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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

During the past 10 years, many epidemiologists have warned that major pathogenic bacteria and parasites are progressively developing resistance to drugs widely used in human medicine. For example, the resistance of Plasmodium falciparum to antimalarial drugs such as chloroquine is increasing at an alarming rate. Similarly, several isolates of Staphylococcus aureus, the opportunistic pathogen responsible for most hospital-acquired infections, resistant to all known antibiotics have been described in the past 2 years. These worrying developments have raised fears that infectious diseases may once again become a major cause of death in industrialized countries. Public health regulations may certainly slow the development of resistance but in the short term, new antimicrobial drugs must be developed to control pathogens that have developed resistance to all the drugs currently in use. It is now widely accepted that the traditional screening methods, based on direct measurements in living cells of the inhibitory capacities of particular compounds, are unlikely to generate many promising molecules. Alternative strategies must therefore be developed to find new drugs. One possible strategy is to identify a molecular target at the outset and then to screen the available libraries of chemical compounds looking for ‘hits’ with potent inhibitory capacities in vitro. For this approach, the identification of a ‘good’ target is vital. It is generally agreed (for a more complete discussion see Meinnel, 1999) that such a target should: (i) be present in most human pathogens (i.e. the inhibitor should have wide-spectrum effects); (ii) be absent from human cells; (iii) be part of an essential pathway in the pathogen; (iv) not be inhibited by widely used antibiotics (i.e. not previously subjected to selection pressure); (v) be easy to assay in vitro and, if possible, in vivo; (vi) be highly specific for the pathogen and non-toxic for humans; and (vii) not result in the rapid acquisition of resistance or by-pass processes if inactivated. Peptide deformylase (PDF) has been suggested as a possible candidate that may fulfil all these criteria. This resulted in renewed interest in this enzyme in the early 1990s and the recent publication of large amounts of data in the field.

Peptide deformylase function and biochemistry

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

Most of our basic knowledge about PDF activity (EC 3.5.1.31) in bacteria comes from pioneering work carried out in the 1960s. It was found that the bacterial initiator tRNA (tRNAfMet) underwent N-formylation after the addition of methionine (Marcker and Sanger, 1964; Adams and Capecchi, 1966). N-formylmethionine was thus shown to be the first amino acid incorporated into bacterial polypeptide chains. The initiator tRNA is not formylated during protein synthesis in the cytoplasm of eukaryotes. However, formylation of the initiator tRNA has been shown to occur in all mitochondria and plastids (for a review see Kozak, 1983). It was also found in the early 1960s that the initial methionine is often removed from mature polypeptide chains (Waller, 1963). Two types of activity were shown to be involved in peptide processing (see Fig. 1): PDF activity, which removes all N-formyl groups from nascent polypeptide chains, and methionine aminopeptidase (MAP) activity, which hydrolyses the initial methionine once the N-formyl group has been removed (Adams and Capecchi, 1966; Fry and Lamborg, 1967). MAP is now known to have a particular substrate specificity and the removal or maintenance of the methionine depends on the nature of the second amino acid of the polypeptide chain (see References in Meinnel et al., 1993a).

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Figure 1. Reaction catalysed by PDF and MAP.

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PDF was first identified in crude extracts of Escherichia coli and Bacillus subtilis. In both cases, PDF activity was found to be highly labile and was lost if fractionation was attempted (Adams, 1968; Livingston and Leder, 1969). The lability of PDF activity accounts for the production of N-formylated proteins by in vitro bacterial translation systems. The difficulties encountered in attempts to purify PDF prevented any further characterization for the next 25 years. The cloning of the E. coli gene (Meinnel and Blanquet, 1993; Mazel et al., 1994) was a turning point, making it possible to purify the protein to homogeneity and to study its great instability. The first attempts to purify the protein to homogeneity generated products with weak activity and kcat/Km values of about 100 M−1 s−1 (Meinnel and Blanquet, 1993). Two fractions with very similar specific activities were separated by hydrophobic interaction chromatography. The major fraction retained one bound zinc ion per polypeptide chain, whereas there was no significant binding of metals in the other fraction (Meinnel and Blanquet, 1995; Ragusa et al., 1998). It was concluded at this point that PDF was a zinc enzyme because the zinc ligands were found to be crucial for PDF activity (Meinnel et al., 1995). However, the weak activity of the zinc enzyme in vitro was not consistent with the strong catalytic activity of PDF in vivo (see data in Ragusa et al., 1998). PDF activity is coupled to protein synthesis, occurring almost immediately after the end of the peptide has emerged from the ribosome. It is therefore considered to be an early cotranslational process (Pine, 1969; Ball and Kaesberg, 1973). Takeda and Webster (1968) attempted to account for the high efficiency of PDF by suggesting that it might be bound to ribosomes. However, no such association was observed in E. coli (C. Lazennec and T. Meinnel, unpublished results).

