IN VITRO ASSAY FOR (1,3)B-GLUCAN SYNTHASE INHIBITORS

(1,3)B-葡聚糖合酶抑制剂的体外测定

• 批准号: 2068807
• 负责人: Claude P Selitrennikoff
• 金额: $ 34.4万
• 依托单位: MYCOLOGICS, INC.
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1997-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2068807
• 关键词: Aspergillus; Candida albicans; antifungal agents; biomedical automation; drug screening /evaluation; enzyme activity; enzyme inhibitors; glucans; microorganism culture

The fungal cell wall protects the organism against a hostile exterior environment and relays signals for invasion and infection of a likely animal or human host. The wall affords a clear and discernible difference between fungi and their hosts, providing an experimental target for antifungal antibiotics. A key step in fungal wall assembly is the synthesis of (1,3)beta-linked glucan and its subsequent incorporation into the cell wall. The observation that humans lack (1,3)beta-glucan makes (1,3)beta-glucan synthesis an attractive target for antifungal antibiotics. In Phase I, a rapid, high throughput in vitro enzyme assay suitable as a screen to detect inhibitors of (1 ,3)beta-glucan synthase activity of Neurospora crassa was developed. The Specific Aims of this Phase II proposal are: 1. Development of optimum in vitro conditions for the assay of (1,3)beta- glucan synthase of Candida albicans and Aspergillus fumigatus. 2. Development of a semi-automated in vitro assay for (1,3)beta-glucan synthase activity. 3. Screening of bona fide samples for enzyme inhibitors. 4. Follow-up testing of positive samples using whole-cell assays. The development of a high throughput semi-automated in vitro assay of (1,3)beta-glucan synthase activity of human pathogenic fungi will permit the mass screening of samples for enzyme inhibitors. PROPOSED COMMERCIAL APPLICATION: This assay will permit the screening of compounds on a large scale for inhibitors of fungal (1,3)beta-glucan synthase using human pathogens as enzyme sources. Antifungal drugs for human therapeutic use have significant market potential.

真菌细胞壁保护生物体免受敌对外部的侵害 环境并传递可能的入侵和感染信号 动物或人类宿主。墙壁提供了清晰可辨的差异 真菌及其宿主之间的关系，提供了一个实验目标 抗真菌抗生素。真菌墙组装的关键步骤是 (1,3)β-连接葡聚糖的合成及其随后掺入 细胞壁。人类缺乏 (1,3)β-葡聚糖的观察使得 (1,3)β-葡聚糖合成是抗真菌药物的一个有吸引力的靶点 抗生素。 在第一阶段，快速、高通量的体外酶测定适合作为 筛选检测 (1,3)β-葡聚糖合酶活性抑制剂 粗糙脉孢菌被开发出来。第二阶段的具体目标 提案是： 1. 开发 (1,3)β- 测定的最佳体外条件 白色念珠菌和烟曲霉的葡聚糖合酶。 2. (1,3)β-葡聚糖半自动体外测定的开发 合酶活性。 3. 筛选真实样品中的酶抑制剂。 4. 使用全细胞检测对阳性样本进行后续检测。 高通量半自动体外检测方法的开发 (1,3)人类病原真菌的β-葡聚糖合酶活性将允许 大规模筛选样品中的酶抑制剂。 拟议的商业应用：该测定将允许筛选 大规模用于真菌 (1,3)β-葡聚糖抑制剂的化合物 使用人类病原体作为酶源的合酶。抗真菌药用于 人类治疗用途具有巨大的市场潜力。