Essential Bacterial Enzyme Research Articles

Identification of Borinic Esters as Inhibitors of Bacterial Cell Growth and Bacterial Methyltransferases, CcrM and MenH

As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.

Gemifloxacin for the Treatment of Respiratory Tract Infections: In Vitro Susceptibility, Pharmacokinetics and Pharmacodynamics, Clinical Efficacy, and Safety

Gemifloxacin is a synthetic fluoroquinolone antimicrobial agent exhibiting potent activity against most gram-negative and gram-positive organisms, such as the important community-acquired respiratory pathogens Streptococcus pneumoniae (including multidrug-resistant S. pneumoniae), Haemophilus influenzae , and Moraxella catarrhalis . The agent's mechanism of action involves dual targeting of two essential bacterial enzymes: DNA gyrase and topoisomerase IV. Gemifloxacin was approved by the Food and Drug Administration in April 2003 for treatment of community-acquired pneumonia and acute bacterial exacerbation of chronic bronchitis. The drug has an oral bioavailability of approximately 71%. Approximately 20-35% of gemifloxacin is excreted unchanged in the urine after 24 hours. The elimination half-life of gemifloxacin is 6-8 hours in patients with normal renal function, supporting once-daily dosing. The 24-hour free-drug area under the plasma concentration-time curve:minimum inhibitory concentration ratio (fAUC(0-24):MIC) associated with efficacy, based on results from in vitro and animal models of infection, is approximately 30. With a mean fAUC(0-24) of approximately 3 microg*hour/ml (35% of total AUC(0-24) of 8.4) and a median S. pneumoniae MIC for 90% of tested strains of 0.03, a fAUC(0-24):MIC ratio of 100 would be expected after standard dosing (320 mg once/day). In clinical studies involving both hospitalized and outpatient populations, gemifloxacin has been highly effective in the treatment of community-acquired pneumonia and acute exacerbation of chronic bronchitis. Clinical success rates ranged from 93.9-95.9% in patients with community-acquired pneumonia and 96.1-97.5% in those with acute exacerbation of chronic bronchitis. Gemifloxacin is well tolerated; the frequency of adverse events with this agent is low. Most adverse events are mild-to-moderate in severity, with diarrhea (< 4%), nausea and rash (< 3%), and headache (< 2%) most commonly reported. Drug interactions with gemifloxacin are not common, although absorption is greatly reduced when given with divalent and trivalent cation-containing compounds, such as antacids. Due to its potent activity against many common gram-positive and gram-negative respiratory pathogens, its proven clinical efficacy, and its favorable safety profile, gemifloxacin is a highly effective empiric treatment for community-acquired lower respiratory tract infections.

The Crystal Structure of a Complex of Campylobacter jejuni dUTPase with Substrate Analogue Sheds Light on the Mechanism and Suggests the “Basic Module” for Dimeric d(C/U)TPases

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised. Members of this family have a novel all-alpha fold and are unrelated to the all-beta dUTPases of the majority of organisms including eukaryotes such as humans, bacteria such as Escherichia coli, archaea like Methanococcus jannaschii and animal viruses. Therefore, dimeric dUTPases can be considered as candidate drug targets. The X-ray structure of the C.jejuni dUTPase in complex with the non-hydrolysable substrate analogue dUpNHp allows us to define the positions of three catalytically significant phosphate-binding magnesium ions and provides a starting point for a detailed understanding of the mechanism of dUTP/dUDP hydrolysis by dimeric dUTPases. Indeed, a water molecule present in the structure is ideally situated to act as the attacking nucleophile during hydrolysis. A comparison of the dUTPases from C.jejuni and Trypanosoma cruzi reveals a common fold with certain distinct features, both in the rigid and mobile domains as defined in the T.cruzi structure. Homologues of the C.jejuni dUTPase have been identified in several other bacteria and bacteriophages, including the dCTPase of phage T4. Sequence comparisons of these proteins define a new superfamily of d(C/U)TPases that includes three distinct enzyme families: (1) dUTPases in trypanosomatides, C.jejuni and several other Gram-negative bacteria, (2) predicted dUTPases in various Gram-positive bacteria and their phages, and (3) dCTP/dUTPases in enterobacterial T4-like phages. All these enzymes share a basic module that consists of two alpha-helices from the rigid domain, two helices from the mobile domain and connecting loops. These results in concert with a number of conserved residues responsible for interdomain cross-talk provide valuable insight towards rational drug design.

Biochemical characterization of an inhibitor of Escherichia coli UDP- N-acetylmuramyl- l-alanine ligase

UDP- N-acetylmuramyl- l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC 50 of 2.3 μM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP- N-acetyl-muramic acid (UNAM) with a K i of 4.5 μM against ATP and 6.3 μM against UNAM. Fluorescence binding experiments yielded a K d of 3.1 μM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding.

