PROBING EBV-LMP-1´S TRANSMEMBRANE ACTIVATION DOMAIN WITH SYNTHETIC PEPTIDE ANTAGO
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 3R21CA138373-02S1 from National Cancer Institute
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?
Relevance: . Many poorly characterized human cancers develop, in part, due to the activity of oncogenic transmembrane proteins which, because of the inability to target protein domains within the membrane bilayer, are inaccessible to molecular probes to study function and are poor targets for current drug treatment methods. This study examines a novel class of rationally engineered peptides capable of interacting with membrane-embedded protein domains for use in the study of oncogenic transmembrane proteins, and potentially for the future development of new therapeutic approaches to the prevention and treatment of such cancers. The Latent Membrane Protein-1 (LMP-1), an oncoprotein expressed by the human tumor virus Epstein-Barr virus (EBV), will be used as a model membrane protein target for the design of such peptide inhibitors, because of its contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and because of EBV´s dependence on LMP-1´s hydrophobic transmembrane domain for transformation
Project start date: 2009-04-10
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: RFA-CA-08-007
3R21CA138373-02S1 (2010): $57679
Sponsored Links Excellgen http://Excellgen.com
PROBING EBV-LMP-1´S TRANSMEMBRANE ACTIVATION DOMAIN WITH SYNTHETIC PEPTIDE ANTAGO
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 3R21CA138373-02S2 from National Cancer Institute
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?
Relevance: . Many poorly characterized human cancers develop, in part, due to the activity of oncogenic transmembrane proteins which, because of the inability to target protein domains within the membrane bilayer, are inaccessible to molecular probes to study function and are poor targets for current drug treatment methods. This study examines a novel class of rationally engineered peptides capable of interacting with membrane-embedded protein domains for use in the study of oncogenic transmembrane proteins, and potentially for the future development of new therapeutic approaches to the prevention and treatment of such cancers. The Latent Membrane Protein-1 (LMP-1), an oncoprotein expressed by the human tumor virus Epstein-Barr virus (EBV), will be used as a model membrane protein target for the design of such peptide inhibitors, because of its contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and because of EBV´s dependence on LMP-1´s hydrophobic transmembrane domain for transformation
Project start date: 2009-04-10
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: RFA-CA-08-007
3R21CA138373-02S2 (2010): $57679
3R21CA138373-01S3 (2009): $54050
3R21CA138373-01S2 (2009): $49535
3R21CA138373-01S1 (2009): $45604
Grants awarded to Hang Yin
PROBING OPIOID-INDUCED GLIAL ACTIVATION WITH PEPTIDE ANTAGONISTS
Hang Yin
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 5R03DA027977-02 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; 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; opiate tolerance; 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-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-DA-09-021
5R03DA027977-02 (2010): $37496
1R03DA027977-01 (2009): $37875
DEVELOPING SMALL-MOLECULE PROBES FOR OPIOID-INDUCED GLIAL ACTIVATION
Hang Yin
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 5R21NS067425-02 from National Institute Of Neurological Disorders And Stroke
