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A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds.

ABSTRACT: Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery.

SUBMITTER: Kubben N

PROVIDER: S-EPMC6317068 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds.

Kubben Nard N Brimacombe Kyle R KR Donegan Megan M Li Zhuyin Z Misteli Tom T

Methods (San Diego, Calif.) 20150901

Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-proge ...[more]

PMID: 26341717

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Project description:BackgroundInflammatory bowel diseases (IBD) are chronic relapsing intestinal inflammations with increasing global incidence, and new drug development remains in urgent demand for IBD management. To identify effective traditional Chinese medicine (TCM) formulae and compounds in IBD treatment, we innovatively combined the techniques of knowledge mining, high-content screening and high-resolution mass spectrometry, to conduct a systematic screening in Zhongjing formulae, which is a large collection of TCM prescriptions with most abundant clinical evidences.MethodsUsing Word2vec-based text learning, the correlations between 248 Zhongjing formulae and IBD typical symptoms were analyzed. Next, from the top three formulae with predicted relationship with IBD, TCM fractions were prepared and screened on a transgenic zebrafish IBD model for their therapeutic effects. Subsequently, the chemical compositions of the fraction hits were analyzed by mass spectrometry, and the major compounds were further studied for their anti-IBD effects and potential mechanisms.ResultsThrough knowledge mining, Peach Blossom Decoction, Pulsatilla Decoction, and Gegen Qinlian Decoction were predicted to be the three Zhongjing formulae mostly related to symptoms typical of IBD. Seventy-four fractions were prepared from the three formulae and screened in TNBS-induced zebrafish IBD model by high-content analysis, with the inhibition on the intestinal neutrophil accumulation and ROS level quantified as the screening criteria. Six herbal fractions showed significant effects on both pathological processes, which were subsequently analyzed by mass spectrometry to determine their chemical composition. Based on the major compounds identified by mass spectrometry, a second-round screen was conducted and six compounds (palmatine, daidzin, oroxyloside, chlorogenic acid, baicalin, aesculin) showed strong inhibitory effects on the intestinal inflammation phenotypes. The expression of multiple inflammatory factors, including il1β, clcx8a, mmp and tnfα, were increased in TNBS-treated fish, which were variously inhibited by the compounds, with aesculin showing the most potent effects. Moreover, aesculin and daidzin also upregulated e-cadherin's expression.ConclusionTaken together, we demonstrated the regulatory effects of several TCM formulae and their active compounds in the treatment of IBD, through a highly efficient research strategy, which can be applied in the discovery of effective TCM formulae and components in other diseases.

Project description:Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.

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A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds.

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A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds.

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