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Dehua Pei - One of the best experts on this subject based on the ideXlab platform.

  • Design and synthesis of macrocyclic peptidyl hydroxamates as Peptide Deformylase inhibitors.
    Bioorganic & medicinal chemistry letters, 2007
    Co-Authors: Gang Shen, Jinge Zhu, Anthony M. Simpson, Dehua Pei
    Abstract:

    Abstract Macrocyclic peptidyl hydroxamates were designed, synthesized, and evaluated as Peptide Deformylase (PDF) inhibitors. The most potent compound exhibited tight, slow-binding inhibition of Escherichia coli PDF ( K I ∗ = 4.4 nM ) and had potent antibacterial activity against Gram-positive bacterium Bacillus subtilis (MIC = 2–4 μg/mL).

  • An improved crystal form of Plasmodium falciparum Peptide Deformylase.
    Protein science : a publication of the Protein Society, 2004
    Co-Authors: Mark A. Robien, Kiet T Nguyen, Dehua Pei, Abhinav Kumar, Stewart Turley, Irwin Hirsh, Wim G. J. Hol
    Abstract:

    An altered version of Peptide Deformylase from Plasmodium falciparum (PfPDF), the organism that causes the most devastating form of malaria, has been cocrystallized with a synthesized inhibitor that has submicromolar affinity for its target protein. The structure is solved at 2.2 A resolution, an improvement over the 2.8 A resolution achieved during the structural determination of unliganded PfPDF. This represents the successful outcome of modifying the protein construct in order to overcome adverse crystal contacts and other problems encountered in the study of unliganded PfPDF. Two molecules of PfPDF are found in the asymmetric unit of the current structure. The active site of each monomer of PfPDF is occupied by a proteolyzed fragment of the triPeptide-like inhibitor. Unexpectedly, each PfPDF subunit is associated with two nearly complete molecules of the inhibitor, found at a protein–protein interface. This is the first structure of a eukaryotic PDF protein, a potential drug target, in complex with a ligand.

  • Structure-Based Design of a Macrocyclic Inhibitor for Peptide Deformylase
    Journal of medicinal chemistry, 2003
    Co-Authors: Kiet T Nguyen, Christophe L. M. J. Verlinde, Wim G. J. Hol, Dehua Pei
    Abstract:

    A macrocyclic, peptidomimetic inhibitor of Peptide Deformylase was designed by covalently cross-linking the P1' and P3' side chains. The macrocycle, which contains an N-formylhydroxylamine side chain as the metal-chelating group, was synthesized from a diene precursor via olefin metathesis using Grubbs's catalyst. The cyclic inhibitor showed potent inhibitory activity toward Escherichia coli Deformylase (K(I) = 0.67 nM) and antibacterial activity against both Gram-positive and Gram-negative bacteria (MIC = 0.7-12 microg/mL).

  • Determination of the ionization state and catalytic function of Glu-133 in Peptide Deformylase by difference FTIR spectroscopy.
    Biochemistry, 2002
    Co-Authors: Hua Deng, Kiet T Nguyen, Jinge Zhu, Robert Callender, Dehua Pei
    Abstract:

    Peptide Deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polyPeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel ...

  • Crystals of Peptide Deformylase from Plasmodium falciparum Reveal Critical Characteristics of the Active Site for Drug Design
    Structure (London England : 1993), 2002
    Co-Authors: Abhinav Kumar, Kiet T Nguyen, Dehua Pei, Sumant Srivathsan, Brad Ornstein, Stewart Turley, Irwin Hirsh, Wim G. J. Hol
    Abstract:

    Peptide Deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum Peptide Deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli Deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

Thierry Meinnel - One of the best experts on this subject based on the ideXlab platform.

  • Mutations in Three Distinct Loci Cause Resistance to Peptide Deformylase Inhibitors in Bacillus subtilis
    Antimicrobial agents and chemotherapy, 2009
    Co-Authors: Yann Duroc, Carmela Giglione, Thierry Meinnel
    Abstract:

    Bacillus subtilis mutants with resistance against Peptide Deformylase inhibitors were isolated. All showed a bypass of the pathway through mutations in three genes required for formylation of Met-tRNAfMet, fmt, folD, and glyA. glyA corresponds to a yet uncharacterized locus inducing resistance. The bypass of formylation caused robust fitness reduction but was not accompanied by alterations of the transcription profile. A subtle adaptation of the enzymes of the intermediary metabolism was observed.