It was not until late 1997 that the purification of a highly active PDF fraction was first reported. It was found that the active enzyme (i) contains a bound ferrous ion and (ii) is very sensitive to atmospheric oxygen (Groche, 1995; Rajagopalan et al., 1997a, b; Groche et al., 1998). It was shown that PDF activity could be protected throughout purification by adding catalase or Tris(2-carboxyethyl)phosphine (TCEP), which both rapidly remove H2O2, a reactive species that arises in buffers containing thiol compounds, O2 and traces of Fe3+. Hence, the ferric form of PDF has only weak activity whereas the ferrous form (Fe–PDF) is fully active (kcat/Km∼ 105 M−1 s−1). The reactivity to oxygen of one of the three metal ligands in PDF, the thiol of the cysteine (Cys90 in E. coli PDF), was also thought to contribute to the instability of the metal ion within the enzyme and to participate in the inactivation equilibrium (Rajagopalan and Pei, 1998). However, zinc was retrieved in most enzyme preparations. Zn–PDF can be separated from Fe–PDF using hydrophobic interaction chromatography (Rajagopalan et al., 1997b). Zn–PDF is very stable in vitro; this is because the association constant for the binding of zinc to PDF is high and because zinc, once bound to the protein, cannot be removed under non-denaturing conditions in vitro. It is unknown whether the zinc and iron forms co-exist in vivo or whether the zinc form is an artefact of the purification procedures (Groche et al., 1998). Neither the PDF of Thermus thermophilus nor that of Bacillus stearothermophilus has been found to contain bound zinc when produced in E. coli. Unlike Zn–PDF, Fe–PDF is active enough to satisfy the deformylation requirements of the cell but is very unstable (t1/2 ∼ 1 min) due to: (i) the weak binding of iron to the enzyme (Kd ∼ 0.6 µM; Groche, 1995) and (ii) the sensitivity of this cation to oxidation. This raises the question of whether the reducing power of the cytoplasm is strong enough to protect a ferrous ion bound to PDF from intracellular reactive oxygen species and whether other metal cations compete for the same binding site in bacterial cells. Indeed, in addition to zinc and iron, nickel, manganese and cobalt cations have been reported to compete for the same binding site in vitro (Groche, 1995; Ragusa et al., 1998; Durand et al., 1999; Rajagopalan et al. 2000). The nickel ion is as efficient as the ferrous ion in PDF catalysis but is insensitive to oxidation (Groche et al., 1998; Ragusa et al., 1998). Interestingly, although nickel concentration is low in aerobically grown E. coli cells, specific channels involved in nickel transport are induced under anaerobic conditions (Silver, 1996). Similarly, anaerobic bacteria such as Helicobacter pylori possess high-affinity nickel transporters (Fulkerson et al., 1998). Thus, iron is a natural ligand of E. coli PDF but, depending on the culture conditions and/or bacterial cell type, it is possible that PDF is also ligated to other cations in vivo.

Many laboratories prefer to work with the nickel form rather than the iron form because it is much more stable and as efficient in vitro. The preparation and assay protocols for PDFs of various origins involve the systematic addition of millimolar concentrations of nickel salts to the buffers because nickel, like iron, is weakly bound to PDF (Kd ∼ 6 µM) (see Groche, 1995; Groche et al., 1998; Ragusa et al., 1998). The availability of this fully active, stable form of PDF has made possible high-throughput screening procedures and studies of the inhibition in vitro of physiologically relevant PDF forms by chemical compounds. In addition various easy, continuous and discontinuous in vitro assays for deformylation activity have recently been described (Lazennec and Meinnel, 1997; Wei and Pei, 1997). All are suitable for use with the automated procedures required for library screening. This has enabled many pharmaceutical companies to develop screening procedures for PDF activity aimed at discovering specific, potent inhibitors (Durand et al., 1999).

