A NOVEL SCREEN FOR ANTIFUNGALS THAT TARGET CHITOSAN BIOSYNTHESIS

针对壳聚糖生物合成的新型抗真菌药物筛选

• 批准号: 8545318
• 负责人: Jennifer K. Lodge
• 金额: $ 39.65万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8545318
• 关键词: Acquired Immunodeficiency Syndrome; Action Potentials; Adverse effects; Anabolism; Antifungal Agents; Antifungal Therapy; Binding; Biological Assay; Cell Line; Cell Wall; Cells; Cessation of life; Chitin; Chitin Synthase; Chitosan; Complex; Country; Cryptococcus; Cryptococcus neoformans; Cryptococcus neoformans infection; Data; Deacetylation; Development; Disease; Ensure; Enzyme Inhibition; Enzymes; Equipment; Family; Future; Genetic; Growth; Homologous Gene; Human; Immunocompromised Host; Individual; Infection; Institutes; Life Cycle Stages; Mammals; Methods; Mycoses; NIH Program Announcements; National Institute of Allergy and Infectious Disease; New Agents; Organ Transplantation; Organelles; Phenotype; Polysaccharides; Preparation; Process; Production; Proteins; Publishing; Reagent; Relapse; Risk; Screening procedure; Specificity; Staining method; Stains; System; Temperature; Testing; Therapeutic; Time; Toxic effect; United States National Institutes of Health; Virulence; assay development; base; calcofluor white; chemotherapy; cytotoxic; design; fungus; high throughput screening; inhibitor/antagonist; interest; killings; meetings; miniaturize; mouse model; novel; novel strategies; patient population; prevent; response

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a pathogenic fungus that is found world-wide and causes meningioencephalitis, particularly in immunocompromised individuals. It is invariably fatal unless treated, and the current antifungals are inadequate to effectively cure this disease, due to inherent toxicities or the inability to kill the fungus and prevent relapse. Recent studies have indicated that there are over 1,000,000 new cases of cryptococcosis in the world each year, which results in over 600,000 deaths. New agents to treat Cryptococcus are needed. We have shown that chitosan, the deacetylated form of chitin, is a critical component of the Cryptococcal cell wall and is absolutely required for virulence using a mouse model of cryptococcosis. Our studies have further identified the key enzymes required for chitosan production. This assay development proposal is based on our findings that deletion strains that eliminate chitosan formation share a common set of phenotypes, termed "chitosan deficiency", which we propose to exploit to identify novel antifungal drugs. The key phenotypes of chitosan deficiency can be combined for the development of robust, miniaturized high throughput screening (HTS) assays. These are reduced growth at elevated temperatures and changes in staining of the cell wall. The proposed assay development strategy is supported by the many genetic deletion strains which we have derived that will enable chitosan deficiency hit selection and profiling. We also show preliminary capabilities to characterize mechanism of action of selected hits through direct enzyme inhibition. The chitin that is deacetylated is primarily generated through the action of a single chitin synthase and a specific chitin synthase regulator, even though Cryptococcus encodes seven other synthases and two other synthase regulators. The deacetylation is also dependent upon a family of three chitin deacetylases that we hypothesize to act in concert with the synthase/regulator. This application is a proposal to develop primary assays that will be suitable for a high-throughput screen (HTS) to identify candidate compounds that will inhibit the biosynthesis of chitosan in cryptococcus. We will also develop secondary screens to rule out non-specific cytotoxic compounds and we present preliminary enzyme assay data that supports our ability to develop the necessary tertiary assays to characterize and validate the screening hits. Our lab has the needed equipment and space to develop the reagents, cell lines and assays outlined in this proposal. We also have considerable industrial HTS expertise in the lab that will ensure that the assays and strategies developed through this proposal will successfully meet the standards of any HTS facility that would implement them. This proposal represents a novel approach to antifungal inhibitor discovery and targets proteins that have no homologies in humans or other mammals.

描述(申请人提供)：新生隐球菌是一种在世界范围内发现的致病真菌，可引起脑膜脑炎，特别是在免疫功能低下的人中。除非得到治疗，否则它总是致命的，而且由于固有的毒性或无法杀死真菌并防止复发，目前的抗真菌药物不足以有效治愈这种疾病。最近的研究表明，世界上每年有100多万新的隐球菌病病例，导致60多万人死亡。需要新的药物来治疗隐球菌。我们已经证明了壳聚糖，甲壳素的脱乙酰化形式，是隐球菌细胞壁的关键成分，并且是使用 隐球菌病小鼠模型。我们的研究进一步确定了壳聚糖生产所需的关键酶。这项试验发展建议是基于我们的发现，即消除壳聚糖形成的缺失菌株具有一组共同的表型，称为“壳聚糖缺乏症”，我们建议利用这些表型来鉴定新的抗真菌药物。壳聚糖缺乏的关键表型可以结合起来，开发出可靠的、小型化的高通量筛选(HTS)检测方法。这些是在高温下生长减慢和细胞壁染色的变化。所提出的检测开发策略得到了我们衍生的许多基因缺失菌株的支持，这些菌株将使壳聚糖缺乏症的选择和分析成为可能。我们还展示了通过直接酶抑制来表征所选HITS的作用机制的初步能力。脱乙酰化的甲壳素主要是通过一个甲壳素合成酶和一个特定的甲壳素合成酶调节剂的作用产生的，即使隐球菌编码另外七个合成酶和两个其他合成酶调节剂。脱乙酰化也依赖于三个甲壳素脱乙酰酶家族，我们假设它们与合成酶/调节因子协同作用。这项应用是一项建议，旨在开发一种适合于高通量筛选(HTS)的初步分析方法，以确定将抑制隐球菌壳聚糖生物合成的候选化合物。我们还将开发二次筛查以排除非特异性细胞毒性化合物，并提供初步的酶分析数据，以支持我们开发必要的三级分析来表征和验证筛选命中的能力。我们的实验室有必要的设备和空间来开发这项提案中概述的试剂、细胞系和分析。我们在实验室中还拥有大量的工业HTS专业知识，将确保通过该提案开发的分析和战略将成功地满足任何HTS设施的标准，这些设施将实施它们。这一建议代表了一种发现抗真菌抑制剂的新方法，并针对在人类或其他哺乳动物中没有同源性的蛋白质。