Characterization of Small Molecule Therapeutic Agents for a Lysosomal Leukodystrophy

溶酶体脑白质营养不良小分子治疗药物的表征

• 批准号: 10208466
• 负责人: Gustavo Henrique Boff Maegawa
• 金额: $ 15.28万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2021-10-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10208466
• 关键词: Affect; Age; Biochemical; Biological Assay; Blood - brain barrier anatomy; Brain; Cell Line; Cells; Demyelinations; Development; Diagnosis; Diffuse; Disabled Persons; Disease; Evaluation; Excision; Galactose; Genetic; Globoid cell leukodystrophy; Goals; Hematopoietic Stem Cell Transplantation; Histologic; Human; Hydrolase; In Vitro; Inherited; Libraries; Lysosomal Storage Diseases; Modeling; Monitor; Mus; Mutation; Myelin; Nature; Neonatal Screening; Neuraxis; Neurologic; Neurologic Symptoms; Newborn Infant; Oligodendroglia; Outcome; Patients; Peripheral Nervous System; Phenotype; Physiological; Positioning Attribute; Prevention; Property; Psychosine; Reducing Agents; Residual state; Schwann Cells; Sphingolipids; Testing; Therapeutic; Therapeutic Agents; Transplant Recipients; Validation; Wheelchairs; analog; base; blood-brain barrier penetration; brain cell; cytotoxic; efficacious treatment; established cell line; galactosylceramidase; high throughput screening; improved; in vivo; leukodystrophy; mortality; mouse model; mutant; neonatal period; nerve stem cell; neurobehavioral; novel; parent grant; pharmacokinetics and pharmacodynamics; prevent; screening; screening program; small molecule; small molecule therapeutics

ABSTRACT Krabbe disease, known as globoid-cell leukodystrophy (GLD), is a classical neurological lysosomal storage disease (LSD) caused by a genetic deficiency of b-galactocerebrosidase (GALC), lysosomal hydrolase responsible for removing galactose residues of galactocebroside and galactosylsphingosine, mostly known as psychosine. This sphingolipid is increased in brain of patients with GLD. Psychosine is physiologically present in most brain cells at low concentrations, however, at high concentrations, it becomes extremely cytotoxic, especially oligodendrocytes resulting in diffuse demyelination and subsequent devastating leukodystrophy. Given the rapidly progressive nature of the neurological manifestations in GLD, small molecule therapies, which are more likely to cross the blood-brain barrier (BBB), become an attractable therapeutic approach. Previously, from newborn brain cortices of GLD murine model, we established a specific brain-derived cell line that recapitulates cellular phenotypic aspects of GLD, in particular the increased psychosine levels, not observed in GLD primary cells. In 2 different small molecule screenings, we identified 2 classes of potential therapeutic molecules for GLD. First, in the parent grant, we developed and implemented a live cell-based throughput LC-MS/MS assay for psychosine and identified small molecules that reduce its levels to almost normal physiological range. Second, expressing a common misfolded GALC-G270D in a brain-derived line, we implemented another cell-based high-throughput screening (HTS) screening and identified molecules able to assist the folding and increased the mutant GALC activity. I propose to test the hypothesis that the psychosine- reducing and GALC-folding assistant molecules are therapeutic agents that reduce psychosine levels resulting in the arrest and even prevention of the rapid demyelination in GLD. Based on our previous studies in GLD and our expertise assembled, our goals are to use induced-neural stem cells (iNSC) from patients with wide GLD spectrum and examine and prioritize the small molecule ‘hits’ that efficaciously reduce psychosine levels; (ii) perform SARs and generate different analogs to optimize their efficacy and improve their access to the central nervous system (CNS); (iii) examine selected small molecules agents in the murine GLD models monitoring established neurobehavioral outcomes and BBB penetration to prioritize the compounds for further studies. Due to initial encouraging results of hematopoietic stem cell transplantation (HSCT), newborn screening (NBS) for GLD has been implemented in several states. However, the HSCT is unable to prevent later onset neurological symptoms 8. As GLD is currently screened in all newborns in several states, the validation of small molecule candidates proposed in this project becomes highly important and urgent. Therefore, a burgeoning number of asymptomatic newborns are now, and will be, diagnosed with GLD throughout these state NBS programs. Most, if not all, of them will eventually benefit from the small molecule therapies coming from the proposed project.

抽象的 Krabbe病，称为Globoid-Cell白细胞营养不良（GLD），是经典的神经溶酶体储存 由B-半乳脑脑化酶（GALC），溶酶体水解酶的遗传缺乏引起的疾病（LSD） 负责去除半乳头骨和半乳糖基肾上腺素的半乳糖残留物，主要称为 Psychosine。 GLD患者的大脑中这种鞘脂增加。 Psychosine在身体上存在 然而，在大多数脑细胞中，在高浓度下，它变得极为细胞毒性， 尤其是少突胶质细胞，导致弥漫性脱髓鞘和随后的毁灭性白细胞营养不良。 鉴于GLD中神经系统表现的快速进行性，小分子疗法， 更可能越过血脑屏障（BBB）成为一种可吸引人的治疗方法。 以前，我们从GLD Murine模型的新生儿脑皮层中建立了特定的脑衍生细胞系 这概括了GLD的细胞表型方面，尤其是心理水平的增加，而不是 在GLD原代细胞中观察到。在2个不同的小分子筛选中，我们确定了2类电位 GLD的治疗分子。首先，在父母赠款中，我们开发并实施了一个基于电池的实时 吞吐量LC-MS/MS分析用于心理碱，并确定了将其水平降低到几乎的小分子 正常生理范围。其次，在脑衍生的线上表达常见的错误折叠的Galc-G270D，我们 实施了另一个基于细胞的高通量筛选（HTS）筛选，并确定的分子可以 协助折叠并增加突变的GALC活性。我建议检验以下假设：Psychosine- 减少和galc折叠助理分子是降低心理水平的热剂 在逮捕甚至预防GLD快速脱髓鞘中。根据我们以前在GLD的研究和 我们的专业知识组装了，我们的目标是使用广泛的GLD患者使用诱导的神经干细胞（INSC） 频谱，检查并确定有效降低心理水平的小分子“命中”； （ii） 执行SARS并产生不同的类似物，以优化其效率并提高他们对中央的访问 神经系统（CNS）； （iii）检查了鼠类GLD模型监测中的小分子剂 已建立的神经行为结局和BBB渗透以确定化合物以进行进一步研究。 由于最初令人鼓舞的造血干细胞移植（HSCT）的结果，新生儿筛查（NBS） 因为GLD已在几个州实施。但是，HSCT无法防止以后发作 神经符号8。由于目前在几个州的所有新生儿中都筛选了GLD，因此验证小 该项目中提出的分子候选者变得非常重要和紧迫。因此，一个新兴的人 现在已经并且将被诊断出在这些状态NBS中被诊断为GLD的无症状新生儿数量 程序。其中大多数（如果不是全部）最终将受益于来自 拟议项目。