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

Mu和Delta阿片受体(MOR，DOR)在体内调节许多相同的大脑过程， 包括对阿片类药物的耐受性和抗伤害性。许多团体发现，抑制 DOR通过多种途径减少吗啡等MOR激动剂的副作用。虽然基础是 这种相互作用是未知的，一个很大的可能性是形成MOR-DOR杂二聚体(MDOR)。许多 研究小组已经证明，MDOR可以在体外异源表达系统中形成，具有独特的 与单体形式比较时的药理学和信号转导概况。德维和同事们 最近开发了一种MDOR选择性抗体，并用这种抗体证明了MDOR在 长期使用吗啡治疗的小鼠的大脑。其他实验表明，MDOR促进了 体内的耐受性、依赖性和药物寻找。虽然MDOR选择性激动剂已经开发出来，但没有 据我们所知，已知的类药物拮抗剂曾被创造出来，限制了我们确定 体内多药耐药。为了解决这一不足，我们创造了一系列新的潜在选择性多肽MDOR 通过连接低亲和力MOR(H-Tyr-Pro-Phe-D1NaL-NH2)和中等亲和力DOR(Tyr-Tic- 具有可变长度(15-42个原子)柔性聚酰胺间隔基的药效团。我们初步测试了这一点 系列体外使用放射性配基结合和35S-GTPγS偶联在拮抗剂模式下使用MOR，DOR和 表达MDOR的细胞系。我们发现了令人信服的证据，我们的初步系列选择性地针对 MDOR，选择性比为我们最好的化合物的~91倍，24原子间隔长度。从这里开始建设 初步成功，我们在目前的提案中旨在探索我们的MDOR结构-活性关系(SAR) 化合物系列，并通过调节药效团来提高MDOR的化合物效力和选择性 亲和力(MOR增加，DOR降低)以及连接子刚性(最低刚性[Gly-Gly-Pro]，中等 刚性[Gly-Pro])。这些研究将在迭代开发过程中进行，使用最好的化合物 从每个系列通知下一个系列，最大限度地减少复合数。在这个特别行政区发展之后，我们将 使用我们最有效和最有选择性的化合物开始测试体内MDOR活性。这将会实现的 小鼠甩尾前脑室(Icv)和鞘内(It)注射MDOR拮抗剂 MOR(DAMGO)、DOR(DSLET)和MDOR(CYM51010)选择性激动剂引起的伤害性感受。这些 研究将证明我们的化合物在体内具有选择性。我们还将用MDOR对小鼠进行预处理 拮抗剂在诱导吗啡耐受和依赖前开始探讨其在体内的作用 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，这一点 最初的优化系列将提供一个有用的工具来询问多药耐药在体内的作用。长期，通过 修饰以提高药效性(糖基化等)，这些化合物可能为选择性 MDOR靶向治疗，以改善阿片类药物治疗的副作用情况。

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.