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: 6099784
• 负责人: James H. McKerrow
• 金额: $ 11.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6099784
• 关键词: Leishmania; Trypanosoma cruzi; antiprotozoal agents; biomedical facility; cell biology; cysteine endopeptidases; drug screening /evaluation; intracellular parasitism; 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.

本节详细介绍了我们将使用的一系列筛选试验， 分析由比尔·赫夫合成的新型抑制剂先导化合物 化学组或预测的计算机建模弗雷德科恩的 组 这些筛选方法包括纯化的抗体的自动化测定。 蛋白酶活性，体外培养的寄生虫阶段，和鼠 感染的模式。 在确定新线索的同时，我们 将对衍生的药物进行初步药代动力学分析。 我们已经证明，假肽可以有效地降低 寄生虫血症。 这些研究旨在优化给药方案 抑制剂，并确定合成可行的修改， 增加半衰期，最大限度地减少毒性，并允许口服生物利用度。 我们已经表明，氟甲基酮衍生的肽，这是 不可逆的，特异性的半胱氨酸蛋白酶抑制剂，将阻止T。 cruzi复制和其生命周期各阶段之间的转化。 围绕锥鞭毛体向无鞭毛体转化的事件 和早期无鞭毛体复制似乎是特别敏感， 抑制蛋白酶。 我们现在将跟进这些观察结果 随着半胱氨酸蛋白酶功能的更详细研究， T. cruzi生命周期，以及对 抑制剂对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 已经产生了纯化的单特异性多克隆抗血清， 重组cruzain。 该抗血清具有优于以前的抗血清的优点， 抗体试剂，因为它只识别蛋白质表位。 这 应该最小化碳水化合物部分之间的交叉反应性， 给之前的本地化尝试蒙上了阴影。 此外，我们还 开发了一种利用生物素化氟甲基 酮肽，其仅不可逆地结合在 酶，并通过链霉亲和素荧光染料衍生物，可用于 确认和补充抗体定位研究。 加上 荧光染料衍生物的用途 细胞器和区域，一个更明确的分析定位 的蛋白酶在每个阶段，并研究其细胞内 可以进行贩卖。 同时，我们将研究 每一代新的抑制剂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 蛋白酶的作用导致组织形态异常 锥鞭毛体与无鞭毛体界面。 这些现在将得到确认， 更详细地分析。