Prajapat Surendra Kumar, Subramani Chandru, Sharma Puja, Vrati Sudhanshu, Kalia Manjula
Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, 121001, Haryana, India.
Autophagy Rep. 2022 Oct 8;1(1):523-536. doi: 10.1080/27694127.2022.2132075. eCollection 2022.
Autophagy is a conserved intracellular degradation pathway that is essential for maintaining cellular homeostasis. Given its critical role in several disease conditions, recent studies are focussed on identifying drugs/small molecules with autophagy modulating capacity for potential clinical applications. Here, we describe the development and characterisation of a quantitative image-based high content screening platform for autophagy flux measurements using the human melanoma A375 cell line that stably expresses the GFP-LC3-RFP probe. The GFP-LC3 is incorporated into autophagosomes, while RFP serves an internal control. The GFP/RFP fluorescence intensity ratio gives an accurate indication of autophagy induction (low ratio) vs blockage of autophagy flux (high ratio), and was validated with the autophagy inducer Torin1 and inhibitor Bafilomycin A. This assay was used to screen the Spectrum collection library comprising of 2560 compounds, to identify autophagy modulators. In addition to known autophagy effectors, several novel autophagy inducers and inhibitors were identified in our study. Further three FDA approved drugs that are widely used in skin-care products: Avobenzone, Guaiazulene and Tioxolone, were validated as potent autophagy inducers that function in an mTOR independent manner.
Baf, Bafilomycin A; LC3, Microtubule-associated protein light chain 3; mTOR, mechanistic target of rapamycin.
自噬是一种保守的细胞内降解途径，对维持细胞内稳态至关重要。鉴于其在多种疾病状态中的关键作用，最近的研究集中于鉴定具有自噬调节能力的药物/小分子，以用于潜在的临床应用。在此，我们描述了一种基于图像的定量高内涵筛选平台的开发与表征，该平台用于使用稳定表达GFP-LC3-RFP探针的人黑色素瘤A375细胞系测量自噬通量。GFP-LC3被整合到自噬体中，而RFP作为内部对照。GFP/RFP荧光强度比准确指示了自噬诱导（低比值）与自噬通量阻断（高比值），并通过自噬诱导剂托瑞司他和抑制剂巴弗洛霉素A进行了验证。该检测方法用于筛选包含2560种化合物的Spectrum文库，以鉴定自噬调节剂。除了已知的自噬效应物外，我们的研究还鉴定了几种新型的自噬诱导剂和抑制剂。此外，三种广泛用于护肤品的FDA批准药物：阿伏苯宗、愈创蓝油烃和噻克索酮，被验证为以不依赖mTOR的方式发挥作用的有效自噬诱导剂。
Baf，巴弗洛霉素A；LC3，微管相关蛋白轻链3；mTOR，雷帕霉素靶蛋白
