The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
David A Scheinberg - One of the best experts on this subject based on the ideXlab platform.
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Structure and activity of human mitochondrial peptide deformylase, a novel cancer target.
Journal of Molecular Biology, 2009Co-Authors: Sindy Escobar-alvarez, Yueming Li, Yehuda Goldgur, Guangli Yang, Ouathek Ouerfelli, David A ScheinbergAbstract:Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor Actinonin (1.7 A) identified the substrate-binding site. A defined S1' pocket, but no S2' or S3' substrate-binding pockets, exists. A conservation of PDF-Actinonin interaction across PDFs was observed. Despite the lack of true S2' and S3' binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2'and P3' positions of a formylated peptide substrate to turnover.
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human mitochondrial peptide deformylase a new anticancer target of Actinonin based antibiotics
Journal of Clinical Investigation, 2004Co-Authors: Michael J Soskis, Christopher Borella, Jeffrey R Gardner, Paula Hayes, Benzon M Dy, Mark L Heaney, Mark R Philips, William G Bornmann, Francis M Sirotnak, David A ScheinbergAbstract:Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that Actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of Actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of Actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral Actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of Actinonin-based antibiotics.
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a new human peptide deformylase inhibitable by Actinonin
Biochemical and Biophysical Research Communications, 2003Co-Authors: Christophe Antczak, David A Scheinberg, William G Bornmann, Francis M Sirotnak, Yueming LiAbstract:Abstract Peptide deformylases (PDFs) have been investigated as potential specific targets for antibiotics, but the possible existence of a functional human PDF (HsPDF) presents a potential hurdle to the design of specific drugs. We have expression cloned a HsPDF that has deformylase activity, although it is a slower and catalytically less active enzyme than bacterial or plant PDFs. A cobalt-substituted form of HsPDF (but not nickel or zinc) is active, and the enzyme appears to be active at a pH between 6.0 and 7.2, a temperature range of 25–50 °C, and in a low KCl ionic strength buffer. Actinonin inhibits HsPDF activity with an IC50 of 43 nM and kills Daudi and HL60 human cancer cell lines with an LC50 of 5.3 and 8.8 μM, respectively. The inhibition of HsPDF may provide an explanation for the mechanism by which Actinonin is cytotoxic against various human tumor cell lines.
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antitumor activity of Actinonin in vitro and in vivo
Clinical Cancer Research, 1998Co-Authors: Yang Xu, Janice Gabrilove, David A ScheinbergAbstract:Actinonin, an antibiotic and CD13/aminopeptidase N (APN) inhibitor, has been shown to be cytotoxic to tumor cell lines in vitro. We investigated the antiproliferative effects of Actinonin on human and murine leukemia and lymphoma cells. Actinonin inhibited growth of NB4 and HL60 human cell lines and AKR mouse leukemia cells in vitro with an IC50 of about 2-5 micrograms/ml. The inhibitory effect on CD13-positive cells was not blocked by pretreatment with the anti-CD13/APN monoclonal antibody F23, which binds with high affinity to the active site of CD13/APN and blocks its enzymatic activity. Moreover, F23 alone was not inhibitory to CD13-positive cells. Furthermore, a similar inhibitory IC50 of Actinonin was seen in the CD13-negative cell lines RAJI and DAUDI human lymphoma. These data suggest that the inhibitory effect of Actinonin is not mediated by inhibition of CD13/APN. Cell cycle analysis showed that Actinonin induces a G1 arrest in HL60 and NB4 cells; apoptosis was observed in 20-35% of the cells as measured by intracellular flow cytometry. To assess whether these effects could be seen in vivo, the effect of Actinonin on the syngeneic AKR mouse leukemia model was evaluated. Actinonin showed dose-dependent antitumor effects on AKR leukemia in vivo, resulting in a survival advantage. In conclusion, apoptosis, growth inhibition, and therapeutic effects in vivo are induced by Actinonin and are not likely to be mediated by CD13/APN.
Joaquim Trias - One of the best experts on this subject based on the ideXlab platform.
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Resistance of Streptococcus pneumoniae to Deformylase Inhibitors Is Due to Mutations in defB
Antimicrobial Agents and Chemotherapy, 2001Co-Authors: Peter S Margolis, C J Hackbarth, Wen Wang, Zhengyu Yuan, Richard G. White, Sara L. Lopez, Mita Maniar, Joaquim TriasAbstract:Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due to inactivation of transformylase activity. Knockout experiments in Streptococcus pneumoniae R6x indicate that the transformylase (fmt) and deformylase (defB) genes are essential and that a def paralog (defA) is not. Actinonin-resistant mutants of S. pneumoniae ATCC 49619 harbor mutations in defB but not in fmt. Reintroduction of the mutated defB gene into wild-type S. pneumoniae R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to Actinonin.
