High Throughput Screening for Modulators of the TRPML1 Ion Channel

描述（由申请人提供）：人粘脂蛋白瞬时受体电位1 （TRPML1）突变导致IV型粘脂病（ML4），这是幼儿中一种毁灭性的神经退行性疾病。ML4患者表现为运动缺陷、智力迟钝和视网膜变性。目前还没有治疗ML4的方法。通过开发膜片钳法直接测量TRPML蛋白在晚期核内体和溶酶体（LEL）中的功能，我们发现TRPML1介导Ca2+和Fe2+从核内体和溶酶体流出，并且PI(3,5)P2是一种低丰度的内溶酶体特异性磷酸肌肽，结合并特异性激活TRPML。在生理条件下，trpml通过将PI(3,5)P2水平的信息转导到细胞器旁Ca2+的变化来调节膜运输，从而在内吞晚期途径（内溶酶体运输）中触发膜融合/裂变事件。此外，TRPML1介导晚期核内体和溶酶体释放铁离子，这是细胞铁代谢所必需的。因此，受损的离子（Ca2+和Fe2+）稳态是溶酶体功能障碍和ML4表型的基础。在ML4细胞中，问题是双重的。首先，由于trpml1缺乏导致Ca2+依赖的膜运输缺陷导致溶酶体中脂质和其他生物材料的积累。第二次打击来自于溶酶体铁超载（由于TRPML1的Fe2+电导率受损），它将积累的物质转化为溶酶体中不可降解（抵抗溶酶体降解）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似乎是溶酶体离子稳态的重要调节因子。我们特别假设，使用合成激动剂刺激TRPML1溶酶体中的Fe2+/Ca2+通道活性可以减轻与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），使用针对MLSCN（分子文库筛选中心网络）化合物收集的Ca2+成像试验，鉴定新的TRPML1小分子激活剂和抑制剂。我们的第二个目标是利用Ca2+成像和电生理分析来表征和优化从HTS中鉴定出的TRPML1激活剂和抑制剂。我们的第三个目标是利用来自ML4和NPC疾病患者的细胞系验证候选化合物的转运拯救和储存减少功能。总的来说，证明溶酶体Fe2+/Ca2+外排缺陷是ML4的原因，因此获得可以操纵TRPML1通道活性的药物具有重要意义。我们提出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.