Screening And Study On The Microorganism Produced Active Compounds That Inhibite The VCAM-1 Gene Expression

For the purpose of screening bioactive compounds that regulating the target gene expression of VCAM-1, we have established recombinant human cell line based high throughput screening assay system by suing luciferase as a reporter gene in 96-well plate to identify the metabolites of microorganisms that regulate the target gene expression.The assay system is the recombinant N1-14 cell line, which was come from the human umbilical vein endothelial cell line ECV304 and was used for the screening of bioactive compounds that prevent the expression of VCAM-1 gene for the purpose of search new drugs of treating immunological diseases such as rheumatoid arthritisr etc. The assay system is stable transfected cell line and containing the VCAM-1 transcription regulating element respectively refused to the luciferase reporter gene.In the course of screening of 10318 samples produced by 5159 strain of microorgannism, total of 414 primary screened samples and 152 secondary screened samples have showed positive activity. Based on the above research results, two of strain and their metabolites have been studies further more.From the metabolites of fungus FO-5897, one compound Ascofuranone have been isolated and showed very interesting bioactivity of inhibiting the VCAM-1 reporter gene expression. This compound was first reported has hypolipidemic and antitumor activity, but its inhibitory activity to the VCAM-1 reporter gene expression was first found. The other novel compound Zelkovamycin, which was produced by soil actinomycete K-670, is elucidated to be a cyclic peptide, comprising six unusual amino acid and two usual amino acid by spectroscopic studies including ¹H-¹H COSY, ¹³C-¹H COSY, ¹³C-¹H HMQC, ¹³C-¹H HMBC, 15N-¹H HMQC, 15N-¹H HMBC experiments. This compound showed very good selective inhibitory activity to the VCAM-1 reporter gene expression with IC50 of about 9.5μg/ml. Based on the taxonomic properties studies, strain K-670 was considered to belong to the genus streptomyces.