Development of allosteric modulators of the MC4 receptor

描述(申请人提供)：黑素皮质素-4受体(MC4R)是能量平衡的关键调节器，控制食物摄入量和能量消耗。在人类中，MC4R基因突变导致了高达5%的早发性肥胖症。因此，MC4R已成为主要制药公司开发MC4R激动剂治疗肥胖症的目标。然而，有效的MC4R激动剂的临床试验，所有这些都是正畸性质的，由于不想要的副作用而失败。我们的目标是开发MC4R的变构调节剂，它将有效地治疗肥胖症，而不会引起危险的副作用。根据目前关于变构调节剂的知识，我们假设以变构方式增强MC4R信号的分子不仅比直接的正构激动剂更特异、更有效，而且更安全。事实上，变构调节剂不太可能引起副作用，因为它们不直接激活靶受体，而是以更生理的时空模式增强其对内源性激动剂的反应，在这种情况下是1-MSH。此外，由于变构位点的保守性较低，变构调节剂通常比直接激动剂更具特异性。为了识别MC4R的这种调节剂，我们使用Vanderbilt化合物库(160,000种化合物)和基于Promega pGLO技术的系统运行了高通量筛选(HTS)，该系统允许实时记录细胞中的cAMP反应和变构调节剂的最终鉴定。通过筛选确定的Hit在类似的Gs偶联受体(22-肾上腺素能受体)上的活性被反筛选，以消除非MC4R特有的化合物。通过这一过程，从文库(166)中鉴定出0.1%的化合物为MC4R特异性的正变构调节剂。这项应用的第一个目标将在于使用基于细胞的药理学和生物化学方法来表征这些先导化合物。然后，最有希望的化合物将被推进到化学优化，使用技术支持的合成来制造和测试大量选定HITS的类似物。第二个目标将在于利用肥胖的MC4R基因敲除小鼠模型在体内测试最好的现有化合物以及最有希望的化合物，这些化合物将通过化学优化产生。事实上，WT、MC4R和MC4R-/-小鼠将接受载体或所选化合物的治疗，同时将监测食物摄入量、氧气消耗和体重。因此，这项培训拨款的科学目标将是开发小分子类药物化合物，并使用它们在体内测试阳性变构MC4R调节剂可能对治疗综合征和可能的饮食性肥胖有用的假设。培训目标是通过在药物发现技术、药物和化学生物学、小动物和转基因/基因敲除畜牧业以及小动物生理和行为分析方面的广泛培训来补充候选人在分子药理学方面的现有技能。应聘者将获得领先的黑素皮质素受体和肥胖症实验室(Mentor实验室)、一个有成功经验的学术药物发现计划(范德比尔特化学生物学研究所)，以及由NIH资助的领先的小鼠表型来源(范德比尔特小鼠代谢表型中心)，以提供实现计划目标所需的培训。 公共卫生相关性：黑素皮质素-4受体(MC4R)的单倍体蛋白充足是导致高达5%的严重早发性肥胖的原因。在这项申请中提出的MC4R的有效变构调节剂的开发将为这种常见的孟德尔综合征的治疗提供一种治疗方法。还将研究这些化合物在常见饮食肥胖方面的潜在应用。

DESCRIPTION (provided by applicant): The Melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and controls food intake and energy expenditure. In humans, mutations in the MC4R are responsible for up to 5% of early onset obesity. Consequently, MC4R has been a target of the major pharmaceutical companies for the development of MC4R agonists to treat obesity. However, clinical trials of potent MC4R agonists, all of which have been orthosteric in nature, failed due to unwanted side-effects. Our goal is to develop allosteric modulators of the MC4R that will efficiently treat obesity without causing dangerous side effects. Based on the current knowledge on allosteric modulators, we hypothesize that a molecule that would potentiate MC4R signaling in an allosteric fashion would not only be more specific and more potent, but also safer than a direct orthosteric agonist. Indeed, allosteric modulators are less likely to cause side effects since they do not directly activate the target receptor but potentiate its response to endogenous agonist, in this case 1-MSH, in a more physiological temporo-spatial pattern. In addition, due to the lower conservation of allosteric sites, allosteric modulators are usually more specific than direct agonists. In order to identify such modulators of the MC4R, we have run a high-throughput screen (HTS) using the Vanderbilt compound library (160,000 compounds) and a system, based on the promega pGLO technology, that allows real time recording of cAMP responses in cells and the definitive identification of allosteric modulators. Hits identified by the screen were counterscreened for their activity at a similar Gs-coupled receptor (22-adrenergic receptor) in order to eliminate compounds that are not specific to the MC4R. 0.1% of the compounds from the library (166) were identified via this process as MC4R specific positive allosteric modulators. The first aim of this application will consist in characterizing these lead compounds using cell based pharmacological and biochemical methods. The most promising compounds will then be advanced to chemical optimization using technology-enabled synthesis to make and test large numbers of analogues of selected hits. The second aim will consist in testing in-vivo the best existing compounds as well as the most promising compounds that will arise from chemical optimization using the MC4R knockout mouse model of obesity. Indeed, WT, MC4R and MC4R-/- mice will be treated with vehicle or with the selected compounds while food intake, oxygen consumption and body weight will be monitored. Thus, the scientific goals of this training grant will be to develop small molecule drug-like compounds and use them to test in vivo the hypothesis that positive allosteric MC4R modulators may be useful for the treatment of syndromic, and possibly dietary obesity. The training goal will be to complement the candidate's existing skills in molecular pharmacology with extensive training in drug discovery technology, medicinal and chemical biology, small animal and transgenic/knockout husbandry, and small animal physiological and behavioral analysis. The candidate will have access to a leading laboratory in melanocortin receptors and obesity (mentor's laboratory), an academic drug discovery program with a proven record of success (Vanderbilt Institute for Chemical Biology), and leading NIH-funded source for mouse phenotyping (Vanderbilt Mouse Metabolic Phenotyping Center) to provide the training necessary to accomplish the program goals. PUBLIC HEALTH RELEVANCE: Haploin sufficiency of the melanocortin-4 receptor (MC4R) is responsible for up to 5% of severe early onset obesity. The development of potent allosteric modulators of the MC4R, proposed in this application, would provide a therapeutic approach to the treatment of this common Mendelian syndrome. The potential application of these compounds to common dietary obesity will also be examined.