Peptide Deformylase as an Antibacterial Drug Target: Assays for Detection of Its Inhibition in Escherichia coli Cell Homogenates and Intact Cells (original) (raw)
Related papers
Characterization of a Human Peptide Deformylase: Implications for Antibacterial Drug Design †
Biochemistry, 2003
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-aminoacid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co 2+ -substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.
Peptide Aldehyde Inhibitors of Bacterial Peptide Deformylases
Archives of Biochemistry and Biophysics, 1999
Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gramnegative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with K i values of 26.0 and 55.6 M, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with K i values for calpeptin of 9.5 and 12.4 M, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity.
The Journal of antimicrobial chemotherapy, 2012
OBJECTIVES: Bacterial drug resistance is a worrying public health problem and there is an urgent need for research and development to provide new antibacterial molecules. Peptide deformylase (PDF) is now a well-described intracellular target selected for the design of a new antibiotic group, PDF inhibitors (PDFIs). The initial bacterial susceptibility to an inhibitor of a cytoplasmic target is directly associated with the diffusion of the compound through the membrane barrier of Gram-negative bacteria and with its cytosolic accumulation at the required concentration. METHODS: We have recently demonstrated that the activity of different PDFIs is strongly dependent on the accumulation of the active molecules by using permeabilizing agents, efflux inhibitors or efflux-mutated strains. In this work we assessed various combination protocols using different putative inhibitors (PDFIs, methionine aminopeptidase inhibitors etc.) to improve antibacterial activity against various resistant Gr...
Deformylase as a novel antibacterial target
Drug Discovery Today, 2001
Bacterial genomics has revealed a plethora of previously unknown targets of potential use in the discovery of novel antibacterial drugs. However, so far little has emerged from this approach. Peptide deformylase is an interesting target that was discovered more than 30 years ago, but was not exploited until recently. The reawakening of interest in this target resulted from an improved understanding of the enzyme, making it a more tractable and attractive target. Information on the properties of the enzyme, such as its three-dimensional structure, the activity of inhibitors, its resistance and suitability as a target are discussed. deformylase, a distinctive enzyme of eubacterial translation. EMBO J.
Theoretical study of Escherichia coli peptide deformylase inhibition by several drugs
In Silico Biology, 2006
Because peptide deformylase (PDF) is essential for the initiation of translation in eubacteria but not in eukaryotes, it is a potentially interesting target for antibiotics. Computer simulation using docking software can be used to model protein-ligand interactions, and in this brief report we describe its use in optimizing the design in PDF-directed inhibitors.
Peptide deformylase: a target for novel antibiotics?
Emerging Therapeutic Targets, 2001
Peptide deformylase (PDF) catalyses the hydrolytic removal of the N-terminal formyl group from nascent ribosome-synthesised polypeptides. Its activity is essential and it is present in all eubacteria. It is also present in the organelles of some eukaryotes. PDF represents a novel class of mononuclear iron protein, utilising an Fe 2+ ion to catalyse the hydrolysis of an amide bond. Due to its extreme lability, isolation and characterisation of PDF was not possible until very recently. This review will discuss the recent progress in the elucidation of the the structure and function of PDF, evaluating its suitability as a target for antibiotic design and summarising the current approaches to designing drugs that target PDF.
Enzymatic properties of Escherichia coli peptide deformylase
Journal of bacteriology, 1995
Since its discovery in crude extracts in the late sixties, Escherichia coli peptide deformylase activity could not be further characterized because of an apparent extreme instability. We show that this behavior was caused by an inadequate activity assay, involving substrate concentration inhibition and substrate precipitation in crude extracts. The homogeneous protein, as it was previously purified (T. Meinnel and S. Blanquet J. Bacteriol. 175:7737-7740, 1993), had actually retained its initial activity. The influence on the deformylation reaction of several factors was studied and used to improve the activity assay. Pure peptide deformylase proves to act only on peptide substrates with an N-formylmethionyl moiety. In agreement with the occurrence of zinc in the enzyme, peptide deformylase activity is inhibited by 1,10-phenanthroline.
Ligand-Induced Changes in the Structure and Dynamics of Escherichia coli Peptide Deformylase
Biochemistry, 2009
Peptide deformylase (PDF) is an enzyme that is responsible for removing the formyl group from nascently synthesized polypeptides in bacteria, attracting much attention as a potential target for novel antibacterial agents. Efforts to develop potent inhibitors of the enzyme have progressed based on classical medicinal chemistry, combinatorial chemistry, and structural approaches. Yet, the validity of PDF as an antibacterial target hangs, in part, on the ability of inhibitors to selectively target this enzyme in favor of structurally related metallohydrolases. We have used 15 N NMR spectroscopy and isothermal titration calorimetry to investigate the high-affinity interaction of EcPDF with actinonin, a naturally occurring potent EcPDF inhibitor. Backbone amide chemical shifts, residual dipolar couplings, hydrogen-deuterium exchange, and 15 N relaxation reveal structural and dynamic effects of ligand binding in the immediate vicinity of the ligand binding site as well as at remote sites. A comparison of the crystal structures of free and actinonin-bound EcPDF with the solution data suggests that most of the consequences of ligand binding on the protein are lost or obscured during crystallization. The results of these studies improve our understanding of the thermodynamic global minimum and have important implications for structure-based drug design. Peptide deformylase (PDF) is an essential and highly conserved enzyme that functions in protein maturation by removing the N-formyl group from the methionine of nascently synthesized polypeptides in bacteria, protists and eukaryotic organelles (1-9). Because protein translation in bacteria is initiated with N-formyl-methionine, PDF has emerged as a target of efforts to develop novel antibacterial agents (10-16). The extensively characterized enzyme from E. coli (EcPDF) exhibits some substrate selectivity in vitro (4,6,17-21); this property has guided the design of several effective substrate analog inhibitors (18-23). Current work seeks to identify compounds with broad-spectrum activity against bacterial PDF while avoiding inhibition of other cellular targets, including the recently identified human mitochondrial protein (7,9,24).