Discovery of new allosteric modulators that convert antagonists to agonists

描述（由申请人提供）：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，会产生许多不良反应，包括耐受性和依赖性的发展。镇痛和呼吸抑制反应之间的差异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，这是一个主要关注的问题。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镇痛药，即具有最小副作用的镇痛药，包括耐受性和依赖性。我们没有开发特异性的正构配体来激活单个受体，而不管受体的寡聚状态如何，我们已经寻求了一种新的方法来开发一种可以被阿片拮抗剂激活的阿片受体突变体。这种方法是基于我们偶然发现的，在第4跨膜区域的mu阿片受体（OPRM1） Ser196残基突变导致阿片生物碱拮抗剂如纳洛酮和纳曲酮激活受体的能力，而不改变配体亲和力或激动剂活性。这种拮抗剂的活性在体内通过S196A敲入小鼠系和腺相关病毒介导的突变受体递送到疼痛通路的各个部位得到了证实。重要的是，长期服用纳洛酮激活这种突变受体不会导致耐受性的发展。OPRM1突变体的成功使我们假设一定有变构调节剂可以将OPRM1转化为类似于S196A突变所转化的构象。我们将这种调节剂称为激动剂调节剂的拮抗剂或AAMs。AAMs活性存在的“概念证明”是通过我们最近使用基于细胞的测定方法在库中筛选的50,000种化合物中鉴定出10种可能的“命中”来建立的。受这些观察结果的鼓舞，我们建议(1)继续筛选化合物库以寻找存在的其他AAM活性；(2)通过其他OPRM1活性测量，如抑制腺苷酸环化酶活性，通过激活GIRK通道诱导K+电流，以及激活ERK1/2，验证确定的“hit”中的AAM活性。研究表明，“命中”是实际的变构修饰语

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.