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: 6170735
• 负责人: Sherry F Queener
• 金额: $ 18.57万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170735
• 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.

本研究的目的是评价肌苷酸 脱氢酶（IMPDH，EC 1.1.1.205）作为化疗靶点， 卡氏肺孢子虫。 这种酶是嘌呤的限速步骤 生物合成，并且其抑制限制了鸟嘌呤核苷酸的细胞， 除非主动嘌呤补救能克服代谢阻断 两 已知的IMPDH抑制剂（霉酚酸和噻唑弗林）是强的 卡氏肺孢子虫在短期培养中生长的抑制剂;这种和其他 有证据表明，在卡氏肺孢子虫中嘌呤补救可能不足以 克服IMPDH抑制。 待检验的假设为：麦考酚酸 酸和硫唑弗林是卡氏肺孢子虫生长的有效抑制剂， 培养，因为它们和/或它们的代谢产物特异性抑制P. 卡氏菌IMPDH，产生一种不可逾越的代谢阻断， 补救途径不能提供足够的XMP或GMP来支持 生物体的增殖。具体目标是：1）纯化重组蛋白 P. 2）表征重组卡氏毕赤酵母和人IMPDH; IMPDH，以人IMPDH作为参照; 3）评估抑制剂的作用 重组卡氏肺孢子虫IMPDH对卡氏肺孢子虫增殖的影响， 4）评估对卡氏肺孢子虫的摄取和代谢作用， 5）评估IMPDH抑制剂的潜力 通过比较抑制剂对重组E. coliSphi 1101，缺乏天然E.大肠杆菌IMPDH， 含有卡氏肺孢子虫IMPDH或人IMPDH。 为了实现这些目标， 重组卡氏肺孢子虫IMPDH将通过亲和色谱法纯化 并进行了表征，强调了霉酚酸的抑制动力学 酸、噻唑弗林及其12种类似物。 第一个目标2 目的是比较卡氏肺孢子虫IMPDH对人类的催化特性， I型酶，以评估开发 酶的选择性抑制剂。 最后三个目标的目标是 评估IMPDH抑制剂对整个生物体的影响， 揭示需要代谢活化的抑制剂的活性。 在 此外，嘌呤补救将直接测量;各种嘌呤将 还测试了逆转由以下引起的生长抑制的能力： IMPDH抑制剂。建立IMPDH作为P. 卡里尼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。