Identification of human dopamine D1-like receptor agonist using a cell-based functional assay.
OBJECTIVE
To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.


METHODS
Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings.


RESULTS
A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor.


CONCLUSIONS
We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.
Jiang
Nan
N
Shanghai Institute of Brain Functional Genomics and Key Lab of Brain Functional Genomics, Ministry of Education, Science and Technology Commission of Shanghai Municipality, East China Normal University, Shanghai 200062, China.
Ou-Yang
Ke-qing
KQ
Cai
Shao-xi
SX
Hu
Ying-he
YH
Xu
Zhi-liang
ZL
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D004365:Drugs, Chinese Herbal / Q000302:isolation & purification / Q000494:pharmacology*; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D010617:Phenanthridines / Q000494:pharmacology; D010946:Plants, Medicinal / Q000737:chemistry; D017447:Receptors, Dopamine D1 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D050641:Receptors, Dopamine D5 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000235:genetics; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D004365:Drugs, Chinese Herbal; D010617:Phenanthridines; D017447:Receptors, Dopamine D1; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D050641:Receptors, Dopamine D5; C061532:dihydrexidine; D008156:Luciferases