Engineered Opioid Receptors as Therapeutic Agents for Pain Control

工程阿片受体作为控制疼痛的治疗剂

• 批准号: 7749973
• 负责人: PING-YEE LAW
• 金额: $ 41.46万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749973
• 关键词: Acute; Adverse drug effect; Adverse effects; Affect; Agonist; Alkaloids; Amino Acid Sequence Homology; Amino Acids; Analgesics; Area; Biological Assay; Brain; Cell model; Chimera organism; Chronic; Complement; Complementary DNA; Constipation; Data; Dependence; Dependovirus; Development; Docking; Drug usage; Electroporation; Elements; Engineering; Environment; Exhibits; Gene Delivery; Gene Transfer; Generations; Goals; In Vitro; Infection; Injection of therapeutic agent; Intrathecal Injections; Knock-in Mouse; Ligands; Lipids; Medical; Membrane Lipids; Methods; Modeling; Molecular; Morphine; Mus; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Naloxone; Naltrexone; Narcotic Antagonists; Nature; Nausea; Neurons; Nociception; Opioid; Opioid Analgesics; Opioid Receptor; Pain; Pain management; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Physicians; Property; Pruritus; Receptor Activation; Receptor Gene; Research Personnel; Rodent Model; Second Messenger Systems; Sedation procedure; Sequence Homology; Site; Spinal Cord; Spinal cord posterior horn; Structure; Testing; Therapeutic Agents; Transmembrane Domain; Ventilatory Depression; Virus; Wild Type Mouse; addiction; base; chronic pain; design; dorsal horn; drug development; endogenous opioids; engineering design; extracellular; gene therapy; in vitro Assay; in vivo; midbrain central gray substance; mutant; public health relevance; receptor; receptor binding; response; second messenger; social stigma; success

DESCRIPTION (provided by applicant): Opioids have been used very successfully for the treatment of moderate to severe acute and chronic pain. Unfortunately, their uses have been associated with many troublesome side effects such as nausea, constipation, respiratory depression, sedation, pruritus, tolerance and dependence development. Many approaches have been used to alleviate these side effects without diminishing the analgesic effects, with variable success. Notable approaches have been the design of receptor selective ligands that would activate one of the three cloned opioid receptors and co-administration of pharmaceutical agents to block the opioid side effects. Although these approaches and others are viable ones, one of the reasons for their limited success is the high amino acid sequence homology among the cloned receptors. Such homology has slowed the design and development of an opioid drug that will target and activate a specific opioid receptor. In the current studies, we propose to use an alternative approach, i.e., to engineer mutant opioid receptors for pain management. Our approach is based on an accidentally discovered receptor mutation, S4.45(196)L in the u-opioid receptor (MOR), that resulted in the ability of opioid alkaloid antagonists to activate the mutant receptor, both in vitro and in vivo. We hypothesize that, if such a mutant receptor could be delivered to and expressed in the nociceptive neurons, then activation of the mutant receptors by antagonists should produce analgesic responses without eliciting the tolerance responses during chronic treatment with the antagonists. Our overall goal is to develop such receptor mutants as therapeutic agents for pain management. Thus, in the current proposal, we will (1) determine the molecular bases for the activation of receptor mutants by opioid antagonists; (2) validate and refine the receptor model for S4.54 mutation so as to engineer a mutant MOR in which naloxone and naltrexone behave like full agonists; and (3) develop a double stranded adenoassociated virus (dsAAV) for the delivery of the mutant receptor at specific sites of the pain pathway and evaluate the antagonist and agonist efficacies in eliciting antinociceptive responses. We will examine both the acute and chronic responses to opioid agonists and antagonists after dsAAV injection. By developing the receptor model and demonstrating the structural bases for the antagonist activities via receptor mutagenesis studies and generation of mutant mouse lines, we could engineer a mutant receptor in which opioid antagonists behave like full agonists. Since dsAAV has been used successfully in gene therapy, the eventual delivery by dsAAV and expression of the mutant receptor at the nociceptive neurons that normally express MOR should result in analgesic responses after systemic administration of opioid antagonists without tolerance development. Such a mutant opioid receptor gene therapy approach could be a new paradigm for the eventual treatment of chronic pain. PUBLIC HEALTH RELEVANCE: In the treatment of moderate to severe pain, morphine remains the drug of choice. However, the many side-effects of the drugs, notably tolerance and dependence development in prolonged treatment, have reduced the desirability in the use of this opioid analgesic for pain management. It has been the Holy Grail of pharmacologists and pharmaceutical chemists to develop drug molecules or treatment paradigms that elicit the pain relief effects without any side effects. With the discovery of a mutant u-opioid receptor (MOR) that could be activated by opioid antagonists without altering the agonists' properties, we hypothesize that such a mutant receptor could be developed into therapeutic agents for the purpose of pain management. We have demonstrated the feasibility of such approach by "knocking-in" this mutation, S4.54(196)A, into MOR, and generating a mouse line in which opioid antagonists, naloxone and naltrexone produced antinociceptive responses. However, this mutation only resulted in partial agonistic properties observed with these two opioid antagonists. Therefore, in the proposed studies, we will investigate the molecular bases for such antagonistic activities using modeling and mutational analysis. Additional receptor mutations will be identified in order to convert the antagonists into full agonists. We will develop a gene therapy vehicle, initially using dsAAV2 virus, to deliver the mutant receptors into various regions of the pain pathway and to examine the feasibility of using opioid antagonists as antinociceptive agents. The activation of the exogenously introduced mutant MOR, and the inactivation of the endogenous opioid receptors by the antagonists, will provide a unique opportunity to develop a pain treatment paradigm without possible development of tolerance and dependence.

