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

ABSTRACT 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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    • "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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    • "Moreover, inhibiting the Pgam1 protein was shown to attenuate tumor growth [21]. A small-molecule inhibitor of the Pgam1 protein has been developed [22]. "
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    ABSTRACT: Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function. Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload. Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.
    PLoS ONE 08/2013; 8(8):e72173. DOI:10.1371/journal.pone.0072173 · 3.23 Impact Factor
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