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

描述(由申请人提供)：人类粘蛋白瞬时受体潜力1(TRPML1)的突变会导致IV型粘脂沉积症(ML4)，这是一种对幼儿具有破坏性的神经退行性疾病。ML4患者表现为运动障碍、智力低下和视网膜变性。目前还没有针对ML4的治疗方法。通过建立膜片钳方法直接检测晚期内体和溶酶体(LEL)中TRPML蛋白的功能，我们发现TRPML1介导内体和溶酶体的钙和铁的外流，而PI(3，5)P2是一种低丰度的内溶酶体特异的磷脂酰肌醇结合并特异性地激活TRPML。在生理条件下，TRPMLS通过将PI(3，5)P2水平的信息转导到细胞器旁钙离子的变化来调节膜的转运，从而在晚期的内吞途径中触发膜融合/分裂事件(内溶酶体转运)。此外，TRPML1还参与晚期内体和溶酶体内Fe2+的释放，这是细胞铁代谢所必需的。因此，离子(Ca~(2+)和Fe~(2+))动态平衡受损导致溶酶体功能障碍和ML4表型。在ML4细胞中，问题有两个。首先，由于TRPML1缺乏导致的钙离子依赖的膜转运缺陷导致脂类和其他生物物质在溶酶体中积累。第二次冲击来自溶酶体铁超载(由于TRPML1‘S Fe2+电导率受损)，它将溶酶体中积累的物质转化为不可降解(对溶酶体降解具有抵抗力)的脂褐素(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，TRPML1似乎是溶酶体离子动态平衡的重要调节因子。我们特别假设，用合成激动剂刺激溶酶体中TRPML1‘S的Fe2+/Ca2+通道活动可以减轻ML4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)，针对MLSCN(分子图书馆筛选中心网络)化合物集合进行钙成像分析，以确定新的TRPML1小分子激活剂和抑制剂。我们的第二个目标是利用钙离子成像和电生理分析来表征和优化从HTS中鉴定的TRPML1激活剂和抑制剂。我们的第三个目标是使用来自ML4和鼻咽癌患者的细胞系来验证候选化合物的贩运-拯救和减少储存的功能。总之，有缺陷的溶酶体Fe2+/Ca2+外流是ML4的原因，这使得获得能够操纵TRPML1通道活性的药物具有重要意义。我们提议的研究的最终目标是开发ML4的治疗策略。

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.