Abstract: The pharmacological treatment of pain has long been limited by the negative side effects of opioids development of tolerance, dependence, and danger of overdose. A literature has developed linking opiate side effects to their influence on glial cells within the central nervous system (CNS). These effects have been shown to result from toll-like receptor 4 (TLR4)-mediated glial activation, which causes both pain enhancement and opioid tolerance and dependence. As such, there is an urgent need to understand opioid dysregulation via TLR4. The objective of this current proposal is to optimize a small-molecule probe identified from virtual screening, which in turn will be used to study glial activation and its impact on opioid effectiveness. The rationale underlying this research is that optimized small molecule TLR4 inhibitors can serve as highly specific probes to study the molecular mechanism of opioid-induced glial activation. The proposed research is significant because the optimized small molecule agents will preclude the development of novel therapeutics to increase opiate efficacy and safety. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The studies are built on a strong collaborative team with expertise that optimizes its chance to effectively bridge the atomic detail of TLR4 activation with the macroscopic pain management inefficiencies of opioid use. In Aim 1, extensive structure-activity relationship studies will be carried out to optimize the lead compound indentified from in silico screening. Established in vitro biophysical and cellular assays will then be used to evaluate synthesized small molecule agents for their potency in blocking TLR4 activation. Crystallization of TLR4 in complex with the small-molecule ligands will be attempted in a parallel effort to elucidate the structural geometry of TLR4 inhibition. These results will shed light on the molecular recognition in the ligand/receptor association, thereby providing insights for the optimization of the first generation small molecule inhibitors. Aim 2 will test the second working hypothesis, that by inhibiting opioid-induced TLR4 activation, glial activation can also be blocked, thus enhancing analgesia as well as reduce tolerance and dependence on opioids. The proposed studies, if successful, are projected to yield significant novel outcome First, the results will shed light on the mechanism of clinically relevant opioid-induced glial activation. Second, the small molecule antagonists of TLR4 identified from the proposed research will serve as prototypes for potential drug candidates. These inhibitors may be clinically useful in the treatment of opioid side effects, addressing a public heath issue that encompasses opioid addiction, tolerance, and abusing. 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: 3-Hepta, 6-(dimethylamino)-4, 4-diphenyl-; 4-Piperidinecarboxylic acid, 1-methyl-4-phenyl-, ethyl ester; Absence of pain sensation; Absence of sensibility to pain; Adanon; Addiction, Drug; Addiction, Opiate; Address; Adverse effects; Althose; Animal Model; Animal Models and Related Studies; Animal Testing; Arts; Basic Research; Basic Science; Biology; Blood - brain barrier anatomy; Blood-Brain Barrier; Cellular Assay; Central Nervous System; Chemical Dependence; Chemicals; Clinical; Complex; Computer Simulation; Computerized Models; Contin, MS; Crystallization; Demerol; Dependence; Dependence, Drug; Dependence, Opiate; Development; Dihydrohydroxycodei; Dolophine; Drug Addiction; Drug Dependency; Drug Kinetics; Drugs; Effectiveness; Feels no pain; Generations; Glia; Glial Cells; Goals; Hemato-Encephalic Barrier; In Vitro; Infumorph; Isonipecain; Kadian; Kolliker`s reticulum; Lead; Libraries; Ligands; Light; Link; Literature; MSir; Mathematical Model Simulation; Mathematical Models and Simulations; Mediating; Medication; Membrane; Meperidine; Methadone; Methadose; Models, Computer; Molecular; Morphia; Morphinan-3, 6-diol, 7, 8-didehydro-4, 5-epoxy-17-methyl- (5alpha, 6alpha)-; Morphinan-6-one, 4, 5-epoxy-14-hydroxy-3-methoxy-17-methyl-, (5alpha)-; Morphine; Myelogenous; Myeloid; Nerve Cells; Nerve Unit; Nervous System, CNS; Neural Cell; Neuraxis; Neurobiology; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; No sensitivity to pain; Non-neuronal cell; Opiate Addiction; Opiates; Opioid; Oramorph; Oramorph SR; Outcome; Overdose; Oxycodeinon; Oxycodone; Oxycodone SR; Oxycontin; Pain; Pain Control; Pain Therapy; Pain management; Painful; Pb element; Permeability; Pethidine; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacokinetics; Pharmacological Treatment; Photoradiation; Prevention; Property; Property, LOINC Axis 2; Proteins; Receptor