Occurrence of PDF in living cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

PDF in prokaryotes

The first PDF gene to be isolated was that of E. coli (def or fms) (Meinnel and Blanquet, 1993; Mazel et al., 1994). We will use the def nomenclature because it is more consistent. Several other def genes from various Gram-positive and Gram-negative bacteria have since been cloned and characterized genetically and/or biochemically (Meinnel and Blanquet, 1994; Mazel et al., 1997; Meinnel et al., 1997; Belouski et al., 1998; Evans et al., 1998). Sequences significantly similar to those of these PDFs have been retrieved from databases (Dardel et al., 1998; Meinnel, 2000). To date, more than 90 PDF-like sequences have been identified. Most of the PDF sequences retrieved are from bacterial genomes. Bacteria generally possess one def gene, or in some cases, two (Fig. 2). Often, but not systematically, def is expressed as the first cistron of a bi-cistronic unit with the fmt gene (Meinnel and Blanquet, 1993; 1994; Meinnel et al., 1993b). This gene encodes methionyl-tRNAfMet-formyltransferase, the enzyme responsible for the addition of the formyl group onto the first methionine (see References in Meinnel et al., 1993a). The presence of these two genes in the same transcriptional unit is interesting in terms of evolution, but no regulation of expression of either gene has yet been demonstrated (Meinnel et al., 1993b).

image

Figure 2. Similarity of PDF sequences. From the 90 PDF homologue sequences available in the databases, 40 PDF sequences were selected as representative of the sequence diversity of this protein. The sequences were aligned with Clustalx software. The (E) symbol after the abbreviated name indicates that it corresponds to a eukaryotic species and 1 or 2 means that it is one of the two PDF species of this organism. Abbreviations used: A. actinomycetemcomitans, Actinobacillus actinomycetemcomitans;A. aeolicus, Aquifex aeolicus;A. thaliana, Arabidopsis thaliana;B. stearothermophilus, Bacillus stearothermophilus;B. subtilis, Bacillus subtilus;B. burgdorferi, Borrelia burgdorferi;C. trachomatis, Chlamydia trachomatis;C. acetobutylicum, Clostridium acetobutylicum;C. beijerinckii, Clostridium beijerinckii;D. radiodurans, Deinococcus radiodurans;D. melanogaster, Drosophila melanogaster;E.coli, Escherichia coli;E. faecalis, Enterococcus faecalis;H. influenzae, Haemophilus influenzae;H. sapiens, Homo sapiens;L. lactis, Lactobacillus lactis;L. pneumophila, Legionella pneumophila;L. major, Leishmania major;L. esculentum, Lycopersicon esculentum (tomato); M. tuberculosis, Mycobacterium tuberculosis;M. genitalium, Mycoplasma genitalium;M. pneunomiae, Mycoplasma pneumoniae;N. gonorrhoeae, Neisseria gonorrhoeae;P. falciparum, Plasmodium falciparum (causative agent of malaria); P. marinus, Prochlorococcus marinus;P. aeruginosa, Pseudomonas aeruginosa;R. prowazeki, Rickettsia prowazekii;S. aureus, Staphylococcus aureus;S. coelicolor, Streptomyces coelicolor;S. pyogenes, Streptococcus pyogenes;Synechocystis, Synechocystis sp.; T. maritima, Thermotoga maritima;T. thermophilus, Thermus thermophilus;T. pallidum, Treponema pallidum;T. brucei, Trypanosoma brucei;V. cholerae, Vibrio cholerae.

