Hydroxamic Acid Derivatives as Potent Peptide Deformylase Inhibitors and Antibacterial Agents

bGSK1322322 is a novel peptide deformylase (PDF) inhibitor being developed for the intravenous and oral treatment of acute bacterial skin and skin structure infections and hospitalized patients with community-acquired pneumonia. The activity of GSK1322322 was tested against a global collection of clinical isolates of Haemophilus influenzae (n ‫؍‬ 2,370), Moraxella catarrhalis (n ‫؍‬ 115), Streptococcus pneumoniae (n ‫؍‬ 947), Streptococcus pyogenes (n ‫؍‬ 617), and Staphylococcus aureus (n ‫؍‬ 940), including strains resistant to one or more marketed antibiotics. GSK1322322 had an MIC 90 of 1 g/ml against M. catarrhalis and 4 g/ml against H. influenzae, with 88.8% of ␤-lactamase-positive strains showing growth inhibition at that concentration. All S. pneumoniae strains were inhibited by <4 g/ml of GSK1322322, with an MIC 90 of 2 g/ml. Pre-existing resistance mechanisms did not affect its potency, as evidenced by the MIC 90 of 1 g/ml for penicillin, levofloxacin, and macrolide-resistant S. pneumoniae. GSK1322322 was very potent against S. pyogenes strains, with an MIC 90 of 0.5 g/ml, irrespective of their macrolide resistance phenotype. This PDF inhibitor was also active against S. aureus strains regardless of their susceptibility to methicillin, macrolides, or levofloxacin, with an MIC 90 of 4 g/ml in all cases. Time-kill studies showed that GSK1322322 had bactericidal activity against S. pneumoniae, H. influenzae, S. pyogenes, and S. aureus, demonstrating a >3-log 10 decrease in the number of CFU/ml at 4؋ MIC within 24 h in 29 of the 33 strains tested. Given the antibacterial potency demonstrated against this panel of organisms, GSK1322322 represents a valuable alternative therapy for the treatment of infectious diseases caused by drug-resistant pathogens.

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confidence: 99%

Comparative Analysis of the Antibacterial Activity of a Novel Peptide Deformylase Inhibitor, GSK1322322

O’Dwyer

Hackel²,

Hightower

et al. 2013

“…However, recently it has been shown that the natural antibiotic actinonin, a hydroxamic acid pseudopeptide, is a potent inhibitor of PDF (11). In addition, a series of ␤-sulfonyl and ␤-sulfinylhydroxamic acid derivatives have been shown to be potent PDF inhibitors with in vitro antibacterial activity (1). As a result of our previous experience with mammalian matrix metalloproteinases (6), we have accumulated an extensive library of potential metalloenzyme inhibitors.…”

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confidence: 99%

Antibiotic Activity and Characterization of BB-3497, a Novel Peptide Deformylase Inhibitor

Clements¹,

Beckett²,

Brown³

et al. 2001

224

243

Peptide deformylase (PDF) is an essential bacterial metalloenzyme which deformylates the N-formylmethionine of newly synthesized polypeptides and as such represents a novel target for antibacterial chemotherapy. To identify novel PDF inhibitors, we screened a metalloenzyme inhibitor library and identified an N-formylhydroxylamine derivative, BB-3497, and a related natural hydroxamic acid antibiotic, actinonin, as potent and selective inhibitors of PDF. To elucidate the interactions that contribute to the binding affinity of these inhibitors, we determined the crystal structures of BB-3497 and actinonin bound to Escherichia coli PDF at resolutions of 2.1 and 1.75 Å, respectively. In both complexes, the active-site metal atom was pentacoordinated by the side chains of Cys 90, His 132, and His 136 and the two oxygen atoms of N-formyl-hydroxylamine or hydroxamate. BB-3497 had activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, and activity against some gram-negative bacteria. Time-kill analysis showed that the mode of action of BB-3497 was primarily bacteriostatic. The mechanism of resistance was via mutations within the formyltransferase gene, as previously described for actinonin. While actinonin and its derivatives have not been used clinically because of their poor pharmacokinetic properties, BB-3497 was shown to be orally bioavailable. A single oral dose of BB-3497 given 1 h after intraperitoneal injection of S. aureus Smith or methicillin-resistant S. aureus protected mice from infection with median effective doses of 8 and 14 mg/kg of body weight, respectively. These data validate PDF as a novel target for the design of a new generation of antibacterial agents.Ribosome-mediated synthesis of proteins starts with a methionine residue. In prokaryotes, the amino group of the methionyl moiety carried by the initiator tRNA fMet is N formylated by formyltransferase prior to its incorporation into a polypeptide. Consequently, N-formylmethionine is always present at the N terminus of a nascent bacterial polypeptide. However, most mature proteins do not retain the N-formyl group or the terminal methionine residue. Following translation, the formyl group is hydrolyzed by peptide deformylase (PDF), which is necessary for further processing at the N terminus by methionine aminopeptidase (32). Deformylation is therefore a crucial step in bacterial protein biosynthesis, and PDF is essential for bacterial growth (23). The gene encoding PDF (def) is present in all sequenced pathogenic bacterial genomes and has no mammalian counterpart, making it an attractive target for antibacterial chemotherapy. Although the enzyme has been known for 30 years, it has proved difficult to isolate and characterize due to its apparent instability. Recently, two X-ray crystal structures and a solution structure of PDF have been determined (5, 9, 12), identifying PDF as a new class of metalloenzyme related in structure to the metalloproteinase superfamily. ...