  • Structure-activity relationship analysis and therapeutic potential of Peptide Deformylase inhibitors.
    Current opinion in investigational drugs (London England : 2000), 2004
    Co-Authors: Adrien Boularot, Carmela Giglione, Isabelle Artaud, Thierry Meinnel
    Abstract:

    Peptide Deformylase inhibitors (PDFIs) appear to be one of the most exciting classes of antibacterial agents discovered to date. Rapid progress in the development of PDFIs has been possible because Peptide Deformylase is a metalloprotease, and this class of enzymes shows a high degree of structure-function conservation, and because the most potent PDFIs are hydroxamate derivatives, a well known category of pharmacophores. The current challenge in structure-activity relationship analysis is obtaining molecules with potent in vivo antibacterial activity against a range of drug-resistant pathogens. The PDFIs currently in clinical trials target community-based bacterial infections, with a potential major pharmaceutical market.

  • Resistance to anti-Peptide Deformylase drugs.
    Expert opinion on therapeutic targets, 2001
    Co-Authors: Carmela Giglione, Thierry Meinnel
    Abstract:

    Recent work has assessed the potential of Peptide Deformylase (PDF) as a target for broad spectrum antibacterial agents. By using a number of approaches, including proteomics, researchers at Roche have shown that the molecules they had selected in vitro were able to target PDF in vivo. However, the authors, having observed resistance occurring at a rather high frequency and on the basis of the recent discovery of a Deformylase homologue in humans, suggest that PDF ‘may not be an optimal target for broad spectrum antibacterial agents’. We link these data to results published by other laboratories and conclude that PDF deserves to still be considered a valuable target for new antibiotics.

  • Peptide Deformylase as a target for new generation, broad spectrum antimicrobial agents.
    Molecular Microbiology, 2000
    Co-Authors: C. Giglione, M. Pierre, Thierry Meinnel
    Abstract:

    Peptide Deformylase was discovered 30 years ago, but as a result of its unusually unstable activity it was not fully characterized until very recently. The aim of this paper is to review the many recent data concerning this enzyme and to try to assess its potential as a target for future antimicrobial drugs.

  • Peptide Deformylase of Eukaryotic Protists: A Target for New Antiparasitic Agents?
    Parasitology Today, 2000
    Co-Authors: Thierry Meinnel
    Abstract:

    Abstract Peptide Deformylase is found only in Eubacteria, making it a logical target for discovering new antibacterial agents. Although this protein is absent from animal or fungal cells, evidence supports its existence in eukaryotic protists, including the causative agents of malaria, sleeping sickness, Chagas disease and leishmaniosis. Here, Thierry Meinnel discusses the idea that Deformylase inhibitors could be used as very broad-spectrum antibiotics against bacterial infections, as well as parasitic diseases.

Sylvain Blanquet - One of the best experts on this subject based on the ideXlab platform.

  • DESIGN AND SYNTHESIS OF SUBSTRATE ANALOGUE INHIBITORS OF Peptide Deformylase
    Biochemistry, 1999
    Co-Authors: Thierry Meinnel, Stéphane Ragusa, Luc Patiny, Sylvain Blanquet
    Abstract:

    Series of substrates derivatives of Peptide Deformylase were systematically synthesized and studied for their capacities to undergo hydrolysis. Data analysis indicated the requirement for a hydrophobic first side chain and for at least two main chain carbonyl groups in the substrate. For instance, Fo-Met-OCH3 and Fo-Nle-OCH3 were the minimal substrates of Peptide Deformylase obtained in this study, while positively charged Fo-Nle-ArgNH2 was the most efficient substrate (kcat/Km = 4.5 x 10(5) M-1.s-1). On the basis of this knowledge, 3-mercapto-2-benzylpropanoylglycine (thiorphan), a known inhibitor of thermolysin, could be predicted and further shown to inhibit the deformylation reaction. The inhibition by this compound was competitive and proved to depend on the hydrophobicity at the P1' position. Spectroscopic evidence that the sulfur group of thiorphan binds next to the active site metal ion on the enzyme could be obtained. Consequently, a small thiopseudoPeptide derived from Fo-Nle-OCH3 was designed and synthesized. This compound behaved as a competitive inhibitor of Peptide Deformylase with KI = 52 +/- 5 microM. Introduction of a positive charge to this thioPeptide via addition of an arginine at P2' improved the inhibition constant up to 2.5 +/- 0.5 microM, a value 4 orders of magnitude smaller than that of the starting inhibitors. Evidence that this inhibitor, imino[(5-methoxy-5-oxo-4-[[2-(sulfanylmethyl)hexanoyl]amino]pentyl )am ino]methanamine, binds inside the active site cavity of Peptide Deformylase, while keeping intact the 3D fold of the protein, was provided by NMR. A fingerprint of the interaction of the inhibitor with the residues of the enzyme was obtained.