Fluoroquinolones: structure and target sites.

The quinolones are a potent group of drugs that target the essential bacterial enzymes DNA gyrase and topoisomerase IV. DNA gyrase is the primary target of Gram negative organisms however, it is topoisomerase IV that is the primary target of Gram positive organisms. Within these enzymes is a highly conserved region centered round the active site where resistance mutations occur. These mutations are almost always identical, irrespective of organism. In spite of the homology of this region, amino acid sequence analysis shows that there are defined differences between the Gram groups, particularly in topoisomerase IV, and it is speculated that herein lies the origin of target preference. Since the first quinolone nalidixic acid was developed, the quinolones have undergone structural modifications, in particular the addition of a fluorine at position 6, to produce the fluoroquinolones. This has seen their potency and pharmakokinetic profile greatly increase. In vitro selection of resistance mutations has allowed the observation of how resistance is acquired and some of the modifications in newer fluoroquinolones have resulted in the shift of primary target from topoisomerase IV to gyrase with Gram positives. Curiously, purified topoisomerase IV is still more sensitive even if gyrase is the primary target. Gyrase remains the primary target for Gram negatives.

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

Identification, Substrate Specificity, and Inhibition of theStreptococcus pneumoniae β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH)

In the bacterial type II fatty acid synthase system, beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is approximately 41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K(m) values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 microm, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC(50) value of 0.016 microm. SB418011 also inhibited E. coli and H. influenzae FabH with IC(50) values of 1.2 and 0.59 microm, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.

Crystal Structures of Streptococcus pneumoniaeN-Acetylglucosamine-1-phosphate Uridyltransferase, GlmU, in Apo Form at 2.33 Å Resolution and in Complex with UDP-N-Acetylglucosamine and Mg2+ at 1.96 Å Resolution

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33Å resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg2+ at 1.96Å resolution. The protein structure consists of an N-terminal domain with an α/β-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed β-sheet helix (LβH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed β-sheet helix domain. In the crystal, S.pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed β-sheet helix domains. UDP-N-acetylglucosamine and Mg2+ are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg2+ and the side-chain of Lys22.

Identification and cloning of the Mycobacterium avium folA gene, required for dihydrofolate reductase activity

Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state. In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time. It has an open reading frame of 543 bp with a G+C content of 73%. The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases. Recombinant M. avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence.

Fleroxacin Overview

Fleroxacin is a new oral and intravenous trifluorinated 4-quinolone, which acts by inhibiting the essential bacterial enzyme DNA gyrase. Fleroxacin exhibits a broad spectrum of action, characterized by pronounced activity against aerobic gram-negative bacteria, but also against gram-positive pathogens such as staphylococci. Fleroxacin is distinguished by its excellent bioavailability, high concentrations in the plasma and other body fluids, good tissue penetration, and a long half-life of 10-12 h, thus allowing once-a-day administration. A single oral dose of 400 mg fleroxacin is effective in uncomplicated cystitis in women, uncomplicated gonococcal infections, bacterial enteritis, and traveler's diarrhea. A single daily dose of 200 mg administered for 3 days is effective in uncomplicated urinary tract infection (UTI), while longer treatment and higher doses may be required in acute uncomplicated pyelonephritis and complicated UTI. Skin, soft tissue, bone and joint infections, and lower respiratory tract infections including exacerbation of chronic bronchitis and non-pneumococcal pneumonia are further indications for fleroxacin.

Comparative Minimum Inhibitory Concentrations of Danofloxacin and Six Commonly Used Antibacterials Against Pasteurella and Haempophilus From Pneumonic Cattle

Fluoroquinolone antimicrobials exhibit inhibitory and bactericidal activity by blocking DNA gyrase (topoisomerase II), an essential bacterial enzyme.1 These compounds possess excellent antimicrobial activity against bacterial and mycoplasmal pathogens, however few in-vitro studies have been conducted to evaluate the activity of fluoroquinolone antimicrobials against respiratory pathogens of veterinary significance2,3,4 This report summarizes the results of an extensive survey to determine Minimum Inhibitory Concentrations (MIC) of danofloxacin*, a new fluoroquinolone antimicrobial, and six commonly used antibacterials against P. haemolytica, P. multocida, and H.somnus isolated from pneumonic cattle.

