Exploitation of heparanase inhibitors from microbial metabolites using an efficient visual screening system.
In this paper we describe the establishment of an efficient visual method for screening heparanase inhibitors, and we present the results of screening 10,000 microbial culture broths. Heparanase-overexpressing stable clones of the human hepatocellular carcinoma HepG2 cells were established and used as an enzyme source. Digestion of heparan sulfate (HS) was detected using novel HS-containing tablets or SDS-polyacrylamide gel electrophoresis. This method was able to find suramin, a known heparanase inhibitor, from a library of typical enzyme inhibitors. By screening 10,000 culture broths of microorganisms (actinomycetes, fungi, and bacteria) an actinomycete strain, RK99-A234, was found to have heparanase inhibitory activity. RK-682 was identified in the fermentation broth as a heparanase inhibitor, IC50 = 17 microM.
Ishida
Keisuke
K
Antibiotics Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Teruya
Takayuki
T
Simizu
Siro
S
Osada
Hiroyuki
H
D039903:Actinobacteria / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D002999:Clone Cells; D003470:Culture Media; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D005966:Glucuronidase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000473:pathology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D014162:Transfection; D000099175:Heparanase
D003470:Culture Media; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005966:Glucuronidase; D006497:Heparan Sulfate; D010749:Phosphoprotein Phosphatases; D000099175:Heparanase; C094097:RK 682