Protein; Research; Resolution; Roxanol; Roxicodone; Safety; Screening procedure; Signal Pathway; Simulation, Computer based; Statex SR; Structure; Structure-Activity Relationship; TLR4 receptor; Technology; Testing; Therapeutic; Toll-4 receptor; Treatment Side Effects; Validation; Work; addiction; analgesia; base; chemical structure function; clinical efficacy; clinical relevance; clinically relevant; computational chemistry; computational modeling; computational models; computational simulation; computer based models; computerized modeling; computerized simulation; design; designing; drug candidate; drug development; drug discovery; drug/agent; gene product; heavy metal Pb; heavy metal lead; high reward; high risk; improved; in silico; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; insight; interest; membrane structure; model organism; molecular recognition; nerve cement; neurobiological; neuronal; new therapeutics; next generation therapeutics; novel; novel therapeutics; opiate abuse; opioid abuse; opioid addiction; opioid dependence; prevent; preventing; prototype; public health relevance; receptor; receptor function; response; screening; screenings; side effect; small molecule; structure function relationship; therapy adverse effect; toll-like receptor 4; treatment adverse effect; virtual; virtual simulation
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: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-NS-09-003
5R21NS067425-02 (2010): $224978
1R21NS067425-01 (2009): $227250
TRANSFORMING CLINICAL PAIN CONTROL BY TARGETING A NOVEL NON-NEURONAL RECEPTOR
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 5R03DA025740-02 from National Institute On Drug Abuse
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
Keywords: 3-Hepta, 6-(dimethylamino)-4, 4-diphenyl-; 4-Piperidinecarboxylic acid, 1-methyl-4-phenyl-, ethyl ester; Absence of pain sensation; Absence of sensibility to pain; Adanon; Addiction, Drug; Addiction, Opiate; Althose; Analgesics, Opioid; Animal Model; Animal Models and Related Studies; Animal Testing; Basic Research; Basic Science; Binding; Binding (Molecular Function); Biology; Blood; Cell Communication and Signaling; Cell Signaling; Cellular Assay; Chemical Dependence; Chemicals; Chemistry, Pharmaceutical; Clinical; Complex; Contin, MS; Demerol; Dependence; Dependence, Drug; Dependence, Opiate; Development; Dihydrohydroxycodei; Dolophine; Drug Addiction; Drug Dependency; Drugs; Evaluation; Feels no pain; Future; Generations; Genetic Engineering of Proteins; Glia; Glial Cells; Goals; In Vitro; Infumorph; Intracellular Communication and Signaling; Investigation; Isonipecain; Kadian; Kolliker`s reticulum; LY96 protein; Length; Light; Lymphocyte Antigen 96; MD-2 protein; MSir; Measures; Mediating; Medication; Medicinal Chemistry; Membrane; Meperidine; Methadone; Methadose; Molecular; Molecular Interaction; Morphia; Morphinan-3, 6-diol, 7, 8-didehydro-4, 5-epoxy-17-methyl- (5alpha, 6alpha)-; Morphinan-6-one, 4, 5-epoxy-14-hydroxy-3-methoxy-17-methyl-, (5alpha)-; Morphine; Nerve Cells; Nerve Unit; Neural Cell; Neurobiology; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; No sensitivity to pain; Non-neuronal cell; Opiate Addiction; Opioid; Opioid Analgesics; Oramorph; Oramorph SR; Outcome; Oxycodeinon; Oxycodone; Oxycodone SR; Oxycontin; Pain; Pain Control; Pain Therapy; Pain management; Painful; Peptides; Pethidine; Pharmaceutic Chemistry; Pharmaceutic Preparations; Pharmaceutical Chemistry; Pharmaceutical Preparations; Photoradiation; Preclinical Testing; Protein Engineering; Receptor Protein; Regulation; Research; Reticuloendothelial System, Blood; Role; Roxanol; Roxicodone; Series; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Statex SR; Syndrome; System; System, LOINC Axis 4; TLR4 receptor; Testing; Therapeutic; Toll-4 receptor; Validation; Work; analgesia; biological signal transduction; clinical applicability; clinical application; clinical efficacy; clinical relevance; clinically relevant; design; designing; drug development; drug discovery; drug/agent; high reward; high risk; improved; in vitro Assay; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; insight; interest; membrane structure; model organism; nerve cement; neurobiological; neuronal; new approaches; new therapeutic target; new therapeutics; next generation therapeutics; novel; novel approaches; novel strategies; novel strategy; novel therapeutics; opiate abuse; opioid abuse; opioid addiction; opioid dependence; peptidomimetics; prevent; preventing; protein complex; protein protein interaction; prototype; public health relevance; receptor; receptor function; response; small molecule; social role; therapeutic development; toll-like receptor 4; tool
