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

细胞内和细胞内的信号对维持生物体的生理功能非常重要。我们正在通过大规模筛选、分离和鉴定对细胞内和细胞内信号转导有影响的生物活性天然产物来调查影响细胞内和细胞内信号转导的天然产物。5-S-GAD_L(5-S-谷胱甘肽-b-丙氨酰-L-多巴)是从感染大肠杆菌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对成骨细胞具有抑制活性。用酪氨酸酶催化多巴氧化生成邻苯二酚，再与谷胱甘肽及其衍生物的SH基团发生米歇尔加成反应，合成5-S-GAD比较容易。至此，β-丙氨酰-L-多巴部分改为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…更多的谷胱甘肽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。用~(32)&gt；P标记的ATP对vSrc进行自身磷酸化，用重组CSRC对胃泌素的合成多肽Glu-Tyr或Rytie进行底物磷酸化，测定酪氨酸激酶活性。5-S-GAD衍生物的抑硫活性并不完全等同于自身磷化和底物磷化。动力学研究表明，这些多肽类化合物与底物具有竞争性。除了酪氨酸激酶的抑制剂外，人们还利用培养细胞对IL-2和IL-6进行了大规模的筛选工作，这些细胞在ConA或IL-1诱导下释放IL-2和IL-6。对IL-6的生物测定可以同时测量抑制和激活。超过2,000个WAKANYAKU提取物和微生物发酵液的样本被测试了它们对Vero细胞感染单纯疱疹病毒的抑制活性。以毒性与抗病毒活性之比为指标，提交复筛1000份以上。瓦林霉素和刀豆素被鉴定为抗单纯疱疹病毒活性物质，其作用机制目前正在研究中。较少

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