Xu Miao, Liu Ke, Swaroop Manju, Sun Wei, Dehdashti Seameen J, McKew John C, Zheng Wei
1Therapeutics for Rare and Neglected Disease, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
J Biomol Screen. 2014 Jan;19(1):168-75. doi: 10.1177/1087057113501197. Epub 2013 Aug 27.
The lysosome is a vital cellular organelle that primarily functions as a recycling center for breaking down unwanted macromolecules through a series of hydrolases. Functional deficiencies in lysosomal proteins due to genetic mutations have been found in more than 50 lysosomal storage diseases that exhibit characteristic lipid/macromolecule accumulation and enlarged lysosomes. Recently, the lysosome has emerged as a new therapeutic target for drug development for the treatment of lysosomal storage diseases. However, a suitable assay for compound screening against the diseased lysosomes is currently unavailable. We have developed a Lysotracker staining assay that measures the enlarged lysosomes in patient-derived cells using both fluorescence intensity readout and fluorescence microscopic measurement. This phenotypic assay has been tested in patient cells obtained from several lysosomal storage diseases and validated using a known compound, methyl-β-cyclodextrin, in primary fibroblast cells derived from Niemann Pick C disease patients. The results demonstrate that the Lysotracker assay can be used in compound screening for the identification of lead compounds that are capable of reducing enlarged lysosomes for drug development.
溶酶体是一种重要的细胞器，主要作为一个回收中心，通过一系列水解酶分解不需要的大分子。由于基因突变导致的溶酶体蛋白功能缺陷已在50多种溶酶体贮积病中被发现，这些疾病表现出特征性的脂质/大分子积累和溶酶体增大。最近，溶酶体已成为开发治疗溶酶体贮积病药物的新治疗靶点。然而，目前尚无针对患病溶酶体进行化合物筛选的合适检测方法。我们开发了一种溶酶体示踪剂染色检测方法，该方法利用荧光强度读数和荧光显微镜测量来检测患者来源细胞中增大的溶酶体。这种表型检测方法已在从几种溶酶体贮积病患者获得的细胞中进行了测试，并在来自尼曼-皮克C病患者的原代成纤维细胞中使用已知化合物甲基-β-环糊精进行了验证。结果表明，溶酶体示踪剂检测方法可用于化合物筛选，以鉴定能够减少增大的溶酶体的先导化合物，用于药物开发。
