Discovery and Target Identification of an Antiproliferative Agent in Live Cells Using Fluorescence Difference in Two‐Dimensional Gel Electrophoresis

Park, Jong-Min; Oh, Seung-Ha; Park, Seung Bum

doi:10.1002/ange.201200609

Cited by 22 publications

(31 citation statements)

References 37 publications

(13 reference statements)

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“…We recently reported a cell-based target identification method, FITGE 9 . To overcome the limitation of weak binding affinity or low abundance of target proteins, we introduced covalent anchoring of a bioactive small molecule to cellular target proteins in live cells using a photocrosslinker.…”

Section: Potential Icm Targetsmentioning

confidence: 99%

“…To overcome the limitation of weak binding affinity or low abundance of target proteins, we introduced covalent anchoring of a bioactive small molecule to cellular target proteins in live cells using a photocrosslinker. The resolution of the in-gel protein analysis was clearly enhanced by 2D gel electrophoresis with dual-color labeling to differentiate specific target proteins from nonspecific protein binders 9 . On the basis of a structure-activity relationship study for ICM analogs (Supplementary Fig.…”

Section: Potential Icm Targetsmentioning

confidence: 99%

“…Here we report a new anti-neuroinflammatory agent, ICM (1), discovered by cell-based phenotypic screening and the subsequent identification of its molecular target using fluorescence difference in two-dimensional (2D) gel electrophoresis (FITGE) technology 9 .…”

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confidence: 99%

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A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation

et al. 2014

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Because of the critical role of neuroinflammation in various neurological diseases, there are continuous efforts to identify new therapeutic targets as well as new therapeutic agents to treat neuroinflammatory diseases. Here we report the discovery of inflachromene (ICM), a microglial inhibitor with anti-inflammatory effects. Using the convergent strategy of phenotypic screening with early stage target identification, we show that the direct binding target of ICM is the high mobility group box (HMGB) proteins. Mode-of-action studies demonstrate that ICM blocks the sequential processes of cytoplasmic localization and extracellular release of HMGBs by perturbing its post-translational modification. In addition, ICM effectively downregulates proinflammatory functions of HMGB and reduces neuronal damage in vivo. Our study reveals that ICM suppresses microglia-mediated inflammation and exerts a neuroprotective effect, demonstrating the therapeutic potential of ICM in neuroinflammatory diseases.

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Section: Potential Icm Targetsmentioning

confidence: 99%

Section: Potential Icm Targetsmentioning

confidence: 99%

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A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation

et al. 2014

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“…However, if from lysate no target protein can be isolated intact cells should be considered 28. 38a, 45 The use of cell lysates rather than living cells is required if the pulldown probe is membrane impermeable.…”

Section: Approaches To Target Identificationmentioning

confidence: 99%

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

et al. 2013

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Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins.

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“…The authors have successfully accomplished the inter-subunit crosslinking of egg-white avidin tetramer with this probe (Hatanaka et al 1994). Over the next two decades, covalent affinity probes have been extensively employed not only in target identification (Adam et al 2002b;Takaoka et al 2006;MacKinnon et al 2007;Ban et al 2010;Koteva et al 2010;Wurdak et al 2010;Eirich et al 2011;Cisar and Cravatt 2012;Park et al 2012;Shi et al 2012;Park et al 2013;Su et al 2013), but also in affinity labeling of proteins (Dorman and Prestwich 2000;Hatanaka and Sadakane 2002;Chen et al 2003;Hosoya et al 2004;Lee et al 2005;Koshi et al 2008; Tanaka et al Tsukiji et al 2009a;Das 2011;Wacker et al 2011;Wang et al 2011;Hayashi and Hamachi 2012;Tamura et al 2012;Vinkenborg et al 2012;Willems et al 2012) and activity-based proteomic profiling (ABPP) (Kozarich 2003;Chan et al 2004;Ballell et al 2005;Evans and Cravatt 2006;Sadaghiani et al 2007;Salisbury and Cravatt 2007;Cravatt et al 2008;Salisbury and Cravatt 2008;Uttamchandani et al 2008;Nomura et al 2010;…”

Section: Covalent Crosslinkingmentioning

confidence: 96%

Affinity purification in target identification: the specificity challenge

Zheng

2015

Arch. Pharm. Res.

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Since phenotype-based screening directly evaluates capability of small molecules for modulating biology in actual biological systems, it has become an important discover modality in modern pharmaceutical sciences. However, in order to fully elucidate the molecular mechanism underlying the bioactivity of small molecules, identification of their biological targets is an indispensable step. Among the many target identification strategies developed during the past several decades, affinity purification remains to be one of the most important and powerful approaches, as it can directly reveal the physical interactions between small molecules and their biomolecular targets. However, due to the complexity of the proteome and the diversity of small molecule-protein interactions, affinity purification faces the specificity challenge: how to identify the true specific targets from the non-specific background? Focusing on this challenge, in this review, we briefly introduce the history and background of affinity purification, and then we discussed the major technological developments aiming to address this challenge. We have summarized these approaches in two categories: noise reduction and comparative distinction. This review also highlights the importance of choosing an integrated approach combining multiple methods to achieving success in target identification.

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Discovery and Target Identification of an Antiproliferative Agent in Live Cells Using Fluorescence Difference in Two‐Dimensional Gel Electrophoresis

Cited by 22 publications

References 37 publications

A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation

A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

Affinity purification in target identification: the specificity challenge

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