Lee Sunghou, Kim Gun-Do, Park Woo-Kyu, Cho Heeyeong, Yoo Sung-Eun
Department of Biotechnology and Informatics, College of Engineering, Sangmyung University, San 98-20, Anseo-Dong, Cheonan 330-720, Republic of Korea.
J Pharmacol Toxicol Methods. 2006 May-Jun;53(3):242-7. doi: 10.1016/j.vascn.2005.09.001. Epub 2005 Oct 24.
Melanin concentrating hormone is an orexigenic hypothalamic neuropeptide, which plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome.
Although radioligand receptor binding assay has been one of the most powerful tools for receptor research and drug discovery, the limitations of radioisotopes and the problems related to safety and waste disposal limits their application in high throughput screening and has led to a growing interest in alternative, nonradioactive technologies. To develop a sensitive and reproducible assay system for MCH1, the time-resolved fluorescence (TRF) receptor binding assay with AcroWell filter plates was tested and validated.
Comparing to the radioligand receptor binding assay for MCH1, the TRF assay presented higher Z/Z' factors with the lower signal-to-noise ratio. The known high-affinity MCH1 receptor antagonist, SNAP-7941, exhibited an IC50 value of 1.66+/-0.10 nM that is very similar to the IC50 value of MCH in a radioligand binding assay with an excellent correlation coefficient (0.9884).
These results suggest that our TRF receptor binding assay for MCH1 can achieve the desired sensitivity and reproducibility to replace the radioligand receptor assay in a fluorometric system that can be developed for high throughput screening.
黑色素聚集激素是一种下丘脑促食欲神经肽，在由黑色素聚集激素受体亚型1（MCH1）介导的能量平衡和体重的复杂调节中起重要作用。有令人信服的药理学证据表明MCH1信号传导参与食物摄入和能量消耗的调节，这引起了制药公司的极大兴趣，因为MCH1拮抗剂在治疗肥胖症和代谢综合征方面可能具有潜在的治疗益处。
尽管放射性配体受体结合测定一直是受体研究和药物发现的最有力工具之一，但放射性同位素的局限性以及与安全和废物处理相关的问题限制了它们在高通量筛选中的应用，并导致人们对替代的非放射性技术的兴趣日益增加。为了开发一种用于MCHl的灵敏且可重复的测定系统，测试并验证了使用AcroWell滤板的时间分辨荧光（TRF）受体结合测定。
与用于MCH1的放射性配体受体结合测定相比，TRF测定具有更高的Z/Z'因子和更低的信噪比。已知的高亲和力MCH1受体拮抗剂SNAP-7941的IC50值为1.66±0.10 nM，这与放射性配体结合测定中MCH的IC50值非常相似，相关系数极佳（0.9884）。
这些结果表明，我们用于MCH1的TRF受体结合测定可以实现所需的灵敏度和可重复性，以在可用于高通量筛选的荧光系统中替代放射性配体受体测定。
