Study on The Establishment of Bioassay Systems for The Screening to Find Lead Compound for Drug Development

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Inter and Intra-cellular signalings are inportant to maintain physiological function of living organisms. We have investigating natual products affecting inter- and intra-cellular signaling by a large scale screening, isolation and identification of bioactive natural products which have effects upon those signaling. 5-S-GAD_L (5-S-glutathionyl-b-alanyl-L-dopa) was first isolated as an antibiotic from insect infected with E.coli and later it was found that this compound possess inhibitory activity against self phosphorilation of vSrc tyrosine kinase. 5-S-GAD showed inhibitory activity in pit whole test with osteocrast cells. The synthesis of 5-S-GAD was rather easy when tyrosinase was used for the oxidation of dopa moiety to form ortho quinone which underwent Michel addition reaction with SH group of glutathione and its derivatives. Upto now beta-alanyl-L-dopa moiety was altered to beta-alanyl-D-dopa and beta-alanyl-L-methyldopa, and beta-alanyldopamine and dopamine. In the other hand g … More lutathione moiety was also converted into cystein, glycyl-cysteine and glutamyl-cysteine. The tyrosine kinase activity was determined three different assay methods, they are vSrc self phosphorilation with ^<32>P labelled ATP and the other is substrated phosphorilation against synthetic peptide consisting of Glu-Tyr or Rytide, a synthetic derivative of gastrine with recombinant cSrc. Inthibitory activity of 5-S-GAD derivatives are not completly pararel between self phosphorilation and substrate phosphorilation. Kinetic studies revealed these peptide like compounds are competitive against substrates. In addition to the inhibitors against tyrosine kinase, large scale sccreening works have been carried out on interleukin-2 and -6 by using cultured cells which release interleukins upon elicitation with ConA or interleukin 1. Bioassay on interleukins are possible to measure both inhibition and activaation. More than 2,000 samples of WAKANYAKU extracts and microbial cultutre broths are tested for their inhibitory activity against Herpes Simplex Virus infection to vero cells. The ratios of toxicity and anti-viral activity were used as indices and those showed more than 1000 was submitted for second screening. Valinomycine and concanamycins were identified as anti-HSV active substances and their reaction mechanis are now under investigation. Less