IMP DEHYDROGENASE FROM PNEUMOCYSTIS AS A DRUG TARGET

来自肺孢子虫的 IMP 脱氢酶作为药物靶点

• 批准号: 2887615
• 负责人: Sherry F Queener
• 金额: $ 18.03万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887615
• 关键词: Pneumocystis carinii; SDS polyacrylamide gel electrophoresis; affinity chromatography; antiprotozoal agents; autoradiography; drug design /synthesis /production; drug screening /evaluation; enzyme activity; enzyme inhibitors; growth inhibitors; high performance liquid chromatography; inosine monophosphate; laboratory rat; microorganism culture; microorganism growth; molecular cloning; oxidoreductase; polymerase chain reaction

The goal of this research is to evaluate inosine monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) as a target for chemotherapy in Pneumocystis carinii. This enzyme is the rate-limiting step in purine biosynthesis, and its inhibition limits cells for guanine nucleotides, unless active purine salvage can overcome the metabolic blockade. Two known inhibitors of IMPDH (mycophenolic acid and tiazofurin) are strong inhibitors of P. carinii growth in short-term culture; this and other evidence suggests purine salvage in P. carinii may be insufficient to overcome IMPDH inhibition. The hypothesis to be tested is: Mycophenolic acid and tiazofurin are potent inhibitors of P. carinii growth in culture because they and/or their metabolites specifically inhibit P. carinii IMPDH, creating an insurmountable metabolic blockade because salvage pathways cannot supply adequate XMP or GMP to support proliferation of the organism. Specific aims are: 1) Purify recombinant P. carinii and human IMPDH; 2) Characterize recombinant P. carinii IMPDH, with human IMPDH as a reference; 3) Assess effects of inhibitors of recombinant P. carinii IMPDH on proliferation of P. carinii in short term culture; 4) Assess uptake and metabolic effects, on P. carinii and on a mammalian cell line, of IMPDH inhibitors 5) Evaluate potential selectivity by comparing activity of inhibitors against recombinant E. coli Sphi1101, which lacks native E. coli IMPDH, reconstructed to contain P. carinii IMPDH or human IMPDH. To accomplish these goals, recombinant P. carinii IMPDH will be purified by affinity chromatography and characterized, emphasizing kinetics of inhibition with mycophenolic acid, tiazofurin and 12 of their analogs. The goal of these first 2 aims is to compare the catalytic properties of P. carinii IMPDH to human type I enzyme in order to evaluate the potential for developing selective inhibitors of the enzyme. The goal of the last 3 aims is to evaluate effects of IMPDH inhibitors on the whole organism, which will reveal the activity of inhibitors requiring metabolic activation. In addition, purine salvage will be measured directly; various purines will also be tested for the ability to reverse growth inhibition caused by IMPDH inhibitors. Establishing IMPDH as a target for chemotherapy in P. carinii opens the way for developing new agents effective against this important pathogen affecting immunosuppressed patients.

本研究的目的是评估肌苷单磷酸 脱氢酶（IMPDH，EC 1.1.1.205）作为化疗靶点 卡氏肺孢子虫。 该酶是嘌呤的限速步骤 生物合成及其抑制限制了细胞对鸟嘌呤核苷酸的利用， 除非积极的嘌呤补救可以克服代谢障碍。 二 已知的 IMPDH 抑制剂（霉酚酸和噻唑呋林）很强 短期培养中卡氏疟原虫生长的抑制剂；这个和其他 有证据表明，P. carinii 中的嘌呤补救可能不足以 克服IMPDH抑制。 要检验的假设是： 霉酚酸 酸和噻唑呋林是 P. carinii 生长的有效抑制剂 培养物，因为它们和/或其代谢物特异性抑制 P. carinii IMPDH，造成了难以克服的代谢封锁，因为 补救途径无法提供足够的 XMP 或 GMP 来支持 有机体的增殖。具体目标是：1）纯化重组体 P. carinii 和人类 IMPDH； 2) 表征重组 P. carinii IMPDH，以人IMPDH为参考； 3) 评估抑制剂的效果 重组 P. carinii IMPDH 对 P. carinii 增殖的影响 术语文化； 4) 评估对 P. carinii 和 IMPDH 抑制剂在哺乳动物细胞系上的作用 5) 评估潜力 通过比较抑制剂对重组大肠杆菌的活性来确定选择性。 大肠杆菌 Sphi1101，缺乏天然大肠杆菌 IMPDH，被重建为 含有 P. carinii IMPDH 或人 IMPDH。 为了实现这些目标， 重组 P. carinii IMPDH 将通过亲和层析纯化 并进行了表征，强调了霉酚酸的抑制动力学 酸、噻唑呋林及其 12 种类似物。 前 2 个目标 目的是比较 P. carinii IMPDH 与人类的催化特性 I 型酶以评估开发潜力 酶的选择性抑制剂。 最后 3 个目标的目标是 评估 IMPDH 抑制剂对整个生物体的影响，这将 揭示需要代谢激活的抑制剂的活性。 在 另外，直接测定嘌呤残值；各种嘌呤会 还测试了逆转由以下原因引起的生长抑制的能力： IMPDH 抑制剂。建立 IMPDH 作为 P. 化疗的靶点。 carinii 为开发有效对抗这种疾病的新药物开辟了道路 影响免疫抑制患者的重要病原体。