Discovery of new allosteric modulators that convert antagonists to agonists

描述(申请人提供)：阿片类药物是治疗中度至重度疼痛最有效的化合物。然而，长期使用会导致许多不良反应，包括耐受性和依赖性的形成。止痛剂和呼吸抑制反应之间的不同耐受性发展降低了阿片类药物在慢性给药期间的治疗指数，这是一个主要的担忧。为了克服这一障碍，阿片类药物研究的圣杯一直是开发一种理想的止痛药，即具有最小副作用的止痛药，包括耐受性和依赖性形成。我们没有开发特定的邻位配体来激活单个受体，而不是开发一种新的方法来开发一种阿片受体突变体，该突变体可以被阿片拮抗剂激活。这种方法是基于我们偶然发现的，即MU-阿片受体(OPRM1)第4跨膜域的Ser196残基突变导致阿片类生物碱拮抗剂纳洛酮和纳曲酮能够激活该受体，而不改变配体的完整性或激动剂的活性。这种拮抗活性在S196A敲入小鼠体内和腺相关病毒介导的将突变受体传递到疼痛通路的不同位置上也得到了证实。重要的是，长期使用纳洛酮激活这种突变受体并不会导致耐受性的发展。OPRM1突变体的成功使我们假设，一定存在变构调节剂，可以将OPRM1转换为与S196A突变所转换的构象类似的构象。我们称这种调节剂为激动剂调节剂或AAM的拮抗剂。AAMs活性存在的“概念证据”是我们最近利用基于细胞的分析在一个图书馆的50,000种化合物的筛选中确定了10个可能的“命中”。在这些观察的鼓舞下，我们建议：(1)继续筛选化合物库中存在的额外的AAM活性；(2)用其他OPRM1活性测量来验证已鉴定的“HITS”中的AAM活性，例如抑制腺酰环化酶活性，通过激活GIRK通道诱导K电流，以及激活ERK1/2。 OPRM1也将被实施；以及(3)通过测量OPRM1拮抗剂纳洛酮在存在这种“点击”的情况下的抗伤害感受活性来测试体内AAM的活性。目前提出的研究将是我们为OPRM1开发变构调节剂的第一步，并将验证我们的假设，即存在一类新的变构调节剂--AAM。AAM将代表一类新的药物分子，可以在慢性阿片类药物给药期间限制耐受性的发展，从而维持阿片类止痛药治疗范例的治疗指数。

DESCRIPTION (provided by applicant): Opioids are the most efficacious compounds in the treatment of moderate to severe pain. However, with chronic use, many adverse effects including tolerance and dependence development will result. Differential tolerance development between the analgesic and respiration depression responses decreases the therapeutic index of opioids during chronic administration, which is a major concern. In order to overcome this obstacle, the holy grail of opioid research has been the development of an ideal analgesic, i.e., one that has minimal side effects, including tolerance and dependence development. Instead of developing specific orthosteric ligands that will activate a single receptor regardless of the oligomeric state of the receptor, we have pursued a novel approach to develop an opioid receptor mutant that can be activated by the opioid antagonist. This approach was based on our accidental discovery that mutation of Ser196 residue in the 4th transmembrane domain of mu-opioid receptor (OPRM1) results in the ability of opiate alkaloid antagonists such as naloxone and naltrexone to activate the receptor, without altering ligand afinity or agonist activity. This antagonist activity was demonstrated in vivo with a S196A knock-in mouse line and also with the adenoassociated virus- mediated delivery of the mutant receptor to various sites of the pain pathway. Importantly, chronic administration of naloxone in activating this mutant receptor does not result in tolerance development. The success of the OPRM1 mutant leads us to hypothesize that there must be allosteric modulators that can convert OPRM1 into conformations similar to that converted by the S196A mutation. We term such modulators as antagonist to agonist modulators or AAMs. The "proof of concept" for the existence of AAMs activity was established by our recent identification of 10 probable "hits" in our screens of 50,000 compounds in a library using a cell-based assay. Encouraged by these observations, we propose to (1) continue our screens of a chemical compound library for the existing of additional AAM activities; (2) validate the AAM activity in the identified "hits" with other OPRM1 activity measurements, such as inhibition of adenylyl cyclase activity, induction of K+ current via activation of GIRK channels, and activation of ERK1/2. Studies to demonstrate that the "hits" are actual allosteric modifiers of OPRM1 will be carried out also; and (3) to test for in vivo AAM activity by measuring the antinociceptive activity of the OPRM1 antagonist naloxone in the presence of such "hits". The current proposed studies will be the initial steps in our development of allosteric modulators for OPRM1, and will validate our hypothesis that there is a new class of allosteric modulators, AAMs. AAMs will represent a novel class of drug molecules that can limit tolerance development during chronic opioid administration, thereby maintaining the therapeutic index of the opioid analgesic treatment paradigm.