Biomedical subjects

Malcolm G P Page

Publications and source records attributed to Malcolm G P Page.

12 recordsLinked to original sources

PubMed2006

Anti-MRSA beta-lactams in development.

Ceftobiprole medocaril, the most advanced of the anti-MRSA (methicillin-resistant Staphylococcus aureus) beta-lactams in clinical development, has recently completed its first Phase III clinical trial, and has demonstrated non-inferiority to vancomycin. Phase II clinical trials have been initiated with PPI0903, which is, like ceftobiprole medocaril, an injectable pro-drug of a broad-spectrum cephalosporin with anti-MRSA activity, and with RO4908643, a carbapenem with more modest activity against MRSA.

Anti-Bacterial Agents↗

PubMed2006

Discovering new classes of Brugia malayi asparaginyl-tRNA synthetase inhibitors and relating specificity to conformational change.

SLIDE software, which models the flexibility of protein and ligand side chains while docking, was used to screen several large databases to identify inhibitors of Brugia malayi asparaginyl-tRNA synthetase (AsnRS), a target for anti-parasitic drug design. Seven classes of compounds identified by SLIDE were confirmed as micromolar inhibitors of the enzyme. Analogs of one of these classes of inhibitors, the long side-chain variolins, cannot bind to the adenosyl pocket of the closed conformation of AsnRS due to steric clashes, though the short side-chain variolins identified by SLIDE apparently bind isosterically with adenosine. We hypothesized that an open conformation of the motif 2 loop also permits the long side-chain variolins to bind in the adenosine pocket and that their selectivity for Brugia relative to human AsnRS can be explained by differences in the sequence and conformation of this loop. Loop flexibility sampling using Rigidity Optimized Conformational Kinetics (ROCK) confirms this possibility, while scoring of the relative affinities of the different ligands by SLIDE correlates well with the compounds' ranks in inhibition assays. Combining ROCK and SLIDE provides a promising approach for exploiting conformational flexibility in structure-based screening and design of species selective inhibitors.

Animals↗

PubMed2006

Global transcriptome analysis of the responses of a fluoroquinolone-resistant Streptococcus pneumoniae mutant and its parent to ciprofloxacin.

Streptococcus pneumoniae M22 is a multidrug-resistant mutant selected after exposure of capsulated wild-type S. pneumoniae NCTC 7465 (strain M4) to ciprofloxacin. DNA microarray analysis comparing the gene expression profiles of strain M22 with those of strain M4 showed that strain M22 constitutively expressed 22 genes at levels higher than those observed in strain M4 under all conditions studied. These included the genes encoding the enzymes involved in branched-chain amino acid biosynthesis and two genes (patA and patB) with sequences suggestive of ABC transporter proteins. Expression of the patA and patB genes was induced by ciprofloxacin in both strains, but in strain M4 it only reached the levels observed in strain M22 after long incubation with high concentrations of ciprofloxacin. The altered expression profile observed with strain M22 suggested that the mutation or mutations acquired during resistance selection bring the cell into a state in which the expression of critical genes is preemptively altered to correct for the potential effects of ciprofloxacin on gene expression in the parent strain.

Ciprofloxacin↗

PubMed2006

Involvement of the putative ATP-dependent efflux proteins PatA and PatB in fluoroquinolone resistance of a multidrug-resistant mutant of Streptococcus pneumoniae.

The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux proteins belonging to the ABC transporter family. It is shown here that PatA and PatB were strongly induced by quinolone antibiotics and distamycin in fluoroquinolone-sensitive strains. PatA was very important for growth of S. pneumoniae, and it could not be disrupted in strain M22. PatB appeared to control metabolic activity, particularly in amino acid biosynthesis, and it may have a pivotal role in coordination of the response to quinolone antibiotics. The induction of PatA and PatB by antibiotics showed a pattern similar to that exhibited by SP1861, a homologue of ABC-type transporters of choline and other osmoprotectants. A second group of quinolone-induced transporter genes comprising SP1587 and SP0287, which are homologues of, respectively, oxalate/formate antiporters and xanthine or uracil permeases belonging to the major facilitator family, showed a different pattern of induction by other antibiotics. There was no evidence for the involvement of PmrA, the putative proton-dependent multidrug transporter that has been implicated in norfloxacin resistance, in the response to quinolone antibiotics in either the resistant mutant or the fluoroquinolone-sensitive strains.

ATP-Binding Cassette Transporters↗

PubMed2005

Structure-activity relationships of different beta-lactam antibiotics against a soluble form of Enterococcus faecium PBP5, a type II bacterial transpeptidase.

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. beta-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel beta-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different beta-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Delta1-36 rPBP5). The soluble protein, Delta1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH(2)-terminal protein sequence was verified by amino acid sequencing. Using beta-lactams with different R1 side chains, we show that azlocillin has greater affinity for Delta1-36 rPBP5 than piperacillin and ampicillin (apparent K(i) = 7 +/- 0.3 microM, compared to 36 +/- 3 and 51 +/- 10 microM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k(2) = 15 +/- 4 M(-1) s(-1)). Meropenem demonstrates an affinity for Delta1-36 rPBP5 comparable to that of ampicillin (apparent K(i) = 51 +/- 15 microM) but is slower at acylating (apparent k(2) = 0.14 +/- 0.02 M(-1) s(-1)). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a beta-lactam, and suggests that the specific side chain interactions of beta-lactams could be modified to improve inactivation of resistant PBPs.