描述（由申请人提供）：阿片类药物已非常成功地用于治疗中度至重度急性和慢性疼痛。不幸的是，它们的用途与许多麻烦的副作用有关，例如恶心，便秘，呼吸抑郁，镇静，瘙痒，耐受性和依赖性发展。许多方法已被用来减轻这些副作用而不减少镇痛作用，并获得可变的成功。值得注意的方法是受体选择性配体的设计，这些配体将激活三个克隆的阿片类药物受体之一和药物的共同给药以阻止阿片类药物的副作用。尽管这些方法和其他方法是可行的，但成功有限的原因之一是克隆受体之间的高氨基酸序列同源性。这种同源性减慢了阿片类药物的设计和开发，该药物将靶向和激活特定的阿片类药物受体。在当前的研究中，我们建议使用替代方法，即为疼痛管理设计突变的阿片类药物受体。我们的方法是基于意外发现的受体突变，即在U-阿片类受体（MOR）中S4.45（196）L，这导致阿片类生物碱拮抗剂在体外和体内激活突变体受体的能力。我们假设，如果可以在伤害性神经元中递送并表达这种突变受体，那么拮抗剂应激活突变体受体，不应产生镇痛反应，而不会在与拮抗剂慢性治疗期间引起耐受性反应。我们的总体目标是开发这样的受体突变体，例如用于疼痛管理的治疗剂。因此，在当前的建议中，我们将（1）确定阿片类拮抗剂激活受体突变体的分子碱基； （2）验证和完善S4.54突变的受体模型，以设计一种突变体，其中纳洛酮和纳曲酮的行为像全部激动剂一样； （3）开发双链腺关系的病毒（DSAAV），用于在疼痛途径的特定部位传递突变受体，并评估引起抗伤害感受感反应的拮抗剂和激动剂效率。我们将检查DSAAV注射后对阿片类药物激动剂和拮抗剂的急性和慢性反应。通过开发受体模型并通过受体诱变研究和突变小鼠系的产生来证明拮抗剂活性的结构碱基，我们可以设计一种突变体受体，而阿片类拮抗剂的行为像完整的激动剂一样。由于DSAAV已成功用于基因治疗，因此DSAAV最终通过通常表达MOR的伤害感受神经元的表达进行了突变受体的表达，该神经元应在全身施用阿片类拮抗剂而没有耐受性发展后会导致止痛反应。这种突变的阿片类药物受体基因治疗方法可能是最终治疗慢性疼痛的新范式。公共卫生相关性：在治疗中度至重度疼痛时，吗啡仍然是首选药物。但是，药物的许多副作用，尤其是长时间治疗中的耐受性和依赖性发展，已降低了这种阿片类镇痛药对疼痛治疗的使用的可取性。药物学家和药物化学家的圣杯发展了药物分子或治疗范例，这些范例会引起疼痛缓解效果而没有任何副作用。随着发现突变的U-阿片类受体（MOR）可以被阿片类药物拮抗剂激活而不会改变激动剂的性质，我们假设可以将这种突变体受体发展成治疗剂，以进行疼痛管理。我们通过“敲击”这种突变，S4.54（196）A，进入MOR，并生成小鼠系，在该突变中，我们已经证明了这种方法的可行性，而阿片类药物拮抗剂，纳洛酮和纳曲酮产生了抗伤害感受性反应。然而，这种突变只会导致这两个阿片类拮抗剂观察到部分激动特性。因此，在拟议的研究中，我们将使用建模和突变分析研究这种拮抗活性的分子碱基。将确定其他受体突变，以将拮抗剂转化为全部激动剂。我们将开发一种基因治疗工具，最初使用DSAAV2病毒，将突变受体传递到疼痛途径的各个区域，并检查使用阿片类药物拮抗剂作为抗伤害感受剂的可行性。外源引入的突变体的激活以及拮抗剂对内源性阿片类药物受体的失活，将为开发疼痛治疗范式提供独特的机会，而无需发展耐受性和依赖性。