Peptide Deformylase Inhibitors as Potent Antimycobacterial Agents

ABSTRACT Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 μM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of ≤5 × 10−7 in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents.

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A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00723-09 ◽

2009 ◽

Vol 53 (9) ◽

pp. 3972-3980 ◽

Cited By ~ 21

Author(s):

James J. Vecchione ◽

Jason K. Sello

Keyword(s):

Resistance Gene ◽

Resistance Genes ◽

Point Mutations ◽

Trna Synthetase ◽

Sensitive Strain ◽

Antibacterial Drug ◽

Gene Encoding ◽

Trna Synthetases ◽

High Level ◽

Level Resistance

ABSTRACT Indolmycin, a potential antibacterial drug, competitively inhibits bacterial tryptophanyl-tRNA synthetases. An effort to identify indolmycin resistance genes led to the discovery of a gene encoding an indolmycin-resistant isoform of tryptophanyl-tRNA synthetase. Overexpression of this gene in an indolmycin-sensitive strain increased the indolmycin MIC 60-fold. Its transcription and distribution in various bacterial genera were assessed. The level of resistance conferred by this gene was compared to that of a known indolmycin resistance gene and to those of genes with resistance-conferring point mutations.

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Peptide Deformylase Inhibitors as Antibacterial Agents: Identification of VRC3375, a Proline-3-Alkylsuccinyl Hydroxamate Derivative, by Using an Integrated Combinatorial and Medicinal Chemistry Approach

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.1.250-261.2004 ◽

2004 ◽

Vol 48 (1) ◽

pp. 250-261 ◽

Cited By ~ 46

Author(s):

D. Chen ◽

C. Hackbarth ◽

Z. J. Ni ◽

C. Wu ◽

W. Wang ◽

...

Keyword(s):

Bacterial Growth ◽

Medicinal Chemistry ◽

Peptide Deformylase ◽

Pharmacokinetic Studies ◽

Chemical Libraries ◽

Effective Doses ◽

Low Toxicity ◽

Orally Bioavailable ◽

Low Cytotoxicity

ABSTRACT Peptide deformylase (PDF), a metallohydrolase essential for bacterial growth, is an attractive target for use in the discovery of novel antibiotics. Focused chelator-based chemical libraries were constructed and screened for inhibition of enzymatic activity, inhibition of Staphylococcus aureus growth, and cytotoxicity. Positive compounds were selected based on the results of all three assays. VRC3375 [N-hydroxy-3-R-butyl-3-(2-S-(tert-butoxycarbonyl)-pyrrolidin-1-ylcarbonyl)propionamide] was identified as having the most favorable properties through an integrated combinatorial and medicinal chemistry effort. This compound is a potent PDF inhibitor with a Ki of 0.24 nM against the Escherichia coli Ni2+ enzyme, possesses activity against gram-positive and gram-negative bacterial pathogens, and has a low cytotoxicity. Mechanistic experiments demonstrate that the compound inhibits bacterial growth through PDF inhibition. Pharmacokinetic studies of this drug in mice indicate that VRC3375 is orally bioavailable and rapidly distributed among various tissues. VRC3375 has in vivo activity against S. aureus in a murine septicemia model, with 50% effective doses of 32, 17, and 21 mg/kg of body weight after dosing by intravenous (i.v.), subcutaneous (s.c.), and oral (p.o.) administration, respectively. In murine single-dose toxicity studies, no adverse effects were observed after dosing with more than 400 mg of VRC3375 per kg by i.v., p.o., or s.c. administration. The in vivo efficacy and low toxicity of VRC3375 suggest the potential for developing this class of compounds to be used in future antibacterial drugs.

