Kinetic High Throughput Screening for Agonists and Inhibitors of the TRPML1 Ion c

TRPML1 离子 c 激动剂和抑制剂的动力学高通量筛选

• 批准号: 8262514
• 负责人: Haoxing Xu
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-23 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8262514
• 关键词: Aging; Agonist; Binding; Biological Assay; Cell Line; Cells; Child; Childhood; Cholesterol; Clinical; Collection; Defect; Disease; Electrophysiology (science); Employee Strikes; Endosomes; Event; Exhibits; Functional disorder; Ganglioside Sialidase Deficiency Disease; Goals; Homeostasis; Human; Image; Impairment; Ion Channel; Ions; Iron Overload; Kinetics; Lipids; Lipofuscin; Lysosomes; Measures; Mediating; Membrane Fusion; Membrane Protein Traffic; Mental Retardation; Methods; Molecular Bank; Motor; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Outcome; Pathway interactions; Patients; Phenotype; Phosphatidylinositols; Physiological; Pigments; Proteins; Research; Resistance; Retinal Degeneration; Screening procedure; Structure of thyroid parafollicular cell; Supraoptic Vertical Ophthalmoplegia; Therapeutic; high throughput screening; inhibitor/antagonist; insight; iron metabolism; late endosome; novel; patch clamp; receptor; small molecule; trafficking

DESCRIPTION (provided by applicant): Mutations of human Mucolipin transient receptor potential 1 (TRPML1) cause type IV mucolipidosis (ML4), a devastating neurodegenerative disease in young children. ML4 patients exhibit motor defects, mental retardation, and retinal degeneration. Currently there is no treatment for ML4. By developing a patch-clamp method to directly measure the functions of TRPML proteins in the late endosome and lysosome (LEL), we found that TRPML1 mediates Ca2+ and Fe2+ efflux from endosomes and lysosomes, and that PI(3,5)P2, a low-abundance endolysosome-specific phosphoinositide, binds and specifically activates TRPMLs. Under physiological conditions, TRPMLs regulate membrane trafficking by transducing information about PI(3,5)P2 levels into changes in juxtaorganellar Ca2+, thereby triggering membrane fusion/fission events in the late endocytic pathways (endolysosomal trafficking). In addition, TRPML1 mediates release of Fe2+ from late endosomes and lysosomes, which is essential for cellular iron metabolism. Thus impaired ion (Ca2+ and Fe2+) homeostasis underlies lysosomal dysfunction and ML4 phenotypes. In ML4 cells, the problems are twofold. First, defective Ca2+-dependent membrane trafficking due to TRPML1-deficiency causes accumulation of lipids and other bio-materials in the lysosome. The second strike comes from the lysosomal iron overload (due to impairment of TRPML1's Fe2+ conductivity), which converts the accumulated materials into the non-degradable (resistant to lysosomal degradation) lipofuscin (also called aging pigment) in the lysosome. Lipofuscin accumulation dramatically compromises the functions of lysosomes. Thus TRPML1 appears to be an essential regulator of lysosome ion homeostasis. We specifically hypothesize that stimulating TRPML1's Fe2+/Ca2+ channel activity in the lysosome using synthetic agonists can alleviate neurodegeneration associated with ML4. Our first aim is to identify novel small molecule activators and inhibitors of TRPML1 by high throughput screening (HTS) using a Ca2+-imaging assay against the MLSCN (Molecular Libraries Screening Center Network) compound collection. Our second aim is to characterize and optimize the TRPML1 activators and inhibitors identified from HTS using Ca2+ imaging and electrophysiology assays. Our third aim is to validate the trafficking-rescue and storage-reducing functions of candidate compounds using cells lines derived from patients with ML4 and NPC diseases. Overall, the demonstration of defective lysosomal Fe2+/Ca2+ efflux as the cause of ML4 makes it highly significant to obtain pharmacological agents that can manipulate TRPML1 channel activity. The ultimate goal of our proposed research is to develop therapeutic strategies for ML4.

描述（由申请方提供）：人粘脂瞬时受体电位1（TRPML 1）突变导致IV型粘脂沉积症（ML 4），这是一种幼儿毁灭性神经退行性疾病。ML 4患者表现出运动缺陷、智力迟钝和视网膜变性。目前没有治疗ML 4的方法。通过建立膜片钳技术直接测定晚期内体和溶酶体（LEL）中TRPML蛋白的功能，我们发现TRPML 1介导Ca ~（2+）和Fe ~（2+）从内体和溶酶体流出，PI（3，5）P2是一种低丰度的内溶酶体特异性磷酸肌醇，可结合并特异性激活TRPML。在生理条件下，TRPMLs通过将PI（3，5）P2水平的信息转换为近细胞器Ca 2+的变化来调节膜运输，从而触发晚期内吞途径（内溶酶体运输）中的膜融合/分裂事件。此外，TRPML 1介导Fe 2+从晚期内体和溶酶体的释放，这对于细胞铁代谢至关重要。因此，受损的离子（Ca 2+和Fe 2+）稳态是溶酶体功能障碍和ML 4表型的基础。在ML 4细胞中，问题是双重的。首先，由于TRPML 1缺乏导致的Ca 2+依赖性膜运输缺陷导致脂质和其他生物材料在溶酶体中积累。第二次打击来自溶酶体铁过载（由于TRPML 1的Fe 2+电导率受损），其将积累的材料转化为溶酶体中的不可降解（耐溶酶体降解）脂褐素（也称为老化色素）。脂褐素的积累极大地损害了溶酶体的功能。因此，TRPML 1似乎是溶酶体离子稳态的重要调节剂。我们特别假设，使用合成激动剂刺激溶酶体中TRPML 1的Fe 2 +/Ca 2+通道活性可以缓解与ML 4相关的神经退行性变。我们的第一个目标是通过高通量筛选（HTS），使用Ca 2+成像分析对MLSCN（分子库筛选中心网络）化合物集合，以确定新的TRPML 1的小分子激活剂和抑制剂。我们的第二个目标是表征和优化TRPML 1激活剂和抑制剂从HTS使用Ca 2+成像和电生理检测。我们的第三个目标是使用来自ML 4和NPC疾病患者的细胞系验证候选化合物的运输-救援和储存-减少功能。总之，证明缺陷的溶酶体Fe 2 +/Ca 2+流出作为ML 4的原因使得获得可以操纵TRPML 1通道活性的药理学试剂非常重要。我们提出的研究的最终目标是开发ML 4的治疗策略。