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Bacterial PDFs are small monomers composed of about 160–200 residues with few variations in the lengths of their N- and C-terminal extremities. In each case studied, it was shown that the N-terminus was essential for activity in vitro whereas the C-terminus was fully dispensable (Meinnel et al., 1996a; 1997; Durand et al., 1999). An E. coli strain, PAL147Tr, in which the last 21 codons of the def gene were deleted was constructed (Ragusa et al., 1999). PAL147Tr is viable and has a doubling time similar to that of the parental strain in all culture conditions tested (C. Lazennec and T. Meinnel, unpublished results). It was therefore concluded that the C-terminal domain of PDF is not required for deformylase action in vivo. This domain was shown to fold into an α-helix at low temperature in the crystal structure of E. coli PDF but appears to have a much less organized structure at physiological temperatures (30–45°C) in solution. Although bacterial PDFs display significant sequence similarity, they have only a few residues in common. Most of the conserved residues are in three short stretches of amino acids, motifs 1 {GφGφAAXQ}, 2 {EGCφS} and 3 {HEφDH} (where φ is a hydrophobic amino acid and X is any amino acid). These three motifs have been described as the signature sequences of PDFs (Meinnel et al., 1997). These motifs are physically close to each other (Fig. 3), building the three sides of the active site crevice (Dardel et al., 1998). This suggests that the 3-D structure of the active site of PDFs is conserved (Meinnel et al., 1997). The cysteine of motif 2 and the two histidines of motif 3 are involved in metal cation binding (Meinnel et al., 1995; 1996b; Chan et al., 1997; Fig. 3). Extensive site-directed mutagenesis has indicated that the side-chains of the glutamine of motif 1 and of the glutamate of motif 2 (Gln50 and Glu133, respectively, in E. coli PDF; see Fig. 3) are essential for catalysis because they are directly involved in formyl group recognition and the transition state respectively (Becker et al., 1998a, b; Hao et al., 1999; Ragusa et al., 1999). The side-chain of the serine of motif 2 hydrogen bonds with the glutamine side-chain of motif 1. Similarly, the carboxylates of the glutamate of motif 2 and the aspartate of motif 3 hydrogen bond with the side-chain of another strictly conserved residue, a buried arginine located between motifs 2 and 3 (Becker et al., 1998b; Dardel et al., 1998). Without these bonds, PDF is unstable. The last strictly conserved residue is an asparagine, located between motifs 1 and 2. Its side-chain hydrogen bonds with the backbone of an amino acid located at the N-terminus of the protein. The conserved glycine and alanine side-chains of motif 1 have been shown to be essential for catalysis. It has been suggested that the reduced size of these side-chains leaves just enough space within the active site cavity for the binding of the substrate (Ragusa et al., 1999). The alternation of hydrophobic and hydrophilic residues in motif 1 is involved in the formation of the β-1 strand, along which the peptide substrate aligns, while an additional, fourth anti-parallel strand is formed. Other less well-conserved residues are involved in optimization of the packing of the 3-D structure (discussed in Dardel et al., 1998). Overall, this network of hydrogen bonding and hydrophobic interactions makes the structure of PDF very compact and solid. This enables the protein to resist proteolytic attack, folding–unfolding cycles, freezing and high temperature. This solidity of the polypeptide chain contrasts strongly with the fragility of the metal-binding site.

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Figure 3. Cartoon of the structure of the catalytic core of E. coli peptide deformylase.The ribbons diagram of the enzyme is viewed towards the substrate's binding cleft into the catalytic site. The secondary structures are colour coded with α-helical regions as red and yellow and β-sheet regions as light blue, and the remainder as grey. The side-chains of the three ligands of metal (Cys90, His132 and His136) and of Gln50 and Glu133 are shown as green and red respectively. The metal cation is shown as a green sphere. The location of the three conserved motifs, the hydrophobic ‘core’, which buries the end of the active site, and of the hydrophobic S1′/S3′ pocket are indicated. The data were taken from Dardel et al. (1998).

