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: 39.08). 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 · 2.64 Impact Factor
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    • "Such a mechanism can be explored for anticancer therapies. Previous reports describe that targeting PGAM1 by a PGAM1- derived inhibitory peptide or PGAM inhibitor MJE3 attenuates cancer cell proliferation (Engel et al., 2004; Evans et al., 2005). "
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    ABSTRACT: It is unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation in part by controlling intracellular levels of its substrate, 3-phosphoglycerate (3-PG), and product, 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth.
    Cancer cell 11/2012; 22(5):585-600. DOI:10.1016/j.ccr.2012.09.020, · 23.89 Impact Factor
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    • "Using an in situ proteome reactivity profiling approach, Evans et al. [90] showed that MJE3, a compound that inhibits breast cancer cell proliferation, covalently labelled PGM1, reducing its enzymatic activity. These results, together with those reported by Narayanan et al. [91] that underline the importance of PGM B in resveratrol-induced cell growth inhibition of human prostate cancer cells, support the hypothesis that PGM is a regulator of cancer cell viability and suggest a crucial role for PGM as a potential target for new therapeutic strategies. "
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    ABSTRACT: Most cancer cells exhibit elevated levels of glycolysis and this metabolic pathway seems to be related to a greater glucose uptake. This phenomenon, known as the Warburg effect, is considered one of the most fundamental metabolic alterations during malignant transformation. Originally, Warburg hypothesised that the aerobic glycolysis of cancer cells could be just an aspect of a more complex metabolic adaptation. However, this intriguing discovery was partially misinterpreted and disregarded over time. In recent years, the peculiarities of cancer cell metabolism have been re-evaluated in light of new metabolic data that seem to confirm and to widen the original concept of the Warburg effect. In fact, biochemical, molecular, and, above all, proteomic studies on the multifaceted roles of glycolytic enzymes in cancer cells in general, and in cancer stem cells in particular, seem to suggest more complex functional adaptations. These adaptations result in significantly altered protein expression patterns, and they have fundamental implications for diagnosis, prognosis and therapy. Revisiting the Warburg effect in cancer cells with a proteomic approach could deepen our knowledge of cancer cell metabolism and of cancer cell biology in general. Moreover, by identifying useful diagnostic, prognostic and therapeutic targets, it could significantly impact clinical practice.
    PROTEOMICS - CLINICAL APPLICATIONS 02/2010; 4(2):143-58. DOI:10.1002/prca.200900157 · 2.68 Impact Factor
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