| Screening active compound is the beginning and a vital step in innovative drug research. Without screening, novel chemical substances with specific biological activities cannot be discovered and new drug research will be impossible. However, such a basic technical capability is one of the weakest components in China's innovative drug research initiative and a "bottleneck" in the transformation of its pharmaceutical industry. Animal screening is the traditional screening method with hard work and low efficiency. Recently with the development of Biomolecular, pham1acology, automatic etc., high throughput screening (HTS) technology, which has advantages such as the rapid detection of trace quantities with high sensitivity and accuracy, is a effective and early screening method of new drug research and appears with high efficiency using molecular or cell screening targets in minimal form. Neuromedin U (NMU) is a neuropeptide highly expressed in central nervous system. NMU mRNA level is reduced in rat hypothalamus after 48-hr fasting, suggesting that the neuropeptide may involve in feeding behavior. Indeed, ICV injection of NMU in rat brain decreases food intake. Recently, it has been found that an orphan G-protein-coupled receptor, FM-4, is the endogenous receptor for neuromedin U (NMU2R). In situ hybridization analysis of NMU2R indicates that the receptor is strongly expressed in paraventricular-nucleus of the hypothalamus. NMU2R is a Gq coupled receptor. Activation of the receptor in NMU2R-transfected 293 cells by neuromedin U increases intracellular calcium concentration. In order to much more know the physiological function of NMU2R and research the new drug of antiobesity, the author has established the high throughput screening model of agonist for NMU2R, and identified small molecule agonist from extracts of nature products for human NMU2R.This paper has developed a human NMU2R/pcDNA3.1 mammalian expression vector construct and a reporter gene construct what can response to Gs, Gi and Gq coupled receptors. This screening method couples NMU2R receptor to reporter gene, it mean, NMU2R gene and reporter gene are contransfectedHEK293 cell line to establish a stable cell line for screening. When a compound binds to NMU2R, it will activate NMU2R receptor and the signal pathway to change the expression of the second messagers in the cell. Responsive elements respond to this change and cause the expression of reporter gene. The bioactivity of the compound can be evaluated after the expression of the reporter gene was measured.To eliminate effectively the results of false positive and false negative in screening, the negative cell line and another receptors (such as melanocortin 4 receptor, MC4R and muscarinic M1 receptors, M1R) cell lines have been established. To identify and optimize the assay condition, the author examined the effects of some factors by using forskolin and endogenesis agonists of receptors, such as incubation time, cell number in each well, final concentration of DMSO and luciferase's substrate concentration on the assay. The result showed that the author had established some steady cell lines and reliable method for NMU2R, MC4R or M1R agonist screening respectively, the Z'-factor value was 0.75, 0.67 or 0.70 respectively, For all the receptors, the agonist incubation time were from 6 to 8 hour, the cell number per well were from 4×104 to 8×104, the final concentration of DMSO were less than 1%, the luciferase's substrate concentration were 12.5%. This technology was successfully applied to identify agonist for NMU2R. Using this cell line, 211 extracts of nature products were screened and 127 kinds were found to activate NMU2R receptor. The flavone glucosides from nature products might be identified as NMU2R agonists through isolation and purification. The flavone glucosides can bond and active specifically the receptor gene through NMU2R that they did not induce the responses in other receptor-transfected cells, such as M1R, MC4R and the negative cell lines. The results in...
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