Development of a time-resolved fluorometric assay for the high throughput screening of melanin concentrating hormone receptor antagonists

• Published in: Journal of pharmacological and toxicological methods 2006-05, Vol.53 (3), p.242-247
• Main Authors: Lee, Sunghou, Kim, Gun-Do, Park, Woo-Kyu, Cho, Heeyeong, Byung Ho Lee, Yoo, Sung-eun, Jae Yang Kong
• Format: Article
• Language: English
• Subjects: AcroWell; Animals; Biological Assay; Dose-Response Relationship, Drug; Filter plate; Fluorometry - methods; Humans; Inhibitory Concentration 50; Ligands; MCH; Melanin concentrating hormone; Melanins - antagonists & inhibitors; Melanins - genetics; Melanins - physiology; Methods; Mice; Piperidines - pharmacology; Pyrimidines - pharmacology; Radioligand Assay; Rats; Receptor binding assay; Receptors, Pituitary Hormone - analysis; Receptors, Pituitary Hormone - antagonists & inhibitors; Receptors, Pituitary Hormone - genetics; Receptors, Pituitary Hormone - metabolism; Reproducibility of Results; Sensitivity and Specificity; Time Factors; Time-resolved; TRF
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2005.09.001

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 IC 50 value of 1.66 ± 0.10 nM that is very similar to the IC 50 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.