Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling

The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nature Biotechnology (Impact Factor: 41.51). 11/2005; 23(10):1303-7. DOI: 10.1038/nbt1149
Source: PubMed


Chemical genomics aims to discover small molecules that affect biological processes through the perturbation of protein function. However, determining the protein targets of bioactive compounds remains a formidable challenge. We address this problem here through the creation of a natural product-inspired small-molecule library bearing protein-reactive elements. Cell-based screening identified a compound, MJE3, that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were observed exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compounds into chemical genomics screens offers a means to discover targets of bioactive small molecules in living systems, thereby enabling downstream mechanistic investigations.

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    • "First, PGAM activity is up-regulated in many types of cancer (Durany et al. 1997; Ren et al. 2010). Additionally, an inhibitor of PGAM, MJE3, was isolated as the most potent anticancer compound against breast cancer cells (Evans et al. 2005). PGAM knockdown retards cancerous proliferation (Ren et al. 2010; Hitosugi et al. 2012) and provokes premature senescence in primary cells (Kondoh et al. 2005). "
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    ABSTRACT: Substantially high rate of glycolysis, known as the Warburg effect, is a well-known feature of cancers, and emerging evidence suggests that it supports cancerous proliferation/tumor growth. Phosphoglycerate mutase (PGAM), a glycolytic enzyme, is commonly up-regulated in several cancers, and recent reports show its involvement in the Warburg effect. Here, a comprehensive analysis shows that PGAM is acetylated at lysines 100/106/113/138 in its central region, and a member of the Sirtuin family (class III deacetylase), SIRT2, is responsible for its deacetylation. Over-expression of SIRT2 or mutations at the acetylatable lysines of PGAM attenuates cancer cell proliferation with a concomitant decrease in PGAM activity. We also report that the acetyltransferase PCAF (p300/CBP-associated factor) interacts with PGAM and acetylates its C-terminus, but not the central region. As prior evidence suggests that SIRT2 functions as a tumor suppressor, our results would provide support for the mechanistic basis of this activity.
    Genes to Cells 09/2014; 19(10). DOI:10.1111/gtc.12176 · 2.81 Impact Factor
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    • "They are also good at profiling signal transduction pathways in development and differentiation. [17] [18] [19] [20] [21] [22] By using proteomics, profiling and quantification of the proteins from virus-infected cells have been reported. [23] [24] [25] [26] In this study, an MS-based proteomics method was applied to study the proteins of NAC-treated A549 cells infected by A/Puerto Rico/8/1934 (H1N1). "
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    ABSTRACT: The pathology of A/Puerto Rico/8/1934 (H1N1) infection associated with the interaction of virus and its host cells is not clear. N-Acetylcysteine (NAC) is an antioxidant as well as a premier antitoxin and immune support substance. A high dose of NAC was recently reported for a therapy of H1N1 (2009) influenza pneumonia. NAC was used as a small-molecule organic probe to investigate the protein expression of human lung carcinoma cell line (A549) infected by influenza virus A/Puerto Rico/8/1934 (H1N1). Differential proteins were identified from MALDI-TOF MS and Q-TOF MS/MS analyses. The obtained results showed that NAC kept cells away from apoptosis. Virus-infected cells were arrested in G0/G1 phase. The lowest cell population of G0/G1 phase was detected when the cells were treated by 10 mM NAC for one day. Application of MS-based proteomics allowed the identification of the differential proteins. Software analysis showed that four proteins had close relationship. The results indicated that NAC as a small-molecule probe might effect the protein expression of A549 cells infected by the H1N1 virus. Copyright © 2014 John Wiley & Sons, Ltd.
    Rapid Communications in Mass Spectrometry 04/2014; 28(7):741-9. DOI:10.1002/rcm.6840 · 2.25 Impact Factor
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    • "Instead, the structure and functional groups are recognized by specific proteins with similarities at any sequence level, 3D structure, enzyme intermediates, etc (21,22). To capitalize on the properties of small molecules, Cravatt and Sorensen proposed and demonstrated activity-based protein profiling (ABPP) (23-25). ABPP is a chemical proteomic strategy that utilizes small-molecule probes to form covalent bonds at the active site of an enzyme and profile the functional state of the intact enzyme. "
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    ABSTRACT: The recent dramatic improvements in high-resolution mass spectrometry (MS) have revolutionized the speed and scope of proteomic studies. Conventional MS-based proteomics methodologies allow global protein profiling based on expression levels. Although these techniques are promising, there are numerous biological activities yet to be unveiled, such as the dynamic regulation of enzyme activity. Chemical proteomics is an emerging field that extends these types proteomic profiling. In particular, activity-based protein profiling (ABPP) utilizes small-molecule probes to monitor enzyme activity directly in living intact subjects. In this mini-review, we summarize the unique roles of smallmolecule probes in proteomics studies and highlight some recent examples in which this principle has been applied.
    BMB reports 02/2014; 47(3). DOI:10.5483/BMBRep.2014.47.3.264 · 2.60 Impact Factor
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