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Biosynthetic pathways and enzymes involved in the production of phosphonic acid natural products

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa052 ◽

2021 ◽

Vol 85 (1) ◽

pp. 42-52

Author(s):

Taro Shiraishi ◽

Tomohisa Kuzuyama

Keyword(s):

Natural Products ◽

Biosynthetic Pathway ◽

Organophosphorus Compounds ◽

Genome Mining ◽

Biological Molecules ◽

Antibacterial Drug ◽

Gene Encoding ◽

Quarter Century ◽

Lead Compounds ◽

Biosynthetic Machinery

Abstract Phosphonates are organophosphorus compounds possessing a characteristic C−P bond in which phosphorus is directly bonded to carbon. As phosphonates mimic the phosphates and carboxylates of biological molecules to potentially inhibit metabolic enzymes, they could be lead compounds for the development of a variety of drugs. Fosfomycin (FM) is a representative phosphonate natural product that is widely used as an antibacterial drug. Here, we review the biosynthesis of FM, which includes a recent breakthrough to find a missing link in the biosynthetic pathway that had been a mystery for a quarter-century. In addition, we describe the genome mining of phosphonate natural products using the biosynthetic gene encoding an enzyme that catalyzes C–P bond formation. We also introduce the chemoenzymatic synthesis of phosphonate derivatives. These studies expand the repertoires of phosphonates and the related biosynthetic machinery. This review mainly covers the years 2012-2020.

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Antimicrobial and clinical effect of linezolid (ZYVOX), a new class of synthetic antibacterial drug.

Folia Pharmacologica Japonica ◽

10.1254/fpj.120.245 ◽

2002 ◽

Vol 120 (4) ◽

pp. 245-252 ◽

Cited By ~ 2

Author(s):

Kazuhiko IRINODA ◽

Syunji NOMURA ◽

Munehiro HASHIMOTO

Keyword(s):

Clinical Effect ◽

Antibacterial Drug ◽

New Class

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Alfa Thalassemia Intermedia (HbH disease): How the New Information Provided by the Routine Hematology Analysers May Help in Its Differential Diagnosis or Flagging

Blood ◽

10.1182/blood.v120.21.5180.5180 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5180-5180

Author(s):

Susanna Barella ◽

Ramon Simon-Lopez ◽

Nicola Di Gaetano ◽

Renzo Galanello

Keyword(s):