Relevance: 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
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PAS-07-327
5R03DA025740-02 (2010): $189375
OPTIMIZING THE CLINICAL EFFICACY OF OPIOIDS BY TLR4 BLOCKADE
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 5R21DA026950-02 from National Institute On Drug Abuse
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
Keywords: 3-Hepta, 6-(dimethylamino)-4, 4-diphenyl-; 4-Piperidinecarboxylic acid, 1-methyl-4-phenyl-, ethyl ester; Absence of pain sensation; Absence of sensibility to pain; Adanon; Addiction, Drug; Addiction, Opiate; Address; Adverse effects; Althose; Animal Model; Animal Models and Related Studies; Arts; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biology; Blood - brain barrier anatomy; Blood-Brain Barrier; Cell Communication and Signaling; Cell Signaling; Cellular Assay; Chemical Dependence; Chemicals; Chemistry, Pharmaceutical; Clinical; Complex; Contin, MS; Demerol; Dependence; Dependence, Drug; Dependence, Opiate; Development; Dihydrohydroxycodei; Dolophine; Drug Addiction; Drug Dependency; Drugs; Feels no pain; Generations; Glia; Glial Cells; Goals; Hemato-Encephalic Barrier; In Vitro; Infumorph; Intracellular Communication and Signaling; Isonipecain; Kadian; Kolliker`s reticulum; LY96 protein; Length; Ligands; Light; Link; Lymphocyte Antigen 96; MD-2 protein; MSir; Maintenance; Maintenances; Mediating; Medication; Medicinal Chemistry; Membrane; Meperidine; Methadone; Methadose; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Morphia; Morphinan-3, 6-diol, 7, 8-didehydro-4, 5-epoxy-17-methyl- (5alpha, 6alpha)-; Morphinan-6-one, 4, 5-epoxy-14-hydroxy-3-methoxy-17-methyl-, (5alpha)-; Morphine; Nerve Cells; Nerve Unit; Neural Cell; Neurobiology; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; No sensitivity to pain; Non-neuronal cell; Opiate Addiction; Opioid; Opioid Analgesics; Opioid Receptor; Oramorph; Oramorph SR; Outcome; Oxycodeinon; Oxycodone; Oxycodone SR; Oxycontin; Pain; Pain Control; Pain Therapy; Pain management; Painful; Peptide Synthesis; Peptides; Pethidine; Pharmaceutic Chemistry; Pharmaceutic Preparations; Pharmaceutical Chemistry; Pharmaceutical Preparations; Photoradiation; Proteins; Protocol; Protocols documentation; Receptor Activation; Receptor Protein; Receptors, Opiate; Regulation; Research; Rewards; Role; Roxanol; Roxicodone; Services; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Statex SR; Structural Protein; Structure; Syndrome; TLR4 receptor; Technology; Testing; Therapeutic; Toll-4 receptor; Treatment Side Effects; Work; analgesia; assay development; biological signal transduction; chronic pain; chronic painful condition; clinical applicability; clinical application; clinical efficacy; clinical relevance; clinically relevant; conformation; conformational state; design; designing; drug development; drug discovery; drug/agent; gene product; high reward; high risk; improved; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; insight; membrane structure; model organism; molecular recognition; nerve cement; neurobiological; neuronal; neuropathic pain; new therapeutic target; new therapeutics; next generation; next generation therapeutics; novel; novel therapeutics; opiate abuse; opioid abuse; opioid addiction; opioid dependence; painful neuropathy; peptidomimetics; prevent; preventing; protein complex; protein protein interaction; prototype; public health relevance; receptor; receptor function; scaffold; scaffolding; side effect; small molecule; social role; therapy adverse effect; toll-like receptor 4; tool; 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: 2009-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PAR-06-209
