| High throughput screening (HTS) is one of the essential tools in the early stage of drug discovery, and has been made quick progress during recent years. Many methodologies of HTS have been developed. Among them, the reporter gene assay is one of the most important techniques. It has been reported that dopamine D1-like receptor and klotho gene are associated with aging and age related diseases. In this paper, the reporter gene assays was used to screen D1-like receptor agonists and klotho promoter activators. D1-like receptors include D1 and D5, both of them are Gs-coupled receptors. The two receptors share almost identical pharmacological properties. Therefore, it is difficult to pharmacologically distinguish these two receptor subtypes. It has been reported that D1/D5 receptors are involved in Parkinson's disease, drug addiction and memory disorders. Indeed, D1-like receptor agonists have been developed for the treatment of these brain diseases. Klotho is aging suppressor gene. It encodes a β- glycosidase. Klotho gene mutations and down-regulation of its expression will cause symptoms similar to aging. It has been reported that Klotho protein could improve the aging symptoms. Based on the previous results, we reasoned that increasing the expression of Klotho gene in vivo should be able to improve age related disorders. The following are the main contents and results of the present study:(1) A reporter gene system has been established. This system contains 6×CRE (6 tandem cAMP responsive elements) and a TATATA box, which linked to luciferase gene. When intracellular concentration of cAMP changed, it would affect the activity of CRE and regulate the expression of reporter gene luciferase. This system has been used to screen agonists or antagonists of Gs and Gi-coupled receptors.(2) The stable cell lines D5/CRE /TA/ Luci/CHO and D1/CRE /TA/ Luci/CHO were established by co-transfecting CHO cells with D1 or D5 receptors as well as the reporter gene 6CRE/TA/Luci. The cells were used to screen agonists from natural products. This cell-based assay was optimized for high throughput screening. The cells were cultured in 96-well plates overnight, treated with natural product extracts for 8 h, and then the luciferase activity was determined. We have identified several natural product extracts that can activate the reporter gene expression. To test whether the activation was through the D1 receptor, we have established a negative cell line that contains CRE/TA/Luci without D1-like receptors. A number of the natural product extracts were excluded as the receptor agonists, since they can also activate the reporter gene expression in the negative cell line. An extract (SBG492) from an animal corselet was conformed as D1 agonist.(3) A new subtype of D5 receptor (D5B) was identified. It shares 98% identical DNA sequence to D5, and 94% identical to the two D5 pseudogenes. It is interesting to note that the D5B DNA sequences that differ from D5 are the same as the two pseudogenes, while all the sites that are different from pseudogenes are identical to D5. D5B receptor gene encodes a protein that is one amino acid longer than D5 receptor. Pharmacologically, D5B receptor is a Gs-coupled receptor, and it has 2-fold higher affinity with dopamine compare to D5.(4) The klotho promoter was clone into a reporter gene vector pGL3 that contains luciferase gene. Co-transfection of HEK293 cells with this construct and pcDNA3.1, a stable cell line HEK293/KL was established in the presence of G418. The HEK293/KL cells were treated with 0.006 % H2O2 to establish an "aging" cell model. After H2O2 treatment, the klotho promoter was inhibited and cells showed low luciferase activity. This cell model was use to identity klotho promoter activator from natural products. The screening conditions were optimized to make screen method suitable for high throughput mode. After treatment with natural extracts for 6 h, the luciferase activity was detected. Several natural product extracts were identified to ac...
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