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

真菌细胞壁保护有机体免受外界的侵害。 环境和继电器信号的入侵和感染可能 动物或人类宿主。这堵墙提供了一个明显和明显的区别 真菌和它们的寄主之间的关系，为 抗真菌抗生素。真菌壁组装的一个关键步骤是 (1，3)-β-葡聚糖的合成及其在体内的应用 细胞壁。观察到人类缺乏(1，3)β-葡聚糖 (1，3)β-葡聚糖合成诱人的抗真菌靶标 抗生素。 在第一阶段，一种快速、高通量的体外酶分析方法适用于 检测(1，3)β-葡聚糖合成酶活性抑制物的筛选 粗枝脉孢菌(Neuropora Crassa)。第二阶段的具体目标 建议包括： 1.建立测定(1，3)-β-β-半乳糖的最佳体外条件 白念珠菌和烟曲霉的葡聚糖合成酶。 2.(1，3)-β-葡聚糖体外半自动检测方法的建立 合酶活性。 3.酶抑制剂真品的筛选。 4.采用全细胞检测法对阳性标本进行跟踪检测。 一种高通量半自动体外检测方法的建立 (1，3)人类病原真菌的β-葡聚糖合成酶活性将允许 对酶抑制剂的样品进行大规模筛选。 拟议的商业应用：这种检测方法将允许筛选 大规模合成真菌(1，3)β-葡聚糖抑制剂 使用人类病原体作为酶来源的合成酶。抗真菌药物治疗 人类的治疗用途具有巨大的市场潜力。

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.