5R21DA026950-02 (2010): $137227
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
OPTIMIZING THE CLINICAL EFFICACY OF OPIOIDS BY TLR4 BLOCKADE
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 1R21DA026950-01 from National Institute On Drug Abuse
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
Keywords: 3-Hepta, 6-(dimethylamino)-4, 4-diphenyl-; 4-Piperidinecarboxylic acid, 1-methyl-4-phenyl-, ethyl ester; Absence of pain sensation; Absence of sensibility to pain; Adanon; Addiction, Drug; Addiction, Opiate; Address; Adverse effects; Althose; Analgesics, Opioid; Animal Model; Animal Models and Related Studies; Arts; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biology; Blood - brain barrier anatomy; Blood-Brain Barrier; Cell Communication and Signaling; Cell Signaling; Cellular Assay; Chemical Dependence; Chemicals; Chemistry, Pharmaceutical; Clinical; Complex; Contin, MS; Demerol; Dependence; Dependence, Drug; Dependence, Opiate; Development; Dihydrohydroxycodei; Dolophine; Drug Addiction; Drug Dependency; Drugs; Feels no pain; Generations; Glia; Glial Cells; Goals; Hemato-Encephalic Barrier; In Vitro; Infumorph; Intracellular Communication and Signaling; Isonipecain; Kadian; Kolliker`s reticulum; LY96 protein; Length; Ligands; Light; Link; Lymphocyte Antigen 96; MD-2 protein; MSir; Maintenance; Maintenances; Mediating; Medication; Medicinal Chemistry; Membrane; Meperidine; Methadone; Methadose; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Morphia; Morphinan-3, 6-diol, 7, 8-didehydro-4, 5-epoxy-17-methyl- (5alpha, 6alpha)-; Morphinan-6-one, 4, 5-epoxy-14-hydroxy-3-methoxy-17-methyl-, (5alpha)-; Morphine; Nerve Cells; Nerve Unit; Neural Cell; Neurobiology; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; No sensitivity to pain; Non-neuronal cell; Opiate Addiction; Opioid; Opioid Analgesics; Opioid Receptor; Oramorph; Oramorph SR; Outcome; Oxycodeinon; Oxycodone; Oxycodone SR; Oxycontin; Pain; Pain Control; Pain Therapy; Pain management; Painful; Peptide Synthesis; Peptides; Pethidine; Pharmaceutic Chemistry; Pharmaceutic Preparations; Pharmaceutical Chemistry; Pharmaceutical Preparations; Photoradiation; Proteins; Protocol; Protocols documentation; Receptor Activation; Receptor Protein; Receptors, Opiate; Regulation; Research; Rewards; Role; Roxanol; Roxicodone; Services; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Statex SR; Structural Protein; Structure; Syndrome; TLR4 receptor; Technology; Testing; Therapeutic; Toll-4 receptor; Treatment Side Effects; Work; analgesia; assay development; biological signal transduction; chronic pain; chronic painful condition; clinical applicability; clinical application; clinical efficacy; clinical relevance; clinically relevant; conformation; conformational state; design; designing; drug development; drug discovery; drug/agent; gene product; high reward; high risk; improved; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; insight; membrane structure; model organism; molecular recognition; nerve cement; neurobiological; neuronal; neuropathic pain; new therapeutic target; new therapeutics; next generation; next generation therapeutics; novel; novel therapeutics; opiate abuse; opioid abuse; opioid addiction; opioid dependence; painful neuropathy; peptidomimetics; prevent; preventing; protein complex; protein protein interaction; prototype; public health relevance; receptor; receptor function; scaffold; scaffolding; side effect; small molecule; social role; therapy adverse effect; toll-like receptor 4; tool; 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: 2009-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PAR-06-209
1R21DA026950-01 (2009): $136003
PROBING EBV-LMP-1´S TRANSMEMBRANE ACTIVATION DOMAIN WITH SYNTHETIC PEPTIDE ANTAGO
Hang Yin, Assistant Professor
University Of Colorado At Boulder, 572 Ucb, Boulder, Co 80309
Grant 5R21CA138373-02 from National Cancer Institute
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?