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Peptide Deformylase in Staphylococcus aureus: Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: Peter S Margolis, Dawn Chen, C J Hackbarth, Wen Wang, Dennis C. Young, Zhengyu Yuan, Richard G. White, Joaquim TriasAbstract:Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB , were identified in Staphylococcus aureus . The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB , was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB , but not defA , correlated to increased deformylase activity and decreased susceptibility to Actinonin, a deformylase-specific inhibitor. The defB gene could not be disrupted in wild-type S. aureus , suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, the defA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an Actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the Actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to Actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S. aureus Δ fmt strain was also resistant to Actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, the defB gene could be disrupted in an fmt mutant.
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Actinonin a naturally occurring antibacterial agent is a potent deformylase inhibitor
Biochemistry, 2000Co-Authors: Dawn Chen, Dinesh V Patel, C J Hackbarth, Wen Wang, Geoffrey B Dreyer, Dennis Young, Peter S Margolis, Charlotte Wu, Zijie Ni, Joaquim TriasAbstract:Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics. We have identified Actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor. The dissociation constant for this compound was 0.3 × 10-9 M against Ni−PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value. Microbiological evaluation revealed that Actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms. The PDF gene, def, was placed under control of PBAD in E. coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration. The susceptibility of this strain to Actinonin increases with decreased levels of PDF expression, indicating that Actinonin inhibits bacterial growth by targeting this enzyme. Actinonin provides an excellent starting point from which to derive a more potent PDF inhibi...
Vincent Mikol - One of the best experts on this subject based on the ideXlab platform.
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the crystal structures of four peptide deformylases bound to the antibiotic Actinonin reveal two distinct types a platform for the structure based design of antibacterial agents
Journal of Molecular Biology, 2002Co-Authors: J P Guilloteau, Magali Mathieu, Carmela Giglione, Veronique Blanc, A Dupuy, Miline Chevrier, Alain Famechon, Thierry Meinnel, Vincent MikolAbstract:Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic Actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of Actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.
Jian Wu Wang - One of the best experts on this subject based on the ideXlab platform.
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COMPUTATIONAL STUDY OF THE DYNAMICS OF PEPTIDE DEFORMYLASE COMPLEX FROM LEPTOSPIRA INTERROGANS: EXPLORING THE CONFORMATIONAL CHANGES OF THE SUBSTRATE POCKET
Journal of Theoretical and Computational Chemistry, 2020Co-Authors: Qiang Wang, Jian Wu Wang, Wei Ren XuAbstract:The peptide deformylase from Leptospira interrogans (LiPDF) shows many unusual characteristics. The substrate pocket of formate-bound complex adopts an open conformation. However, in the Actinonin-bound LiPDF complex, a slightly open substrate pocket is observed. The opening is not large enough for the inhibitor, because the CD-loop restricts the access to the active site. To explore the conformational changes of the substrate pocket, we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free LiPDF and Actinonin-bound LiPDF. During the molecular dynamics simulations, extensive conformational changes have taken place. The comparison of the two MD results shows that the CD-loop, hydrophilic inhibitor, and hydrophobic cluster are necessary for the reopening of the substrate pocket. In addition, Tyr71 plays an important role in mediating the movements of CD-loop, and the transition of the substrate pocket from open to semi-open only occurs in the presence of an inhibitor, which are consistent with the experiment very well.
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design synthesis and antibacterial activity of novel Actinonin derivatives containing benzimidazole heterocycles
European Journal of Medicinal Chemistry, 2009Co-Authors: Datong Zhang, Wei Ren Xu, Zongheng Wang, Lida Tang, Jian Wu WangAbstract:Abstract A series of novel Actinonin derivatives containing a benzimidazole heterocycle linked as amide isostere have been designed and synthesized. The structures of all the synthesized compounds were confirmed by analytical and spectroscopic methods. All the compounds were evaluated in vitro against Staphylococcus aureus , Klebsiella pneumoniae , and Sarcina lutea . Among them, compound 1a with unsubstituted benzimidazole ring exhibited potent antibacterial activities.
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A molecular dynamics simulation study of peptide deformylase from Leptospira interrogans complex: Exploring the closing mechanism of the substrate pocket
Chinese Chemical Letters, 2008Co-Authors: Qiang Wang, Jian Wu Wang, Wei Ren XuAbstract:Abstract To explore the closing mechanism of the substrate pocket, we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free and Actinonin-bound peptide deformylase from Leptospira interrogans . Our results show that the CD-loop, hydrophilic inhibitor and hydrophobic cluster are necessary for the formation of semi-open conformation, and Tyr71 plays an important role in mediating the movements of CD-loop. The average MD structure of the Actinonin-bound LiPDF complex approaches to the crystal structure. These are consistent with experiment very well.
Thierry Meinnel - One of the best experts on this subject based on the ideXlab platform.
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Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts
Biochimica et Biophysica Acta, 2017Co-Authors: Renata Grzela, Julien Nusbaum, Sonia Fieulaine, Francesco Lavecchia, M. Desmadril, Naima Nhiri, Alain Van Dorsselaer, Sarah Cianférani, Eric Jacquet, Thierry MeinnelAbstract:Abstract Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 PDF specific activity to values similar to those of bacterial PDFs. We next show that Vp16 PDF is as sensitive to the natural inhibitor compound of PDFs, Actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to Actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.