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Amino acid sequence alignments and phylogenic trees (Fig. 2) have revealed the existence of two PDF subfamilies. One is typified by the E. coli enzyme and many PDFs from Gram-negative bacteria (class I) and the second by B. stearothermophilus PDF, including several enzymes from Gram-positive bacteria (streptococci and bacilli) and mycobacteria (class II). Compared with class I, class II PDFs have two sequence insertions at the N-terminal end, just upstream from the α-1 helix (insertion I1) and the β-1 strand in motif 1 (insertion I2). No 3-D structure is currently available for class II PDFs (in contrast to class I PDFs) and therefore the effect of these two insertions on folding is unknown. Insertion I2 is located at the entrance to the active site of the PDFs of many pathogenic bacteria, including Staphylococcus aureus and Enterococcus faecalis (Dardel et al., 1998; Meinnel, 2000). Thus, models of the interaction of PDF with inhibitors should be carried out with PDF structures of both classes. The 3-D models of PDFs with some inhibitors have indeed indicated contact between the side-chains of the ligand and the sequence corresponding to I2, a turn located between the end of the α-1 helix (Fig. 3) and the start of the β-1 strand (Becker et al., 1998b; Ragusa et al., 1999). The C-terminal domains of class II PDFs contain many hydrophobic amino acids whereas those of class I PDFs do not. This suggests that the C-terminal domains of class II PDFs may not fold into an α-helix. The rest of the structure, including the active site crevice, is believed to fold in the same way in all PDFs (Meinnel et al., 1997).

PDF in eukaryotes

No PDF homologues have been found in the genomes of Archaes, Saccharomyces cerevisiae and Caenorhabtidis elegans. This is consistent with the generally accepted notion that PDF is not present in the organelles of animal and fungal cells. Indeed, the proteins synthesized in mammalian mitochondria systematically retain their N-formyl groups (Steffens and Buse, 1976; 1979; von Jagow et al., 1981;Fearnley and Walker, 1986; Yagi and Hatefi, 1988; Walker et al., 1991), providing strong evidence that humans do not possess a PDF activity. Recent analysis of the data produced by the systematic sequencing of other genomes has provided a different view of PDF expression in the eukaryotic kingdom. First, cDNA or genomic sequences encoding homologues of PDF have been identified in several eukaryotic parasites such as the agents of malaria (P. falciparum), Chaga's disease and sleeping sickness (Trypanosoma spp.) (Meinnel, 2000). Similar sequences were also identified in higher plants (Arabidopsis thaliana, tomato, corn, and rice; C. Giglione, M. Pierre and T. Meinnel, unpublished results). Deformylation has been shown to occur in some algal chloroplasts (see References quoted in Meinnel, 2000) and in various plant mitochondria (Braun and Schmitz, 1993; Gabler et al., 1994; Herz et al., 1994) and chloroplasts (Hauska et al., 1988; Schmidt et al., 1992; Shanklin et al., 1995). It therefore seems likely that these PDF are targeted to the mitochondria or plastids by an N-terminal exportation presequence. The sequences obtained from Kinetoplastid protists are significantly less similar to well-characterized PDFs than are other sequences and may therefore constitute a third subclass (Fig. 2). Unexpectedly, two adjacent, PDF-like genes have been very recently identified in the genome of Drosophila melanogaster (GAN AC017652). More surprising again was the discovery of partial Expressed Sequence Tags encoding PDF homologues in mouse and human (Fig. 2). The corresponding, full-length cDNA from human fetus could be cloned in our laboratory, showing that these sequences did not originate from bacterial DNA contamination, as was previously believed (C. Giglione and T. Meinnel; GAN AF239156). Finally, the genomic fragment corresponding to this cDNA was made recently available (GAN AC012040). The intracellular location and exact function of these PDF-like sequences are unknown but their occurrence apparently conflicts with the idea that PDF is not present in animal cells.

In conclusion, PDF activity is more widespread in living organisms than was previously thought, but has not been detected in mammal mitochondria. This and the predicted strong conservation of the structure of its active site crevice, suggest that PDF is indeed a very good target for new wide-spectrum antibiotics. Nevertheless, better characterization of the PDF homologues found in eukaryotes is required in order to know whether they might have important functions, in human for instance.

Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

Deformylation is an essential process in E. coli (Mazel et al., 1994; Meinnel and Blanquet, 1994) and probably in all eubacteria. The reasons for this are unclear but it is possible that deformylation is required partly because MAP activity (see Fig. 1) is strictly required for bacterial cell growth. Indeed, MAP acts on nascent polypeptides and requires the N-formyl group to be removed (Chang et al., 1989; Miller et al., 1989; for a review see Meinnel et al., 1993a; Solbiati et al., 1999). The necessity of deformylation makes PDF an attractive target in the development of new antibacterial agents. It has been demonstrated that the def gene is not required in the absence of formylation (i.e. if the product of the fmt gene is inactivated; Mazel et al., 1994). It should be borne in mind that formylation has been reported to be a non-essential step in several bacteria (Samuel et al., 1972; Guillon et al., 1992; Mazel et al., 1994; Newton et al., 1999). It was therefore anticipated that, if deformylation was blocked, resistance might develop due to mutations inactivating the fmt gene (by-pass of the formylation-deformylation pathway). A recent report from Versicor confirmed this prediction (39th ICAAC meeting, San Francisco; abstract F149.F; http://asm.ctt-inc.com/itinerarybuilder/Login.asp). As fmt bacteria grow very poorly, such resistance is not believed to be a major obstacle to the use of PDF as a target for antimicrobial therapy. Nevertheless, fast-growing mutant fmt bacteria have been isolated (Guillon et al., 1996).

It is not known whether eukaryotic PDFs are essential for cell survival. However, it was suggested that Plasmodium PDF would be active in the apicoplast. This organelle was recently discovered in Apicomplexan protists and is thought to be an excellent drug target because its function is required for cell growth (McFadden and Roos, 1999). The similarity of protein synthesis in organelles and bacteria suggests that deformylation may also be essential for organelle function. It is unclear why human and fungal mitochondria have no deformylation activity, but these organelles each encode about a dozen proteins, whereas plastids and plant mitochondria each encode 50–100 proteins. This reduction in the number of proteins encoded may be the reason why deformylation is no longer required for the few remaining mitochondrion-encoded proteins to be functional and why def genes were deleted from some genomes.

Inhibitors of PDF activity in vitro

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

The identification of PDF as an attractive target for new drugs to treat infectious diseases has led to an extensive search for PDF inhibitors. Despite the efforts of many pharmaceutical companies, no report of a high-throughput screening for inhibitors of PDF activity has yet been published. The results of such screening will certainly provide interesting data in the near future. In particular, it is important to know whether known drugs and antibiotics are potent inhibitors of PDF activity. If so, their structures should be compared with those of well-characterized inhibitors (see Fig. 4).

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Figure 4. What a potent inhibitor of PDF activity should look like. che– is metal cation-chelator function (i.e. thiol, phosphate, carboxylate or hydroxamate). Data were taken from Meinnel et al. (1999). + indicates that the extremity of the side-chain should be positively charged (like the side-chains of arginine or lysine).

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PDF acts as an aminopeptidase because it hydrolyses the peptide bond of a polypeptide (between the N-formyl group and the first methionine) and because at least two consecutive amino acids are required for efficient catalysis (Adams, 1968; Meinnel et al., 1999). Nevertheless, the specificity of N-formyl group recognition is strong, with the conserved glutamine of motif 1 playing a crucial role in this process (Ragusa et al., 1999). The HEXXH signature sequence of the zinc metalloproteases superfamily (Vallee and Auld, 1990a, b; Blundell, 1994) is present within motif 3 of PDFs, suggesting that there may be structural and functional similarities between these molecules (Meinnel and Blanquet, 1993). The side-chains of this motif have a similar function in metal ligation and transition state stabilization, consistent with this idea (Meinnel et al., 1995, 1996b; Chan et al., 1997; Rajagopalan et al., 2000). It has also been shown that the 3-D structure of the E. coli PDF is similar to that of the proteins of the zinc metalloproteases superfamily. It has been suggested that PDF may be part of a distinct, third subfamily, because of differences in its metal binding site, with a cysteine acting as the third metal ligand. Given that metals other than zinc (such as iron) are now thought to be responsible for the full activity of PDF (see part II), we propose changing the name of ‘zinc’ metalloproteases, which is misleading, to ‘HEXXH-containing’ metalloproteases. Thermolysins (TLN) and matrix metalloproteases (also called matricins or metzincins, MMP) are the other two members of this family, and they have glutamate and histidine, respectively, as the third metal ligand. A fourth family has recently been defined, with aspartate as the third metal ligand (Fushimi et al., 1999). The structures of E. coli PDF, TLN and MMP display similarities in their active sites, with a common super-secondary structure involving the three signature sequences (Chan et al., 1997; Dardel et al., 1998). However, unlike TLN and MMP, PDF has a hydrophobic ‘cork’ blocking one end of the active site crevice (Fig. 3). This extremity is open in TLN and MMP, allowing these enzymes to act as endoproteases. The cork is believed to play an important role in restricting the selectivity of PDF such that it functions exclusively as an aminopeptidase. Further analysis of the binding of the substrate within the active site has shown that the alignment of the substrate is similar in PDF, TLN and MMP (Becker et al., 1998a; Hao et al., 1999; Ragusa et al., 1999). In particular, a large hydrophobic binding pocket was revealed at S1′ (Fig. 3). This pocket is large enough to hold the large hydrophobic side-chains from the P1′ and/or P3′ positions of the substrate (Hao et al., 1999). This feature results from the β-stranded structure of the enzyme-bound substrate.