  • Solution structure of nickel-Peptide Deformylase
    Journal of molecular biology, 1998
    Co-Authors: Frédéric Dardel, Sylvain Blanquet, Stéphane Ragusa, Christine Lazennec, Thierry Meinnel
    Abstract:

    In the accompanying paper, we report that zinc is unlikely to be the co-factor supporting Peptide Deformylase activity in vivo. In contrast, nickel binding promotes full enzyme activity. The three-dimensional structure of the resulting nickel-containing Peptide Deformylase (catalytic domain, residues 1 to 147) was solved by NMR using a 13C-15N-doubly labelled protein sample. A set of 2261 restraints could be collected, with an average of 15.4 per amino acid. The resolution, which shows a good definition for the position of most side-chains, is greatly improved compared to that previously reported for the zinc-containing, inactive form. A comparison of the two stuctures indicates however that both share the same 3D organization. This shows that the nature of the bound metal is the primary determinant of the hydrolytic activity of this enzyme. Site-directed mutagenesis enabled us to determine the conserved residues of PDF involved in the structure of the active site. In particular, a buried arginine appears to be critical for the positioning of Cys90, one of the metal ligands. Furthermore, the 3D structure of Peptide Deformylase was compared to thermolysin and metzincins. Although the structural folds are very different, they all display a common structural motif involving an alpha-helix and a three-stranded beta-sheet. These conserved structural elements build a common scaffold which includes the active site, suggesting a common hydrolytic mechanism for these proteases. Finally, an invariant glycine shared by both PDF and metzincins enables us to extend the conserved motif from HEXXH to HEXXHXXG.

  • Structure-function relationships within the Peptide Deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion
    Journal of molecular biology, 1997
    Co-Authors: Thierry Meinnel, Christine Lazennec, Stéphane Villoing, Sylvain Blanquet
    Abstract:

    Thermus thermophilus Peptide Deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli Deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the Peptide Deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a Peptide Deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine Peptide Deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli Peptide Deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of Peptide Deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial Peptide Deformylases adopt a constant overall tertiary structure.

  • A New Subclass of the Zinc Metalloproteases Superfamily Revealed by the Solution Structure of Peptide Deformylase
    Journal of molecular biology, 1996
    Co-Authors: Thierry Meinnel, Sylvain Blanquet, Frédéric Dardel
    Abstract:

    Escherichia coli Peptide Deformylase, a member of the zinc metalloproteases family, is made up of an active core domain composed of 147 residues and of an additional and dispensable C-terminal tail of 21 residues. The three-dimensional structure of the catalytic core could be studied by NMR. 1H and 15N NMR resonances assignments were obtained by two-dimensional and three-dimensional heteronuclear spectroscopy. The structure could be calculated using a set of 1015 restraints for the 147 residues of the enzyme. The overall structure is composed of a series of antiparallel beta-strands which surround two perpendicular alpha-helices. The C-terminal helix contains the HEXXH motif, which is crucial for activity. This helical arrangement and the way the histidines bind the zinc ion clearly are structurally reminiscent of the other members of the metalloprotease family, such as thermolysin or metzincins. Nevertheless, the overall arrangement of secondary and tertiary structures of Peptide Deformylase and the positioning of its third zinc ligand (a cysteine) are quite different from those of the other members of the family. These discrepancies, together with several biochemical differences, lead us to propose that Peptide Deformylase is the first example of a new class of the zinc-metalloproteases family. Studies of the interaction of Peptide Deformylase with either an inhibitor of the reaction or a product of the catalysed reaction, Met-Ala-Ser, as well as comparisons with the structures of other enzymes of the family, have enabled us to delineate the area corresponding to their binding site. The structural basis of the specificity of recognition of the formyl group is discussed in the context of the protease superfamily.

  • Mapping of the active site zinc ligands of Peptide Deformylase.
    Journal of molecular biology, 1995
    Co-Authors: Thierry Meinnel, Christine Lazennec, Sylvain Blanquet
    Abstract:

    Abstract A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of Peptide Deformylase, including the ligands of the zinc ion. In particular, because zinc is usually coordinated by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed. The functional consequence of the substitutions was assessed by complementation of a fms -null strain with the help of vectors expressing the mutated genes. In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132 HEXXH 136 motif of Peptide Deformylase prevented the indicator strain growing. Most enzyme variants were purified to homogeneity in a second step. Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency. The change of Glu88 also led to a significant decrease in catalytic rate. Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency. The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion. Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack. Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of Deformylase. This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily. However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that Peptide Deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.

Kiet T Nguyen - One of the best experts on this subject based on the ideXlab platform.

  • High-throughput screening of Peptide Deformylase inhibitors.
    Methods in molecular medicine, 2008
    Co-Authors: Kiet T Nguyen
    Abstract:

    The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide Deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the diPeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories.