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Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state and upon deformylation inhibition.
Proteomics, 2015Co-Authors: Willy V. Bienvenut, Carmela Giglione, Thierry MeinnelAbstract:A proteome wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of Actinonin, an antibiotic specifically inhibiting peptide deformylase (PDF). A strategy and tool suite (SILProNaQ) was employed to provide large-scale quantitation of N-terminal modifications. In control conditions, more than 1000 unique N-termini were identified with 56% showing initiator methionine removal. Additional modifications corresponded to partial or complete Nα-acetylation (10%) and N-formyl retention (5%). Among the proteins undergoing these N-terminal modifications, 140 unique N-termini from translocated membrane proteins were highlighted. The very early time-course impact of Actinonin was followed after addition of bacteriostatic concentrations of the drug. Under these conditions, 26% of all proteins did not undergo deformylation any longer after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation ratio measured on individual proteins increased with the same trend. Upon early PDF inhibition, two major categories of proteins retained their N-formyl group: a large number of inner membrane proteins and many proteins involved in protein synthesis including factors assisting the nascent chains in early cotranslational events. All MS data have been deposited in the ProteomeXchange with identifiers PXD001979, PXD002012 and PXD001983 (http://proteomecentral.proteomexchange.org/dataset/PXD001979, http://proteomecentral.proteomexchange.org/dataset/PXD002012 and http://proteomecentral.proteomexchange.org/dataset/PXD001983).
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Control of protein life-span by N-terminal methionine excision
The EMBO Journal, 2003Co-Authors: Carmela Giglione, Olivier Vallon, Thierry MeinnelAbstract:Peptide deformylases (PDFs) have been discovered recently in eukaryotic genomes, and it appears that N-terminal methionine excision (NME) is a conserved pathway in all compartments where protein synthesis occurs. This work aimed at uncovering the function(s) of NME in a whole proteome, using the chloroplast-encoded proteins of both Arabidopsis thaliana and Chlamydomonas reinhardtii as model systems. Dis ruption of PDF1B in A.thaliana led to an albino phenotype, and an extreme sensitivity to the PDF- specific inhibitor Actinonin. In contrast, a knockout line for PDF1A exhibited no apparent phenotype. Photosystem II activity in C.reinhardtii cells was substantially reduced by the presence of Actinonin. Pulse–chase experiments revealed that PDF inhibi tion leads to destabilization of a crucial subset of chloroplast-encoded photosystem II components in C.reinhardtii. The same proteins were destabilized in pdf1b. Site-directed substitutions altering NME of the most sensitive target, subunit D2, resulted in similar effects. Thus, plastid NME is a critical mechanism specifically influencing the life-span of photosystem II polypeptides. A general role of NME in modulating the half-life of key subsets of proteins is suggested.
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the crystal structures of four peptide deformylases bound to the antibiotic Actinonin reveal two distinct types a platform for the structure based design of antibacterial agents
Journal of Molecular Biology, 2002Co-Authors: J P Guilloteau, Magali Mathieu, Carmela Giglione, Veronique Blanc, A Dupuy, Miline Chevrier, Alain Famechon, Thierry Meinnel, Vincent MikolAbstract:Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic Actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of Actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.
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Distinctive features of the two classes of eukaryotic peptide deformylases
Journal of Molecular Biology, 2001Co-Authors: Alexandre Serero, Carmela Giglione, Thierry MeinnelAbstract:Peptide deformylases (PDFs) are essential enzymes of the N-terminal protein processing pathway of eubacteria. The recent discovery of two types of PDFs in higher plants, PDF1A and PDF1B, and the detection of PDF1A in humans, have raised questions concerning the importance of deformylation in eukaryotes. Here, we have characterized fully in vitro and compared the properties of the two classes of eukaryotic PDFs, PDF1A and PDF1B, using the PDFs from Arabidopsis thaliana and Lycopersicon esculentum. We have shown that the PDFs of a given class (1A or 1B) all display similar features, independently of their origin. We also observed similar specificity of all plant PDFs for natural substrate peptides, but identified a number of biochemical differences between the two classes (1A or 1B). The main difference lies at the level of the bound cofactor, iron for PDF1B-like bacterial PDFs, and zinc for PDF1A. The nature of the metal cation has important consequences concerning the relative sensitivity to oxygen of the two plant PDFs. Investigation of the specificity of these enzymes with unusual substrates revealed additional differences between the two types of PDFs, enabling us to identify specific inhibitors with a lower affinity against PDF1As. However, the two plant PDFs were inhibited equally strongly in vitro by Actinonin, an antibiotic that specifically acts on bacterial PDFs. Uptake of Actinonin by A. thaliana seedlings was used to investigate the function of PDFs in the plant. Because it induces an albino phenotype, we conclude that deformylation is likely to play an essential role in the chloroplast.
