Practical Studies on Drug Development with The Leads Biological Natural Products

细胞间和细胞内信号对于维持生物体的生理功能至关重要。我们通过大规模筛选、分离和鉴定对这些信号传导有影响的生物活性天然产物，研究了天然产物对细胞间和细胞内信号传导的影响。5-S-GAD_L （5-S-glutathionyl-b-alanyl-L-dopa）是最早从感染E.coil的昆虫中分离到的一种抗生素，后来发现该化合物具有抑制vSrc酪氨酸激酶自磷酸化的活性。5-S-GAD对成骨细胞有抑制作用。利用酪氨酸酶将多巴部分氧化生成邻醌，并与谷胱甘肽及其衍生物的SH基团进行米歇尔加成反应，合成5-S-GAD较为容易。到目前为止，β -丙烯酰- l-多巴被改变为β -丙烯酰-d -多巴和β -丙烯酰- l-甲基多巴，β -丙烯酰多巴胺和多巴胺。另一方面，更多的胱甘肽片段转化为半胱氨酸、甘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检测方法，一种是用^<32>P标记的ATP进行vSrc自磷酸化，另一种是用重组证监会对由Glu-Tyr或Rytide组成的合成肽进行底物磷酸化。5-S-GAD衍生物的抑制活性在自身磷酸化和底物磷酸化之间并不完全平行。动力学研究表明这些肽类化合物对底物具有竞争性。除了针对酪氨酸激酶的抑制剂外，还对白细胞介素-2和-6进行了大规模筛选，方法是使用经ConA或白细胞介素1激发后释放白细胞介素的培养细胞。白细胞介素的生物测定可以测量抑制和激活。WAKANYAKU提取物和微生物培养液的2000多个样品进行了测试，以检测其对vero细胞的单纯疱疹病毒感染的抑制活性。以毒性和抗病毒活性比值为指标，大于1000的进行二次筛选。valinomycin和concanamycin被确定为抗hsv活性物质，其反应机制目前正在研究中。少

Inter and Intra-cellular signalings are inportant to maintain physiological function of living organisms. We have investigating natual products affection 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.coil 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