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 克拉伯病，称为球状细胞脑白质营养不良 (GLD)，是一种经典的神经系统溶酶体贮积病 由 b-半乳糖脑苷脂酶 (GALC)、溶酶体水解酶遗传缺陷引起的疾病 (LSD) 负责去除半乳糖脑苷脂和半乳糖基鞘氨醇的半乳糖残基，通常称为 精神。 GLD 患者大脑中的这种鞘脂含量增加。心理因素是生理上存在的 在大多数脑细胞中，低浓度时，然而，高浓度时，它变得极具细胞毒性， 尤其是少突胶质细胞，导致弥漫性脱髓鞘和随后的破坏性脑白质营养不良。 鉴于 GLD、小分子疗法中神经系统表现的快速进展性， 它们更有可能穿过血脑屏障（BBB），成为一种有吸引力的治疗方法。 此前，我们从GLD小鼠模型的新生脑皮质中，建立了特定的脑源性细胞系 概括了 GLD 的细胞表型方面，特别是增加的精神碱水平，而不是 在 GLD 原代细胞中观察到。在 2 次不同的小分子筛选中，我们确定了 2 类潜在的 GLD 的治疗分子。首先，在家长资助中，我们开发并实施了基于活细胞的 对精神碱进行通量 LC-MS/MS 测定，并鉴定出可将其水平降低至几乎 正常生理范围。其次，在脑源性细胞系中表达常见的错误折叠 GALC-G270D，我们 实施了另一项基于细胞的高通量筛选（HTS）筛选并鉴定出能够 协助折叠并增加突变体GALC活性。我建议检验以下假设：心理因素 还原分子和 GALC 折叠辅助分子是降低精神氨酸水平的治疗剂 阻止甚至预防 GLD 的快速脱髓鞘。根据我们之前对 GLD 的研究 汇集了我们的专业知识，我们的目标是使用来自广泛 GLD 患者的诱导神经干细胞 (iNSC) 谱图并检查和优先考虑有效降低精神碱水平的小分子“命中”； (二) 执行 SAR 并生成不同的类似物，以优化其功效并改善其对中央的访问 神经系统（CNS）； (iii) 检查小鼠 GLD 模型监测中选定的小分子药物 确定了神经行为结果和血脑屏障渗透性，以优先考虑化合物进行进一步研究。 由于造血干细胞移植（HSCT）、新生儿筛查（NBS）的初步令人鼓舞的结果 GLD 已在多个州实施。然而，HSCT 无法预防后期发病 神经系统症状 8. 由于目前在几个州对所有新生儿进行 GLD 筛查，因此验证小 该项目中提出的候选分子变得非常重要和紧迫。因此，一个蓬勃发展的 这些州目前和将来有多少无症状新生儿被诊断患有 GLD NBS 程序。他们中的大多数（如果不是全部）最终将受益于来自小分子疗法 拟议的项目。