Keywords: ATGN; Address; Affect; Affinity; Antigens; Approaches to prevention; Artificial Membranes; Arts; B Cell Proliferation; B blood cells; B-Cell Activation; B-Cells; B-Lymphocytes; Binding; Binding (Molecular Function); Bp50; Burkitt Herpesvirus; Burkitt Lymphoma Virus; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; CD40; CDW40; Cachectin Receptors; Cancer, Oncology; Cancers; Cell Communication and Signaling; Cell Signaling; Cells; Cellular Membrane; Computing Methodologies; Cytoplasm; Dependence; Detergents; Development; Disease; Disorder; Drugs; E-B Virus; EB virus; EBV; Engineering; Engineerings; Environment; Epstein Barr Virus; Epstein-Barr Virus; Epstein-Barr virus LMP-1 protein; Epstein-Barr virus latent membrane protein 1; Family member; Future; Genetic Engineering of Proteins; Germinoblastoma; Goals; HHV-4; Herpesviridae; Herpesvirus 4 (gamma), Human; Herpesvirus 4, Human; Herpesviruses; Homo; Human; Human Herpesvirus 4; Human, General; Hydrogen Oxide; Immune system; In Vitro; Individual; Infectious Mononucleosis Virus; Integral Membrane Protein; Intracellular Communication and Signaling; Intrinsic Membrane Protein; LMP-1 protein, EBV; LMP-1 protein, Epstein-Barr virus; LMP1 protein, EBV; Lead; Libraries; Life; Ligands; Light; Lipid Rafts, Cell Membrane; Lymphoma; Lymphoma (Hodgkin`s and Non-Hodgkin`s); Lymphoma, Malignant; Lymphoproliferative Disorders; MGC9013; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Mediating; Medication; Membrane; Membrane Biology; Membrane Microdomains; Membrane Proteins; Membrane-Associated Proteins; Membranes, Artificial; Memory B Cell; Memory B-Lymphocyte; Methods; Modeling; Molecular Interaction; Molecular Probes; Molecular Target; Oncogene Products; Oncogene Proteins; Oncogenic; Oncogenic Viruses; Oncoproteins; Pathway interactions; Pb element; Peptide Domain; Peptide Synthesis; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphatides; Phospholipids; Photoradiation; Play; Prevention approach; Prevention strategy; Preventive strategy; Process; Protein Biochemistry; Protein Domains; Protein Engineering; Protein/Amino Acid Biochemistry; Proteins; Receptor Signaling; Recombinants; Research; Reticulolymphosarcoma; Role; Sarcoma, Germinoblastic; Screening procedure; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Site; Solutions; Specificity; Sphingolipid Microdomains; Sphingolipid-Cholesterol Rafts; Surface Proteins; Syndrome; TM Domain; TNF Receptor Family Protein; TNF Receptor Superfamily; TNF Receptors; TNFR; TNFRSF5; TNFRSF5 gene; Technology; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Transmembrane Domain; Transmembrane Protein; Transmembrane Region; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor Receptor Family; Tumor Necrosis Factor Receptor Superfamily; Tumor Necrosis Factor Receptor Superfamily Member 5 Gene; Tumor Viruses; VESCL; Vesicle; Viral; Water; Work; Yin; aminoacid sequence of peptide; aminoacid sequence of protein; analog; assay development; base; biological signal transduction; body system, allergic/immunologic; cell transformation; computational methodology; computational methods; computer methods; design; designing; disease/disorder; drug/agent; experiment; experimental research; experimental study; gene product; heavy metal Pb; heavy metal lead; herpes virus; human disease; human herpesvirus 4 group; immunogen; in vitro testing; infected B cell; infected B lymphocyte; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; insight; latent membrane protein 1, Human herpesvirus 4; lipid raft; lymphoproliferative disease; malignancy; membrane structure; molecular recognition; neoplasm/cancer; new approaches; new therapeutic target; novel; novel approaches; novel strategies; novel strategy; novel therapeutic intervention; oncology; organ system, allergic/immunologic; p50; pathway; peptide sequence; prevent; preventing; protein aminoacid sequence; research study; screening; screenings; small molecule; social role; synthetic peptide; tool; transformed cells
Relevance: . Many poorly characterized human cancers develop, in part, due to the activity of oncogenic transmembrane proteins which, because of the inability to target protein domains within the membrane bilayer, are inaccessible to molecular probes to study function and are poor targets for current drug treatment methods. This study examines a novel class of rationally engineered peptides capable of interacting with membrane-embedded protein domains for use in the study of oncogenic transmembrane proteins, and potentially for the future development of new therapeutic approaches to the prevention and treatment of such cancers. The Latent Membrane Protein-1 (LMP-1), an oncoprotein expressed by the human tumor virus Epstein-Barr virus (EBV), will be used as a model membrane protein target for the design of such peptide inhibitors, because of its contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and because of EBV´s dependence on LMP-1´s hydrophobic transmembrane domain for transformation
Project start date: 2009-04-10
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: RFA-CA-08-007
5R21CA138373-02 (2010): $197445
Sponsored Links Excellgen http://Excellgen.com