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

细胞内和细胞间的信号传递对维持生物体的生理功能具有重要意义。我们通过大规模筛选、分离和鉴定对细胞内和细胞间信号传导有影响的具有生物活性的天然产物来研究影响这些信号传导的天然产物。5-S-GAD_L（5-S-glutathionyl-b-alanyl-L-dopa）最初是从大肠杆菌感染的昆虫中分离得到的一种抗生素，后来发现该化合物具有抑制vSrc酪氨酸激酶自身磷酸化的活性。5-S-GAD对破骨细胞的陷窝整体实验有抑制作用。用酪氨酸酶氧化多巴生成邻醌，邻醌与谷胱甘肽及其衍生物的巯基发生Michel加成反应，合成5-S-GAD。到目前为止，β-丙氨酰-L-多巴部分被改变为β-丙氨酰-D-多巴和β-丙氨酰-L-甲基多巴，以及β-丙氨酰多巴胺和多巴胺。另一方面，G ...更多信息 荧光素部分也转化为半胱氨酸、甘氨酰-半胱氨酸和谷氨酰-半胱氨酸。酪氨酸激酶活性的测定采用了三种不同的方法，一种是用~（13）P标记的ATP对vSrc进行自身磷酸化<32>，另一种是用重组cSrc对Glu-Tyr或Rytide（一种合成的胃泌素衍生物）合成肽进行底物磷酸化。5-S-GAD衍生物自身磷酸化和底物磷酸化的抑制活性并不完全平行。动力学研究表明这些肽类化合物对底物具有竞争性。除了抗酪氨酸激酶的抑制剂之外，还通过使用在用ConA或白细胞介素1激发时释放白细胞介素的培养细胞对白细胞介素-2和-6进行了大规模的筛选工作。对白细胞介素的生物测定可以测量抑制和激活。超过2,000个和甘药提取物和微生物培养液样品被测试其对单纯疱疹病毒感染vero细胞的抑制活性。以毒性和抗病毒活性比值为指标，大于1000者进行复筛。缬氨霉素和伴卡那霉素被鉴定为抗HSV活性物质，其作用机制正在研究中。少

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