly contributes to the development of drug addiction and abuse. As an outcome of the proposed investigations, we expect to attain a better understanding of the molecular mechanisms of opioid-induced glial activation, and to clarify novel targets to regulate such activation. The intellectual merit of the proposed work lies not only in its scientific advancements in the field of chemical biology and protein engineering, but also in its potential impact on clinical applications. This work will (1) improve scientific understanding of drug dependence and pain suppression; and (2) allow the development of a new generation of therapeutics
Project start date: 2009-05-15
Project end date: 2011-04-30
Probing EBV-LMP-1´s Transmembrane Activation Domain With Synthetic Peptide Antago
Hang Yin
Chemistry And Biochemistryuniversity Of Colorado At Boulder
Grant 1R21CA138373-01 from National Cancer Institute IRG: ZCA1
Abstract: Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains with high affinity and selectivity. This study utilizes this cutting edge technology to study the activation mechanism of oncogenic Latent Membrane Protein 1 (LMP-1) of Epstein-Barr virus (EBV). EBV is a human tumor virus associated with a number of malignancies and lymphoproliferative syndromes. EBV´s ability to infect and immortalize B lymphocytes underlies its contribution to human disease. EBV´s transforming activity depends on the expression and activity of LMP-1, the viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 functions as a constitutively active Tumor Necrosis Factor Receptor (TNFR) whose activity requires the function of its hydrophobic transmembrane domain. LMP-1 most resembles the TNFR CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1´s activity is constitutive and ligand-independent. Constitutive homo-oligomerization and lipid raft association, activities of LMP-1´s transmembrane domain, play a key role in activation of downstream signaling. This proposal focuses on LMP-1 as a model membrane protein target for the design of peptide inhibitors because of LMP-1´s essential role in EBV-dependent B cell transformation, LMP-1´s contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and EBV´s dependence on LMP-1´s hydrophobic transmembrane domain for activity. This study aims to develop an innovative approach to target LMP-1´s transmembrane domain, using CHAMP-designed anti-peptide antagonists as probes to study the contribution of oligomerization and raft association to LMP-1 activation, with the goal of inhibiting downstream signaling. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes, will reveal the mechanism of LMP-1´s constitutive activation of signaling, and will be applicable to the future development of novel therapeutics targeting diseases dependent on critical transmembrane proteins. Specifically, this proposal addresses the following Aims 1) Can anti-TMD-1 peptides probes be developed that have high affinity and specificity for LMP-1´s TMD-1? 2) Do identified peptides bind specifically and with affinity to LMP-1´s TMD-1 in vitro? and 3) Can peptides that target TMD-1 (identified in Aims 1 and 2) interfere with LMP-1 homo-oligomerization, raft association, and constitutive signaling in intact cells?
Project start date: 2009-04-10
Project end date: 2011-03-31
3R21CA138373-01S3 (2009): $54050
3R21CA138373-01S2 (2009): $49535
3R21CA138373-01S1 (2009): $45604