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，长期使用会导致许多不良反应，包括产生耐受性和依赖性。镇痛和呼吸抑制反应之间的耐受性差异会降低长期给药期间阿片类药物的治疗指数，这是一个主要问题。为了克服这一障碍，阿片类药物研究的目标是开发一种理想的镇痛药，即具有最小副作用（包括耐受性和依赖性发展）的镇痛药。我们没有开发无论受体的寡聚状态如何都会激活单个受体的特定正位配体，而是寻求一种新方法来开发可以被阿片拮抗剂激活的阿片受体突变体。该方法基于我们的偶然发现，即 mu-阿片受体 (OPRM1) 第 4 跨膜结构域中 Ser196 残基的突变导致阿片生物碱拮抗剂（例如纳洛酮和纳曲酮）能够激活受体，而不改变配体亲和力或激动剂活性。这种拮抗剂活性在体内通过 S196A 敲入小鼠系以及腺相关病毒介导的突变受体递送至疼痛途径的各个位点得到证实。重要的是，长期施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推测一定存在变构调节剂可以将OPRM1转化为与S196A突变所转化的构象相似的构象。我们将此类调节剂称为激动剂调节剂的拮抗剂或 AAM。 AAM 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在库中筛选 50,000 种化合物中鉴定出 10 种可能的“命中”而确定的。受到这些观察结果的鼓舞，我们建议 (1) 继续筛选化合物库以查找现有的其他 AAM 活动； (2) 使用其他 OPRM1 活性测量来验证所识别的“命中”中的 AAM 活性，例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。研究证明“命中”是实际的变构调节剂 OPRM1也将进行； (3)通过测量OPRM1拮抗剂纳洛酮在存在此类“命中”的情况下的抗伤害活性来测试体内AAM活性。目前提出的研究将是我们开发 OPRM1 变构调节剂的第一步，并将验证我们的假设，即存在一类新型变构调节剂 AAM。 AAM 将代表一类新型药物分子，可以限制长期阿片类药物给药期间耐受性的发展，从而维持阿片类镇痛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.