  • An improved crystal form of Plasmodium falciparum Peptide Deformylase.
    Protein science : a publication of the Protein Society, 2004
    Co-Authors: Mark A. Robien, Kiet T Nguyen, Dehua Pei, Abhinav Kumar, Stewart Turley, Irwin Hirsh, Wim G. J. Hol
    Abstract:

    An altered version of Peptide Deformylase from Plasmodium falciparum (PfPDF), the organism that causes the most devastating form of malaria, has been cocrystallized with a synthesized inhibitor that has submicromolar affinity for its target protein. The structure is solved at 2.2 A resolution, an improvement over the 2.8 A resolution achieved during the structural determination of unliganded PfPDF. This represents the successful outcome of modifying the protein construct in order to overcome adverse crystal contacts and other problems encountered in the study of unliganded PfPDF. Two molecules of PfPDF are found in the asymmetric unit of the current structure. The active site of each monomer of PfPDF is occupied by a proteolyzed fragment of the triPeptide-like inhibitor. Unexpectedly, each PfPDF subunit is associated with two nearly complete molecules of the inhibitor, found at a protein–protein interface. This is the first structure of a eukaryotic PDF protein, a potential drug target, in complex with a ligand.

  • Structure-Based Design of a Macrocyclic Inhibitor for Peptide Deformylase
    Journal of medicinal chemistry, 2003
    Co-Authors: Kiet T Nguyen, Christophe L. M. J. Verlinde, Wim G. J. Hol, Dehua Pei
    Abstract:

    A macrocyclic, peptidomimetic inhibitor of Peptide Deformylase was designed by covalently cross-linking the P1' and P3' side chains. The macrocycle, which contains an N-formylhydroxylamine side chain as the metal-chelating group, was synthesized from a diene precursor via olefin metathesis using Grubbs's catalyst. The cyclic inhibitor showed potent inhibitory activity toward Escherichia coli Deformylase (K(I) = 0.67 nM) and antibacterial activity against both Gram-positive and Gram-negative bacteria (MIC = 0.7-12 microg/mL).

  • Determination of the ionization state and catalytic function of Glu-133 in Peptide Deformylase by difference FTIR spectroscopy.
    Biochemistry, 2002
    Co-Authors: Hua Deng, Kiet T Nguyen, Jinge Zhu, Robert Callender, Dehua Pei
    Abstract:

    Peptide Deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polyPeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel ...

  • Crystals of Peptide Deformylase from Plasmodium falciparum Reveal Critical Characteristics of the Active Site for Drug Design
    Structure (London England : 1993), 2002
    Co-Authors: Abhinav Kumar, Kiet T Nguyen, Dehua Pei, Sumant Srivathsan, Brad Ornstein, Stewart Turley, Irwin Hirsh, Wim G. J. Hol
    Abstract:

    Peptide Deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum Peptide Deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli Deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

Thomas J. Blacklock - One of the best experts on this subject based on the ideXlab platform.

  • Practical Synthesis of a Peptide Deformylase (PDF) Inhibitor
    Organic Process Research & Development, 2008
    Co-Authors: Yugang Liu, Mahavir Prashad, Lech Ciszewski, Kevin Vargas, Oljan Repic, Thomas J. Blacklock
    Abstract:

    A practical chromatography-free synthesis of an N-formylated hydroxylamine Peptide Deformylase inhibitor LCD320 is described. A diastereoselective Michael reaction of (4S)-3-[2-(cyclobutylmethyl)-1-oxo-2-propenyl]-4-(phenylmethyl)-2-oxazolidinone with O-benzyl hydroxylamine was used to establish the key stereogenic center. We found that traces of residual Li+ from a previous step had a great impact on the diastereoselectivity of this reaction. A very efficient amidation coupling reaction of proline derivative (2S,4R)-4-fluoro-1,2-pyrrolidinedicarboxylic acid 1,1-dimethylethyl ester with weakly nucleophilic 3-pyridazinamine using methanesulfonyl chloride in the presence of 1-methylimidazole in DMF was also developed that proceeded without racemization.

  • β-amino amides from β-lactams : Application to the formal synthesis of a Peptide-Deformylase inhibitor
    Synlett, 2006
    Co-Authors: Xinglong Jiang, Joel Slade, Mahavir Prashad, Oljan Repic, Kapa Prasad, Thomas J. Blacklock
    Abstract:

    A facile and a practical synthesis of Peptide-Deformylase inhibitor 1 is described using an acid-catalyzed aminolysis of )-lactam 12 with pyrrolidine 6 as the key transformation. In addition, simplified conditions for the conversion of a β-hydroxy acid to a β-lactam are reported.