Allosteric Modulators of the Melanocortin-4 Receptor

描述(由申请人提供)：中枢神经系统的黑素皮质素回路是脂肪酶的关键组成部分。这些回路的激活抑制了食物的摄入，刺激了能量消耗，因此黑素皮质素-4受体一直是主要制药公司开发治疗常见肥胖症药物的靶点。两个有效的MC4-R激动剂的临床试验在一些个体中显示出不想要的升压活性。最近，GPCRs的变构调节剂被认为是一种恢复生理系统正常时空活动的方法，而不会产生强大的正构激动剂的毒性。这种MC4R的变构调节剂可能既适用于严重的综合症肥胖，也适用于普通肥胖。在高达5%的病例中，由黑素皮质素信号缺陷引起的严重早发性肥胖与非同义编码突变导致MC4R单倍性不足有关。因此，假设MC4R启动子突变，并最终可能发现该途径中其他基因的突变，预计10%-30%的严重儿童肥胖可能是由黑素皮质素信号缺陷引起的。大多数MC4R突变破坏了受体到细胞表面的运输，而不是与配体的亲和力。与普通肥胖症不同，过度的MC4R刺激可能会导致不必要的副作用，成功治疗由黑素皮质素受体单倍体不足引起的严重肥胖症可能需要将MC4R蛋白水平提高到生理水平，从而潜在地避免副作用。事实上，在MC4R单倍体不足的肥胖症患者中，已经显示出相对低血压。在这项应用中，我们建议从范德比尔特高通量筛选核心的162,000种化合物的高通量筛选开始，识别MC4R的变构调节剂。最初的命中率将得到验证，子集将在细胞培养模型中得到广泛的表征。然后，将与Vanderbilt合成化学核心一起进行化合物优化，以开发临床前先导化合物。我们还建议利用我们提出的两种分析方法，即MC4R功能的电生理切片制备和MC4R单倍体不足的小鼠模型，并表征针对MC4R的抗体，以全面表征屏幕中识别的变构调节剂的作用机制。最后，我们将尝试确定黑素皮质素信号的血清生物标记物，作为分析黑素皮质素信号的变构调节剂的一种侵入性较小的工具。 公共卫生相关性：黑素皮质素-4受体一直是制药行业开发肥胖症治疗药物的靶点，因为该受体调节体重动态平衡，而该受体单倍体功能不全是严重早发性肥胖的最常见原因。然而，有效的正构体MC4-R激动剂的临床试验并不成功，据报道，在一次试验中至少有一些人具有升压活性。在这项应用中，我们将使用Vanderbilt High Throughput筛选设备进行高通量筛选，进行作用机制研究，并利用Vanderbilt合成化学核心开始HITS的优化过程，目标是识别MC4R的变构调节剂，使受体的正常活性最大化，而不会出现快速反应或不受欢迎的副作用。

DESCRIPTION (provided by applicant): The melanocortin circuitry of the CNS is a critical component of the adipostat. Activation of these circuits inhibits food intake and stimulates energy expenditure and thus the melanocortin-4 receptor has been a target of the major pharmaceutical companies for the development of drugs for the treatment of common obesity. Two clinical trials of potent MC4-R agonists exhibited unwanted pressor activity in some individuals. Recently, allosteric modulators of GPCRs have been recognized as a method of restoring normal spatio-temporal activity of physiological systems without the toxicity resulting from potent orthosteric agonists. Such allosteric modulators of the MC4R might have application to both severe syndromic obesity, as well as common obesity. Severe early onset obesity due to defective melanocortin signaling is linked, in up to 5% of cases, with non- synonymous coding mutations causing haploinsufficiency of the MC4R. It would not be unusual to expect that 10-30% of severe childhood obesity may thus result from defective melanocortin signaling, assuming MC4R promoter mutations, and mutations in other genes in the pathway may ultimately be discovered. The majority of MC4R mutations disrupt trafficking of receptors to the cell surface, rather than affinity for ligand. In contrast to common obesity, where excessive MC4R stimulation may cause unwanted side effects, successful treatment of severe obesity due to melanocortin receptor haploinsufficiency may involve increasing MC4R protein levels to physiological levels, thus potentially avoiding side effcets. Indeed, relative hypotension has been demonstrated in MC4R haploinsufficient obese patients. In this application, we propose to identify allosteric modulators of the MC4R, beginning with a high throughput screen of 162,000 compounds with the Vanderbilt High Throughput Screening Core. Initial hits will be validated, and a subset will be extensively characterized in cell culture models. Compound optimization will then be performed with the Vanderbilt Synthetic Chemistry Core, to develop preclinical lead compounds. We also propose to utilize two assays we present here, an electrophysiological slice preparation for MC4R function and a mouse model of MC4R haploinsufficiency, and to characterize antibodies against the MC4R to fully characterize the mechanism of action of allosteric modulators identified in the screen. Finally, we will attempt to identify serum biomarkers for melanocortin signaling as a less invasive tool for analysis of allosteric modulators of melanocortin signaling. PUBLIC HEALTH RELEVANCE: The melanocortin-4 receptor has been a target of the pharmaceutical industry for the development for therapeutics for the treatment of obesity because the receptor regulates body weight homeostasis, and haploinsufficiency of the receptor is the most common cause of severe early onset obesity. Clinical trials of potent orthosteric MC4-R agonists have not been successful, however, and pressor activity has been reported in at least some individuals in one trial. In this application we will conduct a high throughput screen with the Vanderbilt High Throughput Screening Facility, perform mechanism-of-action studies, and begin the process of optimization of hits with the Vanderbilt Synthetic Chemistry Core with the goal of identifying allosteric modulators of the MC4R that may maximize the normal activity of the receptor without tachyphylaxis or unwanted side effects.