Characterization of Small Molecule Therapeutic Agents for a Lysosomal Leukodystrophy

摘要 Krabbe病又称球状细胞脑白质营养不良（GLD），是一种典型的神经系统溶酶体积积症 一种由b-半乳糖苷酶（GALC）、溶酶体水解酶的遗传缺陷引起的疾病（LSD） 负责去除半乳糖头苷和半乳糖鞘氨醇的半乳糖残基，通常称为 精神碱这种鞘脂在GLD患者的大脑中增加。Psychosine在生理上存在 在大多数脑细胞中，低浓度时，然而，在高浓度时，它变得具有极强的细胞毒性， 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神经系统表现的快速进展性质，小分子治疗， 其更可能穿过血脑屏障（BBB），成为一种吸引人的治疗方法。 本研究首先从GLD小鼠模型的新生脑皮质中建立了特异性的脑源性细胞系 它概括了GLD的细胞表型方面，特别是增加的psychosine水平， 在GLD原代细胞中观察到。在2种不同的小分子筛选中，我们确定了2类潜在的 治疗性分子首先，在父母补助金中，我们开发并实施了基于活细胞的 通过LC-MS/MS分析，确定了将其水平降低到几乎 正常生理范围。其次，我们在脑源性细胞系中表达了常见的错误折叠GALC-G270 D 实施了另一种基于细胞的高通量筛选（HTS）筛选，并鉴定了能够 辅助折叠并增加突变体GALC活性。我提议检验精神病的假设- 还原和GALC-折叠辅助分子是治疗剂， 阻止甚至预防GLD的快速脱髓鞘。基于我们以前在GLD和 我们的专业知识集合，我们的目标是使用来自广泛的GLD患者的诱导神经干细胞（iNSC） 光谱和检查并优先考虑有效降低精神病水平的小分子“命中”;（ii） 执行SAR并生成不同的类似物，以优化其功效并改善其进入中枢神经系统的途径。 神经系统（CNS）;（iii）在鼠GLD模型中检查选定的小分子试剂， 建立神经行为结果和BBB渗透，以优先考虑化合物用于进一步研究。 由于造血干细胞移植（HSCT）的初步结果令人鼓舞，新生儿筛查（NBS） 已在几个州实施了一般法律司。然而，HSCT无法预防晚期发作 神经系统症状8.由于GLD目前在几个州的所有新生儿中进行筛查， 因此，本项目中提出的候选分子变得非常重要和紧迫。因此， 在这些州的NBS中，一些无症状的新生儿现在和将来都被诊断出患有GLD 程序.他们中的大多数，如果不是全部，最终将受益于来自世界各地的小分子疗法。 拟议项目。

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.