PURINE BOTTLENECKS FOR THE CHEMOTHERAPY OF TOXOPLASMOSIS

弓形虫病化疗的嘌呤瓶颈

• 批准号: 2075152
• 负责人: ELMER R PFEFFERKORN
• 金额: $ 20.68万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2075152
• 关键词: 5' nucleotidase; AIDS; Toxoplasma gondii; adenosine kinase; antiprotozoal agents; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; guanosine monophosphate; hypoxanthine phosphoribosyltransferase; molecular cloning; mutant; nucleic acid metabolism; opportunistic infections; protein purification; protein structure function; purines; toxoplasmosis; xanthines

DESCRIPTION: (Adapted from Applicant's Abstract): Toxoplasmic encephalitis is a common complication of AIDS. Although the initial response to conventional chemotherapy is generally good, prolonged treatment is often difficult because of hypersensitivity to the drugs, particularly to the sulfonamides. Thus, toxoplasmic encephalitis is a significant factor in the morbidity and mortality of AIDS patients. The long term goal is to develop new drugs to treat this opportunistic infection. Toxoplasma gondii is absolutely dependent upon an exogenous source of preformed purines and usually obtains them from its host cell. Identification of the critical bottleneck steps in purine acquisition by the parasite should define new targets for chemotherapy. Preliminary studies suggest that a parasite ecto-AMPase (or 5'-nucleotidase) is a bottleneck through which all purine nucleotides from the host cell must pass and is thus an inviting target for chemotherapy. The specific aims with respect to T.gondii AMPase are to (1) study this gene by molecular approaches, (2) select enzyme-negative parasite mutants and determine if they become absolutely dependent on the addition of purines to the medium, thus confirming the essential role of ecto-AMPase, (3) purify and characterize the AMPase, and (4) study the antitoxoplasma activity of AMPase inhibitors. Preliminary studies suggest that the purine nucelobases and nucleosides derived from the host cell pool are reconverted to nucleotides within the T.gondii cytoplasm by only two enzymes, adenosine kinase (AK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Thus, these two enzymes represent a double bottleneck in purine acquisition by T.gondii. The specific aims with respect to these two enzymes are to (1) isolate temperature sensitive mutants that will allow both enzymes to be shut down at the restrictive temperature and thus test the double bottleneck hypotheses, (2) determine the antitoxoplasma activity of inhibitors of each enzyme using parasite mutants that lack the other enzyme, and (3) test the anti-wild type-toxoplasma activity of dual drug treatment that blocks both enzymes. Preliminary studies show that 6-thioxanthine is a specific inhibitor of T.gondii because only the parasite can convert this analogue to nucleotide form. The specific aims with respect to 6- thioxanthine are to (1) assay all of the nucleotide interconversion enzymes of T.gondii, several of which have a potential role in the antitoxoplasma activity of 6-thioxanthine, and (2) determine if 6- thioxanthine acts primarily by inhibiting GMP synthesis or by being converted to the toxic GMP analogue, 6-thioguanosine-5-'phosphate. The investigators view the primary goal of this proposal to be the definitive characterization of those steps in toxoplasma purine metabolism that are targets for the chemotherapy of toxoplasmosis.

描述：（改编自申请人的摘要）：弓形虫 脑炎是艾滋病的常见并发症。虽然最初 对常规化疗的反应通常很好，延长 由于对药物过敏，治疗通常很难 特别是磺酰胺。因此，弓形虫脑炎是 艾滋病患者的发病率和死亡率的重要因素。这 长期目标是开发新药来治疗这种机会主义 感染。弓形虫Gondii绝对取决于外源性 预制嘌呤的来源，通常从其宿主细胞中获取。 通过 寄生虫应定义化学疗法的新靶标。初步的 研究表明，寄生虫的寄生虫 - 或5'-核苷酸酶是一个 瓶颈必须通过，所有嘌呤核苷酸都必须来自宿主细胞 通过，因此是化学疗法的诱人靶标。具体目标 关于t.gondii Ampase，是（1）通过分子研究该基因 方法，（2）选择酶阴性寄生虫突变体并确定 如果他们完全取决于向 培养基，从而证实了ecto-ampase的基本作用，（3）净化 并表征放大器，（4）研究抗氧气活性 AMPase抑制剂。初步研究表明嘌呤 源自宿主细胞池的核苷酶和核苷是 仅在T.gondii细胞质内重新连接到核苷酸 酶，腺苷激酶（AK）或低黄嘌呤瓜氨酸 磷酸贝糖基转移酶（HGPRT）。因此，这两种酶代表 T.Gondii在嘌呤采集中的双瓶颈。具体目标 关于这两种酶是（1）分离的温度 敏感的突变体将使这两种酶在 限制性温度，从而测试双瓶颈假设， （2）确定每种酶抑制剂的抗氧气活性 使用缺乏其他酶的寄生虫突变体，（3）测试 双重药物治疗的抗野生型豆腐活性 两种酶。初步研究表明，6-噻思茶是一种特定的 t.gondii的抑制剂，因为只有寄生虫才能转换 类似于核苷酸形式。关于6-的具体目的 硫玉黄嘌呤是（1）测定所有核苷酸互连 T.gondii的酶，其中几种具有潜在的作用 6-噻吩黄嘌呤的抗毒素活性，（2）确定6- 硫嘌呤主要通过抑制GMP合成或存在起作用 转化为有毒的GMP类似物，6-硫代氨酸-5-磷酸盐。这 调查人员认为该提议的主要目标是 这些步骤在弓形虫嘌呤中的确切表征 代谢是弓形虫病化疗的靶标。