Proteomics-based Target Identification

Towbin, Harry; Bair, Kenneth W.; DeCaprio, James A.; Eck, Michael J.; Kim, Sunkyu; Kinder, Frederick R.; Morollo, Anthony A.; Mueller, Dieter; Schindler, Patrick; Song, Hyun Kyu; Oostrum, Jan van; Versace, Richard; Voshol, Hans; Wood, Jeanette M.; Zabludoff, Sonya; Phillips, Penny E.

doi:10.1074/jbc.m309039200

Cited by 144 publications

(97 citation statements)

References 36 publications

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“…Although we have shown that pyridine-2-carboxylic acid derivatives selectively inhibit HsMetAP1 in vitro and block cell proliferation in culture, the causative relationship between these two effects remained to be established. As the first step to assess this relationship, we determined whether pyridine-2-carboxylic acid derivatives are capable of entering cells and inhibiting HsMetAP1 activity in vivo by examining the N-terminal initiator methionine status of a known protein substrate, 14-3-3␥ (11). HeLa cells were incubated with various concentrations of compound 1 for 24 h before they were harvested for Western blot with a monoclonal antibody (clone HS23) specific for the methionylated N-terminal fragment of 14-3-3␥ protein (11).…”

Section: Hsmetap1-specific Inhibitors Block Proliferation Of Tumor Cementioning

confidence: 99%

“…There has also been circumstantial evidence implicating a role of HsMetAP1 in tumor cell proliferation. By using a proteomics-based approach, both human MetAPs were identified as the binding targets of bengamides, a class of marine natural products that inhibit tumor growth in vitro and in vivo (11). Recently, pyridinyl pyrimidines have also been identified as nonselective inhibitors for MetAPs, and inhibit the proliferation of tumor cell lines (12).…”

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confidence: 99%

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Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression

Addlagatta

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Processing of the N-terminal initiator methionine is an essential cellular process conserved from prokaryotes to eukaryotes. The enzymes that remove N-terminal methionine are known as methionine aminopeptidases (MetAPs). Human MetAP2 has been shown to be required for the proliferation of endothelial cells and angiogenesis. The physiological function of MetAP1, however, has remained elusive. In this report we demonstrate that a family of inhibitors with a core structure of pyridine-2-carboxylic acid previously developed for the bacterial and yeast MetAP1 is also specific for human MetAP1 (HsMetAP1), as confirmed by both enzymatic assay and high-resolution x-ray crystallography. Treatment of tumor cell lines with the MetAP1-specific inhibitors led to an accumulation of cells in the G2͞M phase, suggesting that HsMetAP1 may play an important role in G2͞M phase transition. Overexpression of HsMetAP1, but not HsMetAP2, conferred resistance of cells to the inhibitors, and the inhibitors caused retention of N-terminal methionine of a known MetAP substrate, suggesting that HsMetAP1 is the cellular target for the inhibitors. In addition, when HsMetAP1 was knocked down by gene-specific siRNA, cells exhibited slower progression during G2͞M phase, a phenotype similar to cells treated with MetAP1 inhibitors. Importantly, MetAP1 inhibitors were able to induce apoptosis of leukemia cell lines, presumably as a consequence of their interference with the G2͞M phase checkpoint. Together, these results suggest that MetAP1 plays an important role in G2͞M phase of the cell cycle and that it may serve as a promising target for the discovery and development of new anticancer agents.aminopeptidase inhibitors ͉ angiogenesis ͉ apoptosis

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Section: Hsmetap1-specific Inhibitors Block Proliferation Of Tumor Cementioning

confidence: 99%

mentioning

confidence: 99%

Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression

Addlagatta

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 (6)(7)(8). Bengamides inhibit both types of MetAP (9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents.…”

mentioning

confidence: 99%

Structural basis of catalysis by monometalated methionine aminopeptidase

Xie

Qiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.metalloprotease ͉ protein structure ͉ enzyme inhibition ͉ drug discovery ͉ metal occupancy A ll newly synthesized proteins have an amino-terminal methionine residue corresponding to the start codon AUG. In a significant number of cases, this initiating methionine residue is removed, either co-or posttranslationally, by the enzyme methionine aminopeptidase (MetAP) (1). In bacteria, this enzyme is the product of a single gene, and it is absolutely essential for bacterial survival, as demonstrated by gene deletion experiments in Escherichia coli (2) and Salmonella typhimurium (3). The single but critically essential MetAP enzyme of bacteria thus stands out as an attractive target for the design of antibacterial agents (4). Eukaryotes, on the other hand, have two distinct MetAPs, types I and II, arising from different genes (5). The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 (6-8). Bengamides inhibit both types of MetAP (9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial (10-12) or antiangiogenic (13) activities. One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to...

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“…Proteomics-based approaches can also be used to identify the molecular targets of compounds with unknown mechanism of action. For example, the elusive target of LAF-389 was identified by 2-D PAGE approach via molecular changes in expression of cellular proteins following treatment of cell culture with this drug [114,115]. LAF-389 is a synthetic analog of bengamide B possessing antitumor activity.…”

Section: Pharmacoproteomicsmentioning

confidence: 99%

Proteomics in human cancer research

Pastwa

Somiari

Czyż

et al. 2007

Proteomics Clinical Apps

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Proteomics is now widely employed in the study of cancer. Many laboratories are applying the rapidly emerging technologies to elucidate the underlying mechanisms associated with cancer development, progression, and severity in addition to developing drugs and identifying patients who will benefit most from molecular targeted compounds. Various proteomic approaches are now available for protein separation and identification, and for characterization of the function and structure of candidate proteins. In spite of significant challenges that still exist, proteomics has rapidly expanded to include the discovery of novel biomarkers for early detection, diagnosis and prognostication (clinical application), and for the identification of novel drug targets (pharmaceutical application). To achieve these goals, several innovative technologies including 2-D-difference gel electrophoresis, SELDI, multidimensional protein identification technology, isotope-coded affinity tag, solid-state and suspension protein array technologies, X-ray crystallography, NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulation have evolved, and are being used in different combinations. This review provides an overview of the field of proteomics and discusses the key proteomic technologies available to researchers. It also describes some of the important challenges and highlights the current pharmaceutical and clinical applications of proteomics in human cancer research.

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Proteomics-based Target Identification

Cited by 144 publications

References 36 publications

Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression

Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression

Structural basis of catalysis by monometalated methionine aminopeptidase

Proteomics in human cancer research

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