“…This highthroughput standard assay is based on the measurement of the hydrolysis of formyl-methionine-alanine-serine (f-MAS; K m , 4 to 10 mM) by recombinant purified E. coli PDF via detection of the formation of the new free amino group of MAS by reaction with fluorescamine (2). To further characterize the compounds, a PDF-specific TC-TL assay was developed that simulates the physiological conditions much better than the assay with the isolated enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently described the identification, optimization, and biological characterization of new PDF inhibitors (2). Since the antibacterial activities of these inhibitors were lower than expected from the inhibition at the enzyme level, we have performed several studies in order to better understand this discrepancy (3).…”

mentioning

confidence: 99%

Peptide Deformylase as an Antibacterial Drug Target: Assays for Detection of Its Inhibition in Escherichia coli Cell Homogenates and Intact Cells

Apfel

Evers

Hubschwerlen

et al. 2001

Self Cite

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35 S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.All nascent polypeptides in eubacteria are synthesized with N-formyl-methionine at the N terminus, using the formyl-methionyl-tRNA for initiation (11). During elongation of the polypeptide chain, the formyl group is removed by the enzyme peptide deformylase (PDF; EC 3.5.1.27) (1, 5). The N-terminal methionine is removed by methionine aminopeptidase (MAP; EC 3.4.11.18) if the penultimate residue is small and uncharged, e.g., alanine, cysteine, glycine, proline, serine, threonine, and valine. N-blocked methionine polypeptides are not a substrate for the MAP, making deformylation a prerequisite for protein maturation (6,13,18). PDF and MAP are enzymes essential for growth in wild-type bacteria, as demonstrated by gene deletion in Escherichia coli (6,12,14). PDF mutants can only be constructed in an fmt (coding for formyltransferase; EC.2.1.2.9) mutant background (12). Formyltransferase formylates the methionyl-tRNA f Met , and mutants for the corresponding gene show a strongly decreased growth rate (9).We have recently described the identification, optimization, and biological characterization of new PDF inhibitors (2). Since the antibacterial activities of these inhibitors were lower than expected from the inhibition at the enzyme level, we have performed several studies in order to better understand this discrepancy (3). As part of this effort, we developed new specific assays to determine the PDF activity in crude E. coli homogenates as well as in intact bacteria. The assay conditions reflect the physiological conditions in bacteria much more closely than an assay using the isolated enzyme, making them valuable tools for a meaningful assessment of the PDF-inhibitory activities of antibacterial compounds. MATERIALS AND METHODSBacterial strains, plasmids, enzymes, and chemicals. The E. coli strains used in this study were XL2-blue and BL21 (DE3) carrying pLysS (Stratagene, Basel, Switzerland). The strain was grown in Luria-Bertani medium (Difco Laboratories, Detroit, Mich.) with aeration at 37°C. The restriction enzymes were from New England Biolabs (Beverly, Mass.) or Amersham Pharmacia Biotech (Dübendorf, Switzerland) and were used in accordance with the specifications of the manufacturer. All other chemicals, including actinonin (Ro 06...