Prevention of Bacterial Resistance in Urinary Tract Infections

Recurrences of urinary tract infection (UTI) are frequent in many segments of the population. Most women with recurrent UTI have normal genitourinary tracts, and infection is thought to emanate from the fecal bacterial reservoir, with subsequent vaginal and periurethral colonization. The fluoroquinolones, e.g., norfloxacin, work by inhibiting the A-subunit of DNA gyrase, an essential bacterial enzyme. Plasmid-mediated resistance to the fluoroquinolones has not been reported to occur, but bacterial persistence, which is often an unstable form of resistance, may occur. Norfloxacin is able to decontaminate the anal area selectively and, while being administered, remove potential pathogens from the periurethral area for periods of up to 1 year. Additionally, preliminary data suggest that norfloxacin may prevent catheter-associated gram-negative bacilluria for an average of 17 days. Data concerning 1,130 infected patients treated with norfloxacin showed the development of resistance to be infrequent (1.7%).

DNA gyrase: structure and function.

DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.

Mode of Action of the Quinolone Antimicrobial Agents: Review of Recent Information

Recent studies concerning the mechanism of action of quinolones against DNA gyrase are reviewed. DNA gyrase is an essential bacterial enzyme known to be a primary target of quinolone agents. Quinolone-resistant alleles of both the gyrA and gyrB genes of DNA gyrase have been sequenced, and domains that affect the action of quinolones have been identified within the amino terminus of the gyrase A peptide and the midportion of the gyrase B peptide. In addition, an ATP-induced structural transition of DNA complexed with DNA gyrase was shown to be blocked by norfloxacin, but the means by which quinolones effect this change and the molecular site of quinolone binding remain unclear. Studies of structure-activity relationships of the quinolone molecule have been expanded and have included effects of quinolones on DNA gyrase. Stereochemical effects at positions 1 and 7 have been found. Substitutions at position 7 that improve potency against gram-positive bacteria have also been identified. Novel mono- and three-ring structures and an isothiazolo substitution at position 3 have broadened the range of structures known to have activity. Studies of bacterial killing by quinolones have revealed additional correlations with markers of DNA damage and additional alterations in bacteria and growth conditions that affect bacterial killing. The exact events responsible for quinolone-mediated lethality, however, remain undefined.

Mode of action of the quinolone antimicrobial agents.

The newer quinolone antimicrobial agents are more potent antibacterial agents than is the older analog nalidixic acid. For many of the quinolone agents, increased antibacterial potency correlates with increased potency in inhibiting the essential bacterial enzyme DNA gyrase. Studies with mutants resistant to drug action have identified the A subunit of this enzyme as a drug target. Other drug-resistance mutations are associated with pleiotropic drug resistance and decreased amounts of porin outer-membrane proteins and appear to involve alterations in permeability of the bacterium to the drug. Structure-activity studies of a large number of quinolone analogs have identified the importance to drug action of substituents at positions 1, 3, 4, 6, and 7 of the quinolone ring. Uncertainties remain, however, about the details of the molecular interactions of quinolones, DNA gyrase, and DNA and about the specific events that lead to quinolone-mediated bacterial killing.

Effects of novobiocin, coumermycin A1, clorobiocin, and their analogs on Escherichia coli DNA gyrase and bacterial growth.

Novobiocin, coumermycin A1, and clorobiocin, structurally related compounds that antagonize the B subunit of the essential bacterial enzyme DNA gyrase, were compared with 18 of their analogs for the inhibition of Escherichia coli DNA gyrase supertwisting activity in vitro and of bacterial multiplication. This family of compounds has a 4-hydroxy-8-methylcoumarin core substituted in the 7 and 3 positions. Important for enzyme inhibition in vitro is a 7 ether linkage to a 3'-substituted noviose sugar. The 3'-ester-linked 5-methylpyrrole, found in the coumermycin series, conferred at least 10-fold more inhibitory activity than did the similarly linked amide, found in the novobiocin series; lack of the pyrrole and amide results in the loss of inhibitory activity. Of many aryl and alkyl substituents linked as an amide at the 3 position, the 4-hydroxyl-3-(3-methyl-2-butenyl)benzoic acid moiety, found in novobiocin and clorobiocin, and the reduplication of the coumarin-noviose-5-methylpyrrole, found in coumermycin A1, were most effective in gyrase inhibition. In vivo, the ability of these compounds to inhibit the growth of E. coli varied greatly. The enhanced inhibition of gyrase in vitro conferred by a 5-methylpyrrole relative to an amide in the 3'-noviose position was reflected in inhibition of bacterial multiplication. Several substitutions at the 3 position of the coumarin core conferring similar antagonism of gyrase in vitro resulted in substantially different inhibitory activities for E. coli, suggesting that these moieties at the 3 position affect drug access to the intracellular target. This target was shown for isobutyryl PNC-NH2 (PNC-NH2 is 3-amino-4-hydroxy-8-methyl-7-[3-O-(5-methyl-2-pyrrolylcarbonyl)noviosyloxy] coumarin) and confirmed for novobiocin, coumermycin A1, and clorobiocin to be in the B subunit of DNA gyrase.