T GONDII--PYRIMIDINE SYNTHESIS AS A CHEMOTHERAPY TARGET

弓形虫——嘧啶合成作为化疗靶点

• 批准号: 2887589
• 负责人: DAVID J BZIK
• 金额: $ 25.9万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887589
• 关键词: Toxoplasma gondii; aminohydrolases; biosynthesis; dihydrofolate reductase; enzyme activity; enzyme inhibitors; gene deletion mutation; gene expression; laboratory mouse; microorganism growth; microorganism metabolism; molecular cloning; nucleic acid sequence; pentosyltransferase; pyrimidines; thymidine kinase; thymidylate synthase; uracil

DESCRIPTION: (Adapted from Applicant's Abstract) The goal of the proposed studies is to develop the basis for new chemotherapy that will depend on inhibition of pyrimidine biosynthesis. This pathway is a likely target for chemotherapeutic intervention since some of the parasite enzymes are markedly different from mammalian cells. The investigator presents preliminary evidence that T. gondii is dependent upon its de novo pathway of pyrimidine synthesis. Thus, elimination of the de novo pathway by irreversible genetic deletion of a key enzyme should block replication of the parasite and confirm the investigator's hypothesis that the parasite cannot make effective use of host cell pyrimidines. In the first specific aim, the investigator intends to genetically delete the T. gondii gene for carbamoyl phosphate synthetase (CPSII), the first committed step of pyrimidine synthesis in order to produce an auxotroph that will be completely dependent on salvage of uracil. A defined uracil auxotroph will confirm the validity of the de novo pyrimidine synthesis as a chemotherapeutic target. The effect of selected inhibitors of de novo pyrimidine synthesis on the intracellular growth of T. gondii will be studied in cell culture. Inhibitors of intracellular replication will represent useful lead compounds for further development. Uracil auxotrophs will be tested for in vivo growth and virulence in a mouse model. In the second specific aim, the investigator will expand the parasite's salvage repertoire by expression of cytosine deaminase and thymidine kinase in T. gondii. This will allow the use of these salvage enzymes for positive and negative selection. The linker region of dihydrofolate reductase-thymidylate synthetase (DHFR-TS) will be developed as a locus that can be expanded to express genes of interest in T. gondii.

描述：（改编自申请人的摘要） 研究的目的是为新的化疗奠定基础， 抑制嘧啶生物合成。 这条途径可能是 化疗干预，因为一些寄生虫酶 与哺乳动物细胞有很大不同。 调查员介绍 初步证据表明T.弓形虫依赖于它的从头途径， 嘧啶合成 因此，消除从头途径， 一个关键酶的不可逆基因缺失应该会阻止 并证实了研究人员的假设，即寄生虫 不能有效利用宿主细胞嘧啶。 在第一个具体的 为了达到这个目的，研究者打算从基因上删除T。弓形虫基因 氨甲酰磷酸合成酶（CPSII）， 嘧啶合成，以产生营养缺陷型， 完全依赖于尿嘧啶的补救。 确定的尿嘧啶营养缺陷型将 证实了从头嘧啶合成作为一种 化疗靶点 选择的从头抑制剂的作用 嘧啶合成对T.弓形虫将是 在细胞培养中研究。 细胞内复制的抑制剂将 代表用于进一步开发的有用的先导化合物。 尿嘧啶营养缺陷型 将在小鼠模型中测试体内生长和毒力。 在 第二个具体目标，研究人员将扩大寄生虫的救助， 通过T. 刚地。 这将允许使用这些补救酶进行阳性和阴性的检测。 负选择 二氢叶酸的接头区 还原酶-胸苷酸合成酶（DHFR-TS）将被开发为 可以在T.刚地。