TLN and MMP have long been studied and many inhibitors of their activity have been described. In addition, the structure of the complexes of these enzymes with their targets is known in many cases. This knowledge should be useful for identifying PDF inhibitors. Furthermore, the particular and crucial role of the metal ion in catalysis (see part II) has been used to design PDF inhibitors (discussed in Meinnel et al., 1999). Analysis of these data showed that a peptide derivative with the characteristics shown in Fig. 4 should act as a potent inhibitor of PDF activity. Systematic analysis of the substrate specificity of PDF was useful in this respect (Hu et al., 1999; Meinnel et al., 1999). The most potent inhibitors fully characterized to date have binding constants in the micromolar range (Hu et al., 1998; Durand et al., 1999; Meinnel et al., 1999). These inhibitors are unlikely to have bacteriostatic or bactericidal effects, for which binding constants in the nanomolar range are usually required. However, the combination of all the effective chemical groups in a single molecule has yet to be described. For instance, the effect of adding a peptide chain with an aromatic ring at P3′ has not been tested in the context of the thiol derivative. In addition, the effects on PDF of hydroxamic derivatives, known to be very potent inhibitors of MMP (Browner et al., 1995), have yet to be studied. While this work was completed and under final revision, it was shown indeed that the naturally occurring antibacterial agent actinonin, a pseudo-peptide hydroxamate, acts as a very potent inhibitor of PDF (Kd = 0.3 nM) (Chen et al. 2000).

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

These data indicate that PDF is a very promising target in the search for new antibacterial agents. The discovery that PDF is present in several human parasites suggests that it may also be a good target for antiparasitic agents (Meinnel, 2000). This raises the question of whether a single inhibitor of PDF activity could have potent activity as both an antiparasitic and an antibacterial drug.

There is little doubt that very potent inhibitors of PDF activity will be discovered in the next few years. However, such inhibitors are likely to be derived from well-known families of peptide inhibitors. Such compounds are already widely used in human medical treatment. The major questions to be addressed are therefore: (i) whether such inhibitors will be specific in vivo for PDF activity only and (ii) whether they will inhibit other cellular functions important in humans, such as MMP activity. It is important to set suitable conditions for assays aiming to assess these problems. For instance, given that deformylation is not essential in a fmt background and that def– fmt– and def+ fmt–cells grow, albeit slowly (Guillon et al., 1992; Mazel et al., 1994), we could investigate whether a putative inhibitor compound inhibits the growth of a wild-type strain but not that of the corresponding fmt derivative. In terms of toxicity, it would be useful to check in vitro, before any clinical trial, whether the selected compounds inhibit well-characterized members of the ‘zinc’ metalloproteases superfamily. Fortunately, many simple in vitro assays are available for these enzymes and most are commercially available.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References

We wish to thank everyone at the Ecole Polytechnique (Palaiseau, France) who has been involved in the deformylase project over the past 6 years. This work was supported by an ATIPE grant from the C.N.R.S. to T.M. and by the Fondation pour la Recherche Médicale. C.G. is supported by a postdoctoral fellowship from the Association pour la Recherche sur le Cancer (ARC, Villejuif, France).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Peptide deformylase function and biochemistry
  5. Occurrence of PDF in living cells
  6. Deformylation as an essential function: anticipating acquired resistance due to deformylase inhibition
  7. Inhibitors of PDF activity in vitro
  8. Concluding remarks
  9. Acknowledgements
  10. References
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