Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.
Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.
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DOI：
10.1021/acschemneuro.1c00804
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发表时间：
2022-05-18
影响因子：
5
通讯作者：
Kao AW
中科院分区：
文献类型：
--
作者：
Chin MY;Ang KH;Davies J;Alquezar C;Garda VG;Rooney B;Leng K;Kampmann M;Arkin MR;Kao AW
溶酶体是负责细胞内多种大分子降解的细胞内细胞器。溶酶体酶的最佳功能需要高酸性 pH 值。溶酶体腔内酸度的丧失会破坏细胞蛋白质稳态，并与神经退行性疾病等年龄相关疾病有关。使用新型稳健的溶酶体 pH 生物传感器 (FIRE-pHLy)，我们开发了一种用于高通量筛选 (HTS) 的细胞荧光测定法，并将其应用于分化的 SH-SY5Y 神经母细胞瘤细胞。这项研究的目标有两个：(1) 筛选酸化溶酶体 pH 值的小分子；(2) 确定调节溶酶体 pH 值的分子靶标和途径。我们对 1835 种生物活性化合物进行了筛选，并附有注释的目标信息，以识别溶酶体 pH 调节剂（酸化剂和碱化剂）。使用基于群体和基于对象的数据的组合分析方法，45 种化合物通过了初始命中选择标准。在剂量反应测定中重新测试了 23 种化合物，其中 OSI-027 和 PP242 两种化合物被确定为最有效的酸化剂。总体而言，来自表型 HTS 筛选的数据可用于探索溶酶体 pH 调节的新调节途径。此外，OSI-027 和 PP242 可以作为有用的工具化合物，使自噬激活和溶酶体酸化的机制研究成为神经退行性疾病的潜在治疗途径。
Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.
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影响因子：
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作者：
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通讯作者：
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