Universal positive allosteric modulators of CCK1R without intrinsic agonist activity for the treatment of obesity

项目摘要/摘要 肥胖已经达到了流行的程度，导致了2型糖尿病和其他疾病的类似增加。 多种并存，导致巨大的费用、痛苦和早期死亡。改变饮食习惯 生活方式，虽然对急性减肥有效，但不耐用，减肥手术，虽然对 病态肥胖是不能满足这种需求的。因此，治疗肥胖的新的安全有效的药物疗法 都是需要的。这项建议是对PAR-16-374征求发现这种疗法的化验的响应。 我们的目标是鉴定具有活性的分子作为1型的正变构调节器(PAM) CCK1R不显示固有的激动剂活性，可以是安全有效的 所有潜在的患者，从那些倾向于增加体重的人到患有病态肥胖症的人 代谢综合征。这种药物现在已经不存在了。它将增加信号响应以 当更大的生物反应可以增加饱腹感时，餐后会释放内源性激素，而 克服完全激动剂引起的副作用和潜在毒性。它还将具有独特的属性 纠正刺激-活动耦合的缺陷现在被认为存在于这些患者的一部分中。 连续的双重筛选策略将针对自然野生型(WT)CCK1R以及 膜胆固醇升高引起的异常构象中的受体，后者 被CCK1R突变体模仿。增强和/或重新校准CCK1R以正常响应CCK 这些设置可以以一种温和而安全的方式强调荷尔蒙对饱腹感的影响。这表示一个 梅奥诊所的米勒博士在CCK1R的分子药理学方面具有专业知识，他与米勒博士建立了合作关系。 Sergienko在Sanford Burnham Prebys工作，拥有高通量筛选(HTS)和化学品方面的专业知识 基因组学。有三个特定的目标：(I)利用细胞内钙离子初步建立小分子文库 表达WT CCK1R的CHO细胞在两种模式下的反应分析以检测(A)内源性激动剂 活性和(B)PAM活性，增强对CCK的反应性。命中率将在正交IP中确认- 1使用亲本CHO细胞进行检测和反筛选；(2)HIT验证和功能分析将 对表达模拟异常的突变CCK1R的CHO细胞进行类似的分析 这种受体在高胆固醇环境中的构象。此外，还将进行化验以确保 化合物不刺激CCK1R内化，干扰自然激素结合和生物 活性或引起细胞毒性；以及(3)将进行铅的鉴定和功能鉴定 使用一种自然表达CCK1R、CHP212细胞生理密度的神经细胞系，以确保 缺乏内在的激动剂和营养活性，并再次确认增强对CCK的反应的能力。 其他特征将包括研究CCK-58和CCK1R上不同物种和 关于CCK2R，以及一组GPCR，以及对信号响应和ADME/Tox分析的广泛调查。

PROJECT SUMMARY/ABSTRACT Obesity has reached epidemic proportions, contributing to similar increases in type 2 diabetes mellitus and its multiple co-morbidities, accounting for immense expense, suffering, and early mortality. Modification of diet and lifestyle, while effective for acute weight loss, is not durable, and bariatric surgery, while effective for morbid obesity, is not scalable for this need. Therefore, new safe and effective pharmacotherapies for obesity are needed. This proposal is responsive to PAR-16-374 soliciting assays for discovery of such therapeutics. Our OBJECTIVE is to identify molecules with activity as positive allosteric modulators (PAMs) of the type 1 cholecystokinin receptor (CCK1R) that do not exhibit intrinsic agonist activity and that can be safe and effective across the full spectrum of potential patients, from those tending to gain weight to the morbidly obese with metabolic syndrome. This type of drug does not now exist. It would increase signaling responses to endogenous hormone released after a meal, when a greater biological response can enhance satiety, while overcoming side effects and potential toxicity caused by full agonists. It would also have the unique property of correcting a defect in stimulus-activity coupling now recognized to be present in a subset of these patients. Sequential dual screening strategies will be directed toward the natural wild type (WT) CCK1R, as well as this receptor in an abnormal conformation induced by elevated cholesterol in the membrane, with the latter mimicked by a CCK1R mutant. Enhancing and/or recalibrating CCK1R to be normally responsive to CCK in these settings could accentuate the hormonal effect on satiety in a gentle and safe manner. This represents a partnership between Dr. Miller at Mayo Clinic, with expertise in molecular pharmacology of CCK1R, and Dr. Sergienko at Sanford Burnham Prebys, with expertise in high throughput screening (HTS) and chemical genomics. There are 3 specific AIMS: (i) Primary HTS of a small molecule library using intracellular calcium response assays in CHO cells expressing WT CCK1R, performed in two modes to examine (a) intrinsic agonist activity and (b) PAM activity that enhances the responsiveness to CCK. Hits will be confirmed in orthogonal IP- 1 assays and counter-screened using parental CHO cells; (ii) Hit validation and functional profiling will be performed using analogous assays with CHO cells expressing a mutant CCK1R that mimics the abnormal conformation of this receptor in a high cholesterol environment. Also, assays will be performed to ensure that compounds do not stimulate CCK1R internalization, interfere with natural hormone binding and biological activity, or cause cytotoxicity; and (iii) Lead identification and functional characterization will be performed using a neuronal cell line that naturally expresses a physiologic density of CCK1R, CHP212 cells, to ensure absence of intrinsic agonist and trophic activity, and to reconfirm ability to enhance the responses to CCK. Other characterization will include studying effects on CCK-58, and those at CCK1R from various species and on CCK2R, as well as a panel of GPCRs, and a broad survey of signaling responses and ADME/Tox assays.