CORE--INHIBITOR SCREENS, CELL BIOLOGY, AND PARASITOLOGY

核心——抑制剂筛选、细胞生物学和寄生虫学

• 批准号: 6268163
• 负责人: JUAN Carlos ENGEL
• 金额: $ 13.29万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6268163
• 关键词: Trypanosoma cruzi; biomedical facility; cysteine endopeptidases; drug screening /evaluation; host organism interaction; light microscopy; pharmacokinetics; protease inhibitor; recombinant proteins

This section details the series of screening assays we will use to analyze new inhibitor leads synthesized by Bill Roush's synthetic chemistry group or predicted by the computer modeling of Fred Cohen's group. These screening methods include an automated assay of purified protease activity, in vitro culture of the parasite stages, and a murine model of infection. In parallel with identification of new leads, we will carry out initial pharmacokinetic analysis of the derivatized pseudopeptides we have already shown to be effective in lowering parasitemia. These studies will be aimed at optimizing dosing schedules for inhibitors, and identifying synthetically feasible modifications to enhance half-life, minimize toxicity, and allow oral bioavailability. We have shown that fluoromethyl ketone-derivatized peptides, which are irreversible, specific inhibitors of cysteine proteases, will arrest T. cruzi replication and transformation between stages of its life cycle. The events surrounding the transformation of trypomastigote to amastigote and early amastigote replication appear to be especially sensitive to inhibition of the protease. We will now follow up on these observations with a more detailed study of the function of the cysteine protease at different stages of the T. cruzi life cycle, and an analysis of the specific effects of inhibitors on parasite morphology at both the light and electron microscopic levels. As a foundation to these studies, we have produced monospecific, polyclonal antisera to the purified recombinant cruzain. This antisera has the advantage over previous antibody reagents in that it recognizes only protein epitopes. This should minimize cross-reactivity between carbohydrate moieties that may have clouded previous attempts at localization. Furthermore, we have developed a localization assay utilizing a biotinylated fluoromethyl ketone peptide which irreversibly binds only at the active site of the enzyme and, via a streptavidin fluorochrome derivative, can be used to confirm and supplement antibody localization studies. Coupled with the use of fluorochrome derivatives that can identify specific subcellular organelles and locales, a more definitive analysis of the localization of the protease at each stage, and studies of its intracellular trafficking can be carried out. Concurrently, we will study the effects of each new generation of inhibitors on parasite morphology at the light and ultrastructural level. Our previous work has suggested inhibition of the protease results in morphologic abnormalities at the trypomastigote to amastigote interface. These will now be confirmed and analyzed in more detail.

本节详细介绍我们将使用的一系列筛查分析 分析Bill Roush合成的新缓蚀剂先导化合物 化学组或由弗雷德·科恩的计算机模型预测的 一群人。这些筛选方法包括对纯化的 寄生虫期的体外培养的蛋白酶活性，以及一只小鼠 感染模式。在发现新线索的同时，我们 将对衍生品进行初步药代动力学分析 我们已经证明伪肽能有效地降低 寄生虫血症。这些研究将旨在优化给药计划。 用于抑制剂，并确定合成上可行的修饰 提高半衰期，将毒性降至最低，并允许口服生物利用度。 我们已经证明了氟甲基酮衍生的多肽，它们是 不可逆的半胱氨酸蛋白酶的特异性抑制剂将阻止T. 克鲁兹病毒在其生命周期各阶段之间的复制和转化。 围绕着类鞭毛体向无鞭毛体转变的事件 而早期的无鞭毛体复制似乎对 抑制该蛋白水解酶。我们现在将对这些观察结果进行跟进 更详细地研究半胱氨酸蛋白酶的功能，请访问 克氏锥虫生活史的不同阶段，并对其进行了分析 抑制剂对两种光照条件下寄生虫形态的特异性影响 和电子显微镜水平。作为这些研究的基础，我们 产生了针对纯化的单特异性多克隆抗血清 重组克鲁萨因。该抗血清比以往的抗血清更具优势。 抗体试剂，因为它只识别蛋白质表位。这 应将碳水化合物部分之间的交叉反应降至最低 给之前的本地化尝试蒙上了一层阴影。此外，我们还拥有 开发了一种利用生物素化氟甲基进行定位分析的方法 仅不可逆地结合在活性部位的酮肽 酶，并通过链霉亲和素荧光色素衍生物，可用于 确认和补充抗体定位研究。再加上 可识别特定亚细胞的荧光色素衍生物的使用 细胞器和场所，更明确的本地化分析 各阶段的蛋白水解酶及其胞内活性的研究 可以进行人口贩运。同时，我们将研究这些影响 每一代新的抑制剂在光照下对寄生虫形态的影响 和超微结构水平。我们之前的研究表明，抑制 蛋白水解酶的作用会导致组织形态异常 类鞭毛体到无鞭毛体的界面。这些现在将得到确认，并 更详细地分析了。