Development of a Selective Mu-Delta Opioid Receptor Heterodimer Antagonist Using a Linked Bivalent Pharmacophore Approach

μ和δ阿片受体（莫尔，DOR）在体内调节许多相同的脑过程， 包括对阿片类药物的耐受性和抗伤害感受。许多研究小组发现， DOR通过各种方式降低了莫尔激动剂如吗啡的副作用。虽然基础 这种相互作用是未知的，一种很大的可能性是MOR-DOR异二聚体（MDOR）的形成。许多 研究组已经表明，MDOR可以在体外异源表达系统中形成，具有独特的 药理学和信号转导谱。Devi和同事们 最近开发了一种MDOR选择性抗体，并使用该抗体证明MDOR上调， 长期注射吗啡的老鼠的大脑其他实验表明，MDOR促进 耐受性、依赖性和体内药物寻求。虽然已经开发了MDOR选择性激动剂，但没有 据我们所知，已知的药物样拮抗剂已经被创造出来，限制了我们确定药物的作用的能力。 体内MDOR。为了解决这一问题，我们创造了一系列新的潜在选择性肽MDOR， 通过连接低亲和力莫尔（H-Tyr-Pro-Phe-D1 Nal-NH 2）和中等亲和力DOR（Tyr-Tic-D1 Nal-NH 2）， OH）药效团与可变长度（15-42个原子）的柔性聚酰胺间隔基。我们初步测试了 使用放射性配体结合和35 S-GTP γS偶联的体外系列，拮抗剂模式使用莫尔、DOR和 MDOR表达细胞系。我们发现了令人信服的证据，表明我们的初步系列选择性地针对 MDOR，对于我们最好的化合物，24个原子间隔长度，具有约91倍的选择性比率。在此基础上构建 初步的成功，我们的目标是在目前的建议，探索MDOR的结构活性关系（SAR），我们的 化合物系列，并通过调节药效团提高化合物的效力和MDOR的选择性 亲和力（增加的莫尔，降低的DOR）以及连接体刚性（最小刚性[gly-gly-pro]，中等刚性[gly-gly-pro]） 刚性[gly-pro]）。这些研究将在迭代开发过程中进行，使用最佳化合物 从每一个系列，以通知下一个系列，最大限度地减少化合物数量。特区发展后，我们会 使用我们最有效和选择性的化合物开始测试MDOR的体内活性。这将是完成 小鼠侧脑室（icv）和鞘内（it）注射MDOR拮抗剂， 由莫尔（DAMGO）、DOR（DSLET）和MDOR（CYM 51010）选择性激动剂诱发的伤害感受。这些 研究将证明我们的化合物在体内的选择性。我们还将用MDOR预处理小鼠， 拮抗剂在诱导吗啡耐受和依赖之前就开始探索体内作用 在这些阿片类药物副作用中的MDOR，这是文献所建议的。短期内这 初步优化系列将提供一个有用的工具，询问体内MDOR的作用。长期，通过 修饰以改善可药用性（糖基化等），这些化合物可以提供选择性的基础 MDOR靶向治疗以改善阿片类药物治疗的副作用特征。

The mu and delta opioid receptors (MOR, DOR) modulate many of the same brain processes in vivo, including tolerance and anti-nociception in response to opioid drugs. Many groups have found that inhibiting the DOR through various means decreases the side effects of MOR agonists like morphine. While the basis for this interaction is unknown, one strong possibility is the formation of a MOR-DOR heterodimer (MDOR). Many groups have shown that the MDOR can form in heterologous expression systems in vitro, with a unique pharmacology and signal transduction profile when compared to the monomeric forms. Devi and colleagues recently developed an MDOR selective antibody, and used this antibody to demonstrate MDOR upregulation in the brains of mice chronically treated with morphine. Other experiments suggested that the MDOR promotes tolerance, dependence, and drug seeking in vivo. While MDOR selective agonists have been developed, no known drug-like antagonist has ever been created to our knowledge, limiting our ability to determine the role of MDOR in vivo. To address this lack, we created a novel series of potential selective peptide MDOR antagonists by connecting low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2) and moderate affinity DOR (Tyr-Tic- OH) pharmacophores with a variable length (15-42 atom) flexible polyamide spacer. We tested this preliminary series in vitro using radioligand binding and 35S-GTPγS coupling in antagonist mode using MOR, DOR, and MDOR expressing cell lines. We found compelling evidence that our preliminary series selectively targets the MDOR, with a selectivity ratio of ~91 fold for our best compound, the 24 atom spacer length. Building from this initial success, we aim in the current proposal to explore the MDOR structure-activity relationship (SAR) of our compound series, and improve compound potency and selectivity at the MDOR by modulating pharmacophore affinity (increased MOR, decreased DOR) as well as linker rigidity (minimally rigid [gly-gly-pro], moderately rigid [gly-pro]). These studies will be performed in an iterative development process, using the best compound from each series to inform the next series, minimizing compound number. After this SAR development, we will use our most potent and selective compound to begin to test MDOR activity in vivo. This will be accomplished by intracerebroventricular (icv) and intrathecal (it) injection of MDOR antagonist into mice prior to tail-flick anti- nociception evoked by MOR (DAMGO), DOR (DSLET), and MDOR (CYM51010) selective agonists. These studies will demonstrate the selectivity of our compound in vivo. We will also pre-treat mice with MDOR antagonist prior to the induction of tolerance and dependence with morphine to begin to explore the in vivo role of the MDOR in these opioid side effects, which has been suggested by the literature. In the short term, this initial optimized series will provide a useful tool to interrogate the role of the MDOR in vivo. Long term, through modifications to improve drugability (glycosylation, etc.), these compounds may provide the basis for selective MDOR targeted therapeutics to improve the side effect profile of opioid therapy.