Development of allosteric modulators of the MC4 receptor

描述（由申请人提供）：黑皮质素-4受体（MC 4 R）是能量稳态的关键调节剂，控制食物摄入和能量消耗。在人类中，MC 4 R的突变导致高达5%的早发性肥胖。因此，MC 4 R已成为主要制药公司开发MC 4 R激动剂以治疗肥胖症的目标。然而，有效的MC 4 R激动剂的临床试验由于不希望的副作用而失败，所有这些激动剂在性质上都是正构的。我们的目标是开发MC 4 R的变构调节剂，有效治疗肥胖而不会引起危险的副作用。基于目前对变构调节剂的了解，我们假设以变构方式增强MC 4 R信号传导的分子不仅更特异、更有效，而且比直接的正构激动剂更安全。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（在这种情况下为1-MSH）的响应。此外，由于变构位点的保守性较低，所以变构调节剂通常比直接激动剂更具特异性。为了鉴定这种MC 4 R的调节剂，我们使用范德比尔特化合物文库（160，000种化合物）和基于Promega pGLO技术的系统进行了高通量筛选（HTS），该系统允许真实的时间记录细胞中的cAMP应答和变构调节剂的确定性鉴定。通过筛选鉴定的命中物针对其在相似的Gs偶联受体（22-肾上腺素能受体）上的活性进行反筛选，以消除对MC 4 R不特异的化合物。来自文库的0.1%的化合物（166）通过该方法鉴定为MC 4 R特异性正变构调节剂。本申请的第一个目的在于使用基于细胞的药理学和生物化学方法表征这些先导化合物。然后，最有前途的化合物将使用技术支持的合成进行化学优化，以制造和测试大量选定命中的类似物。第二个目标将包括在体内测试现有的最佳化合物以及最有前途的化合物，这些化合物将使用MC 4 R敲除小鼠肥胖模型从化学优化中产生。实际上，WT、MC 4 R和MC 4 R-/-小鼠将用媒介物或用所选化合物处理，同时监测食物摄入、氧消耗和体重。因此，该培训资助的科学目标将是开发小分子药物样化合物，并使用它们在体内测试阳性变构MC 4 R调节剂可能用于治疗综合征和可能的饮食性肥胖的假设。培训目标将是补充候选人在分子药理学方面的现有技能，并在药物发现技术，药物和化学生物学，小动物和转基因/基因敲除饲养以及小动物生理和行为分析方面进行广泛的培训。候选人将有机会进入黑皮质素受体和肥胖症的领先实验室（导师的实验室），一个具有成功证明记录的学术药物发现计划（范德比尔特化学生物学研究所），以及NIH资助的小鼠表型分析的领先来源（范德比尔特小鼠代谢表型分析中心），以提供完成计划目标所需的培训。 公共卫生相关性：黑皮质素-4受体（MC 4 R）的单倍蛋白不足导致高达5%的严重早发性肥胖。 本申请中提出的MC 4 R的有效变构调节剂的开发将提供治疗这种常见孟德尔综合征的治疗方法。 这些化合物对常见饮食性肥胖的潜在应用也将被研究。

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.