Acylation↗

PubMed2004

Crystallographic and biophysical analysis of a bacterial signal peptidase in complex with a lipopeptide-based inhibitor.

We report here the crystallographic and biophysical analysis of a soluble, catalytically active fragment of the Escherichia coli type I signal peptidase (SPase Delta2-75) in complex with arylomycin A2. The 2.5-A resolution structure revealed that the inhibitor is positioned with its COOH-terminal carboxylate oxygen (O45) within hydrogen bonding distance of all the functional groups in the catalytic center of the enzyme (Ser90 O-gamma, Lys145 N-zeta, and Ser88 O-gamma) and that it makes beta-sheet type interactions with the beta-strands that line each side of the binding site. Ligand binding studies, calorimetry, fluorescence spectroscopy, and stopped-flow kinetics were also used to analyze the binding mode of this unique non-covalently bound inhibitor. The crystal structure was solved in the space group P4(3)2(1)2. A detailed comparison is made to the previously published acyl-enzyme inhibitor complex structure (space group: P2(1)2(1)2) and the apo-enzyme structure (space group: P4(1)2(1)2). Together this work provides insights into the binding of pre-protein substrates to signal peptidase and will prove helpful in the development of novel antibiotics.

Bacterial Outer Membrane Proteins↗

PubMed2004

Essential role of histidine 20 in the catalytic mechanism of Escherichia coli peptidyl-tRNA hydrolase.

The peptidyl-tRNA hydrolase (Pth) enzyme plays an essential role in recycling tRNA from peptidyl-tRNA that has prematurely dissociated from the ribosome. In this study of Escherichia coli Pth, the critical role of histidine 20 was investigated by site-directed mutagenesis, stopped-flow kinetic measurements, and chemical modification. The histidine residue at position 20 is known to play an important role in the hydrolysis reaction, but stopped-flow fluorescence measurements showed that, although the His20Asn Pth mutant enzyme was unable to hydrolyze the substrate, the enzyme retained the ability to bind peptidyl-tRNA. Chemical modification of Pth with diethyl pyrocarbonate (DEPC) showed that a residue, with a pK(a) value of 6.3, was essential for substrate hydrolysis and that the stoichiometry of inhibition was 0.70 +/- 0.06 mol of DEPC/mol of enzyme, indicating that modification of only a single residue by DEPC was responsible for the loss of activity. Parallel chemical modification studies with the His20Asn and Asp93Asn mutant enzymes showed that this essential residue was His20. These studies indicate that histidine 20 acts as the catalytic base in the hydrolysis of peptidyl-tRNA by Pth.

Asparagine↗

PubMed2004

Impact of specific pbp5 mutations on expression of beta-lactam resistance in Enterococcus faecium.

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.

Ampicillin Resistance↗

PubMed2004

Cephalosporins in clinical development.

Bacterial resistance to established classes of antibiotics in clinical use is continuing to increase, making the need for new agents that can be used to treat the newly multi-drug resistant organisms steadily more urgent. Cephalosporins have been a successful group of antibiotics since they were first introduced to combat drug-resistant organisms, including staphylococci. The history of cephalosporins has emphasised an improvement of their stability towards beta-lactamases, thus expanding their spectrum of activity against important Gram-negative pathogens. New cephalosporins that have potent activity against multi-resistant Gram-positive bacteria, including methicillin-resistant staphylococci and penicillin-resistant pneumococci have recently entered clinical development. At least two of these, BAL-5788 and S-3578, also have Gram-negative activity, which is comparable to that of the third-and fourth-generation cephalosporins, making them broad-spectrum agents that could be used in hospital infections where methicillin-resistant staphylococci is likely to be present.

Anti-Bacterial Agents↗

PubMed2003

A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii beta-lactamase revealed by infrared spectroscopy and molecular dynamics simulations.

Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions. The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.

Acylation↗

PubMed2002

Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay.

Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly used fluorophores--fluorescein, DDAO and resorufin--are evaluated. Experimental evidence is given that S9 liver homogenate contains a heat-labile, reversible fluorophore-binding activity and therefore, significantly reduces fluorescence intensities. We have worked out simple solutions to overcome the S9 related interference in order to be able to establish a robust bacterial genotoxicity assay.

Bacteria↗

PubMed2002

Development of a sensitive and specific enzyme-linked immunosorbent assay for detecting and quantifying CMY-2 and SHV beta-lactamases.

Polyclonal rabbit antibodies against SHV-1 and CMY-2 beta-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC beta-lactamase tested. The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC beta-lactamases of Enterobacter aerogenes, Morganella morganii, and Citrobacter freundii. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of E. coli possessing TEM-1, SHV-1, K-1, or OXA-1 beta-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC beta-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC beta-lactamases of M. morganii, C. freundii, E. coli, and E. cloacae). As the homology to CMY-2 beta-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV beta-lactamases, and the results were confirmed with PCR amplification of bla(AmpC) and bla(SHV) genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC beta-lactamases. The ELISA format can facilitate the identification of AmpC and SHV beta-lactamases and can be used to quantify relative amounts of beta-lactamase enzymes in clinical and laboratory isolates.

Antibodies, Bacterial↗