Research Funding ◽

Roc Analysis ◽

Dna Analysis ◽

Point Mutations ◽

Control Group ◽

Thalassemia Intermedia ◽

Gene Encoding ◽

New Information ◽

Best Parameters ◽

Hbh Disease

Abstract Abstract 5180 Alpha-thalassemia (α-thalassemia) has two clinically significant forms: hemoglobin Bart hydrops fetalis (Hb Bart) syndrome and hemoglobin H (HbH) disease. HbH disease is characterized by microcytic hypochromic hemolytic anemia, hepatosplenomegaly, mild jaundice, and sometimes thalassemia-like bone changes. Diagnostic of Alfa Thalassemia: Classic testing for α-thalassemia includes: hematologic testing of red blood cell indices, peripheral blood smear, supravital stain to detect RBC inclusion bodies, and qualitative and quantitative hemoglobin analysis. HBA1, the gene encoding α1-globin, and HBA2, the gene encoding α2-globin, are the two genes most commonly associated with α-thalassemia. Molecular genetic testing of HBA1 and HBA2 detects deletions in about 90% and point mutations in about 10% of affected individuals. Objective: Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV, @RSF, @MAF, @LHD% and many morphological parameters for RBC and Reticulocytes calles Cell Population Data. All this parameters may be used to create flagging for laboratory use only (LUO) or Research use only (RUO). The purpose of this study is to investigate the possible use or utility of this new information for the screening/flagging of Alfa Thalassemia. Patient and Methods: We have collected 129 patients with Alfa Thalassemia Intermedia (HbH disease). All of them were confirmed by red cell morphology, Hgb Electroforesis, cromatography in liquid phase in human whole blood for the determination of Hemoglobin A2, F, A1c, and identification of abnormal hemoglobins and DNA analysis (DNA Analysis by GAP-PCR). We have compared these patients with a control group (184 individuals) and with other anemias (see Table 1). Results: Using ROC analysis, the best parameters differentiating the HbH Disease from the normals were: RDW (AUC 1. 000), @LHD(AUC 1. 000), @MAF(AUC 1. 000), @MCNRET (AUC 1. 000), MCV (AUC 0. 999), @MCRET (AUC 0. 999), @RSF (AUC 0. 998), HGB (AUC 0. 996), @MSCV (AUC 0. 995). Using ROC analysis, the best parameters differentiating the HbH Disease from other anemias (excluding normals) were: @LHD(AUC 0. 957), @MCNRET (AUC 0. 946), @MCRET (AUC 0. 902), @MAF(AUC 0. 873), MCV (AUC 0. 869). Using logistic regression we found a discrminant function that permits to differentiate/flag perfectly the patients with HbH disease from other anemias, and of course from normals: AUC 0. 996) Sensitivity: 91. 47% Specificity 94. 68% with a percent of cases correctly classified of: 93. 67 %. Disclosures: Simon-Lopez: Beckman Coulter: @LHD, @MAF, @RSF, @LHD, @MAF, @RSF Patents & Royalties, Employment. Di Gaetano:Instrumentation Laboratory spa: Work for a distributor of Beckman Coulter Instruments in Italy Other. Galanello:Novartis: Research Funding, Speakers Bureau; Apopharma: Research Funding, Speakers Bureau; Ferrokin: Research Funding.

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ChemInform Abstract: Amidinohydrazones as Guanidine Bioisosteres: Application to a New Class of Potent, Selective and Orally Bioavailable, Non-amide-based Small-Molecule Thrombin Inhibitors.

ChemInform ◽

10.1002/chin.200018172 ◽

2010 ◽

Vol 31 (18) ◽

pp. no-no

Author(s):

Richard M. Soll ◽

Tianobao Lu ◽

Bruce Tomczuk ◽

Carl R. Illig ◽

Cynthia Fedde ◽

...

Keyword(s):

Small Molecule ◽

Thrombin Inhibitors ◽

New Class ◽

Orally Bioavailable

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1053-1057.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1053-1057 ◽

Cited By ~ 10

Author(s):

Christian M. Apfel ◽

Stefan Evers ◽

Christian Hubschwerlen ◽

Wolfgang Pirson ◽

Malcolm G. P. Page ◽

...

Keyword(s):

Drug Target ◽

Peptide Deformylase ◽

Translation System ◽

Antibacterial Drug ◽

Bacterial Cells ◽

Methionine Aminopeptidase ◽

Intact Cells ◽

Physiological Conditions ◽

A Cell ◽

Antibacterial Drug Target

ABSTRACT 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 [35S]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.

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N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide Deformylase Inhibitors with Antibacterial Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.9.2752-2764.2002 ◽

2002 ◽

Vol 46 (9) ◽

pp. 2752-2764 ◽

Cited By ~ 78

Author(s):

Corinne J. Hackbarth ◽

Dawn Z. Chen ◽

Jason G. Lewis ◽

Kirk Clark ◽

James B. Mangold ◽

...

Keyword(s):

Antibacterial Activity ◽

Antimicrobial Agents ◽

Enzyme Inhibitor ◽

Structural Information ◽

Hydroxamic Acids ◽

Parallel Synthesis ◽

Peptide Deformylase ◽

New Class ◽

Relationship Analysis ◽

Starting Point

ABSTRACT Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P1′ site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P1′ site. Compounds with MICs of ≤4 μg/ml against gram-positive and gram-negative pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae, have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC50s) for Escherichia coli Ni-PDF were ≤0.1 μM, demonstrating the specificity of the inhibitors. In addition, these compounds were very selective for PDF, with IC50s of consistently >200 μM for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 Å. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

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