Identification of novel human high-density lipoprotein receptor Up-regulators using a cell-based high-throughput screening assay.
Scavenger receptor class B type I (SR-BI) is the high-affinity high-density lipoprotein (HDL) receptor, and CLA-1 is the human homologue of the murine SR-BI. CLA-1/SR-BI receptor has been suggested as a new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in overall HDL cholesterol (HDL-C) metabolism and its antiatherogenic activity in vivo. To search for active compounds that can increase CLA-1 transcription, a novel cell-based assay was developed for application in high-throughput screening (HTS). Human hepatoma HepG2 cells were transfected with a CLA-1-promoter-luciferase reporter gene construct, and the stable transfected cell line was selected and named CLAp-LUC HepG2. With rosiglitazone as a positive control, this stable cell line was used to establish a specific CLA-1 gene expression assay in a 96-well microplate format. The evaluating parameter Z' value of 0.64 showed that this cell-based HTS assay was robust and reliable. Screening of 6000 microbial secondary metabolite crude extracts identified 8 positive strains. Between 2 identified CLA-1 up-regulators produced by actinomycete strain 04-4776, 4776B may stimulate not only the expression of CLA-1 on the transcriptional and translational levels but also the activity of CLA-1 to uptake the HDL-C in HepG2 cells. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerosis agents.
Yang
Yuan
Y
School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
Zhang
Zhongbing
Z
Jiang
Wei
W
Gao
Lei
L
Zhao
Guiyu
G
Zheng
Zhihui
Z
Wang
Min
M
Si
Shuyi
S
Hong
Bin
B
D000191:Actinomycetaceae / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002232:Carbocyanines; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D005285:Fermentation; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry; D007529:Isoflavones / Q000302:isolation & purification / Q000494:pharmacology*; D008075:Lipoproteins, HDL / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D047495:PPAR gamma / Q000819:agonists; D018110:Receptors, Lipoprotein / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000077154:Rosiglitazone; D051122:Scavenger Receptors, Class B / Q000235:genetics / Q000378:metabolism*; D045162:Thiazolidinediones / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007529:Isoflavones; D008075:Lipoproteins, HDL; D047495:PPAR gamma; D018110:Receptors, Lipoprotein; D011993:Recombinant Fusion Proteins; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D045162:Thiazolidinediones; C031616:high density lipoprotein receptors; D000077154:Rosiglitazone; D017665:Hydroxyl Radical; C024286:3,3'-dioctadecylindocarbocyanine; C543998:4'-methoxy-3',5,7-trihydroxyisoflavone; D008156:Luciferases