CB1 Allosteric Modulators: Molecular, Cellular and In Vivo Pharmacology

 描述(申请人提供)：大麻素受体CB1是一种G蛋白偶联受体(GPCR)，调节神经传递和其他生理过程。CB1被内源性配体(如花生四烯酰乙醇胺(AEA)和2-花生四烯酰甘油(2-AG))以及与受体邻位构型结合的各种合成和植物来源的配体激活。这些CB1正构体配体主要通过Gi/o偶联将信号转导到下游效应器，但Gs和arrestin偶联也是可能的。生物反应涉及许多治疗应用，如物质成瘾、疼痛和炎症、记忆、摄食和能量平衡。最近，GPCRs的变构调节剂被发现。正性变构调节剂(PAM)增强正构化激动剂的功能效应，负性变构调节剂(NAMS)非竞争性降低受体的活性。这些调节剂在拓扑上与不同于正构体配体结合部位的GPCR区结合，从而以高度亚型特异性的方式发挥其调节作用。CB1NAMS为开发药物成瘾和代谢综合征的治疗方法提供了令人兴奋的机会，PAM开辟了一条治疗疼痛相关疾病的新途径，减少了不良精神药物影响的发生率。然而，CB1变构调节的潜在机制，包括变构调节偏向的下游信号，仍然难以捉摸。我们设计和合成了高效的PAM，如LDK1256(Kb=89 nM)，并具有高变构，如LDK1258(=24.5)，用于增强激动剂的结合。我们还确定了NAMS是唯一已知的减少激动剂与CB1结合的调节剂。我们发现CB1变构调节器ORG27569与arrestin信号通路偶联，这代表了CB1偏置变构调节器的第一个例子。在这个项目中，我们将通过优化已鉴定的变构调节剂的性质来合成化合物，开发新的支架材料，并鉴定可提高平衡解离常数和协作性因子的药效基团。这些化合物将测试对激动剂和反向激动剂结合的影响。我们还将阐明它们的G蛋白和arrestin偶联及其下游分子水平的活性。在迭代过程中，关键的CB1调节剂将通过结构-活性关系(SAR)分析进行微调。我们将评估有效的变构调节剂在体内对CB1药理反应的影响。这项工作包括在功能分析中评估关键的CB1变构调节剂与邻位化合物，包括在大麻素四分体和药物鉴别分析中。我们还将在神经病理性疼痛、进食和尼古丁奖励的小鼠模型中测试铅配体。这项工作的总体目标是确定新的PAM和NAM，阐明它们的分子水平分布，并在动物模型中测试铅配体。这些努力对于阐明CB1变构调节的基础至关重要 因此，最终可以通过治疗剂获得高度特异性的反应。

 DESCRIPTION (provided by applicant): The cannabinoid receptor CB1 is a G-protein coupled receptor (GPCR) that regulates neural transmission and other physiological processes. CB1 is activated by endogenous ligands (e.g. arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG)) and various synthetic and plant-derived ligands that bind to the receptor orthosteric site. These CB1 orthosteric ligands transduce signals to downstream effectors primarily via Gi/o coupling, but Gs and arrestin coupling are also possible. The biological responses are implicated in many therapeutic applications, such as substance addiction, pain and inflammation, memory, and feeding and energy balance. Recently, allosteric modulators for GPCRs were discovered. Whereas positive allosteric modulators (PAMs) enhance the functional efficacy of orthosteric agonists, negative allosteric modulators (NAMs) non-competitively decrease the activity of the receptor. These modulators bind GPCR regions topographically distinct from the orthosteric ligand binding sites and thereby exert their modulatory effects in a highly subtype specific manner. CB1 NAMs offer exciting opportunities for developing therapeutics for drug addiction and metabolic syndromes and PAMs open a new pathway to treat pain-related conditions with a reduced incidence of unwanted psychotropic effects. However, the underlying mechanisms for CB1 allosteric modulation, including for allosteric-modulation biased downstream signaling, remain elusive. We have designed and synthesized highly potent PAMs, such as LDK 1256 (KB = 89 nM), and with high allostery, such as LDK1258 (=24.5), for enhanced agonist binding. We also have identified NAMs that are the only known modulators for reducing agonist binding to CB1. We have discovered that the CB1 allosteric modulator ORG27569 is coupled to an arrestin signaling pathway, which represents the first example of a CB1 biased allosteric modulator. In this project, we will synthesize compounds through optimizing the properties of identified allosteric modulators, develop new scaffolds and identify pharmacophoric groups that improve the equilibrium dissociation constants and cooperativity factors. These compounds will be tested for impact on agonist and inverse agonist binding. We will also elucidate their G-protein and arrestin coupling and downstream molecular-level activities. In an iterative process, key CB1 modulators will be fine tuned via structure-activity relationship (SAR) analysis. We will evaluate potent allosteric modulators for their impact on CB1 pharmacological responses in vivo. This effort involves assessing key CB1 allosteric modulators versus orthosteric compounds in functional assays including in the cannabinoid tetrad and drug discrimination assays. We will also test lead ligands in mouse models of neuropathic pain, feeding, and nicotine reward. The overall goal of this work is to identify new PAMs and NAMs, elucidate their molecular level profiles, and test lead ligands in animal models. These efforts are critical for elucidating the basis of CB1 allosteric modulation so that ultimately highly specific responses can be attained via therapeutic agents.