Structure-activity relationships of actively FhuE transported rifabutin derivatives with potent activity against Acinetobacter baumannii.

Hospital-acquired infections are on the rise and represent both, a clinical and financial burden. With resistance emerging and an ever-dwindling armamentarium at hand, infections caused by Acinetobacter baumannii are particularly problematic, since these bacteria have a high level of resistance and resilience to traditional and even last-resort antibiotics. The antibiotic rifabutin was recently found to show potent in vitro and in vivo activity against extensively drug resistant A. baumannii. Building on this discovery, we report on the synthesis and activity of rifabutin analogs, with a focus on N-functionalization of the piperidine ring. The antimicrobial testing uncovered structure activity relationships (SAR) for A. baumannii that were not reflected in Staphylococcus aureus. The cellular activity did not correlate with cell-free transcription inhibition, but with bacterial intracellular compound accumulation. Mass spectrometry-based accumulation studies confirmed the involvement of the siderophore receptor FhuE in active compound translocation at low concentrations, and they showed a strong impact of the culture medium on the accumulation of rifabutin. Overall, the study underlines the structural feature required for strong accumulation of rifabutin in A. baumannii and identifies analogs as or more potent than rifabutin against A. baumannii.

Pharmacokinetic-Pharmacodynamic Evaluation of Ertapenem for Patients with Hospital-Acquired or Ventilator-Associated Bacterial Pneumonia.

Ertapenem provides activity against many pathogens commonly associated with hospital-acquired and ventilator-associated bacterial pneumoniae (HABP and VABP, respectively), including methicillin-susceptible Staphylococcus aureus and numerous Gram-negative pathogens with one major gap in coverage, Pseudomonas aeruginosa Pharmacokinetic-pharmacodynamic (PK-PD) target attainment analyses were conducted to evaluate ertapenem against the most prevalent Enterobacteriaceae causing HABP/VABP. The objective of these analyses was to provide dose selection support for and demonstrate the appropriateness of ertapenem to empirically treat patients with HABP/VABP when administered with murepavadin, a novel targeted antimicrobial exhibiting a highly specific spectrum of activity against P. aeruginosa A previously developed population pharmacokinetic model, a total-drug epithelial lining fluid (ELF) to free-drug serum penetration ratio, contemporary in vitro surveillance data for ertapenem against Enterobacteriaceae, and percentage of the dosing interval for which drug concentrations exceed the MIC value (%T>MIC) targets associated with efficacy were used to conduct Monte Carlo simulations for five ertapenem regimens administered over short or prolonged durations of infusion. Overall total-drug ELF percent probabilities of PK-PD target attainment based on a %T>MIC target of 35% among simulated patients with HABP/VABP arising from Enterobacteriaceae based on pathogen prevalence data for nosocomial pneumonia ranged from 89.1 to 92.7% for all five ertapenem regimens evaluated. Total-drug ELF percent probabilities of PK-PD target attainment ranged from 99.8 to 100%, 97.9 to 100%, 10.6 to 74.1%, and 0 to 1.50% at MIC values of 0.06, 0.12, 1, and 4 µg/ml, respectively (MIC90 values for Escherichia coli, Serratia marcescens, Enterobacter species, and Klebsiella pneumoniae, respectively). Results of these analyses provide support for the evaluation of ertapenem in combination with murepavadin for the treatment of patients with HABP/VABP.

Pharmacokinetics and Pharmacodynamics of Murepavadin in Neutropenic Mouse Models.

Murepavadin (POL7080) represents the first member of a novel class of outer membrane protein-targeting antibiotics. It specifically interacts with LptD and inhibits lipopolysaccharide (LPS) transport. Murepavadin is being developed for the treatment of serious infections by Pseudomonas aeruginosa We determined the plasma protein binding and the pharmacokinetics of murepavadin in plasma and epithelial lining fluid (ELF; pulmonary) in infected animals, and we determined the exposure-response relationship. Treatment of CD-1 neutropenic mice was started 2 h after infection using murepavadin at different dosing frequencies for 24 h, and the number of CFU per lung was determined. The sigmoid maximum-effect model was used to fit the dose-response, and the pharmacodynamic index (PDI) response was used to determine the PDI values, resulting in a static effect and 1-log kill reduction. Using R2 as an indicator of the best fit, the area under the concentration-time curve for the unbound fraction of the drug (fAUC)/MIC ratio correlated best with efficacy. The mean AUC required to provide a static effect was 36.83 mg h/liter (fAUC = 8.25 mg h/liter), and that to provide a 1-log reduction was 44.0 mg h/liter (fAUC = 9.86 mg h/liter). The mean static fAUC/MIC was determined to be 27.78, and that for a 1-log reduction was 39.85. These data may serve to determine doses in humans that are likely to be efficacious.

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif.

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.

A three-dimensional model of endothelin-converting enzyme (ECE) based on the X-ray structure of neutral endopeptidase 24.11 (NEP).

Endothelin-converting enzyme 1 (ECE-1, EC 3.4.24.71) is a zinc-dependent type II mammalian membrane protein comprising the active site in the ectodomain. It exists in multiple splice variants that all catalyze the last and rate-limiting step in the activation of preproendothelin to the highly potent vasoconstrictor endothelin. There is high interest in finding small and potent inhibitors for this enzyme that could be used in numerous indications, e.g. hypertension. Since there is no structural information available for this important enzyme, we built a model of the complete ectodomain using the recently solved structure of human NEP as template. The naturally derived metalloproteinase inhibitor phosphoramidon was docked in the active site of this model and comparisons with the respective NEP complex were made.

Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis.

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.

Roles of aconitase in growth, metabolism, and morphological differentiation of Streptomyces coelicolor.

The studies of aconitase presented here, along with those of citrate synthase (P. H. Viollier, W. Minas, G. E. Dale, M. Folcher, and C. J. Thompson, J. Bacteriol. 183:3184-3192, 2001), were undertaken to investigate the role of the tricarboxylic acid (TCA) cycle in Streptomyces coelicolor development. A single aconitase activity (AcoA) was detected in protein extracts of cultures during column purification. The deduced amino acid sequence of the cloned acoA gene constituted the N-terminal sequence of semipurified AcoA and was homologous to bacterial A-type aconitases and bifunctional eukaryotic aconitases (iron regulatory proteins). The fact that an acoA disruption mutant (BZ4) did not grow on minimal glucose media in the absence of glutamate confirmed that this gene encoded the primary vegetative aconitase catalyzing flux through the TCA cycle. On glucose-based complete medium, BZ4 had defects in growth, antibiotic biosynthesis, and aerial hypha formation, partially due to medium acidification and accumulation of citrate. The inhibitory effects of acids and citrate on BZ4 were partly suppressed by buffer or by introducing a citrate synthase mutation. However, the fact that growth of an acoA citA mutant remained impaired, even on a nonacidogenic carbon source, suggested alternative functions of AcoA. Immunoblots revealed that AcoA was present primarily during substrate mycelial growth on solid medium. Transcription of acoA was limited to the early growth phase in liquid cultures from a start site mapped in vitro and in vivo.

Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris.

Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP-phosphoramidon binary complex and the apo enzyme.

A vibrational structure of 7,8-dihydrobiopterin bound to dihydroneopterin aldolase.

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7, 8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and glycolaldehyde. An inhibitor of the enzyme, 7,8-dihydrobiopterin, free in solution and bound in its complex with the enzyme has been studied by Raman difference spectroscopy. By using isotopically labeled 7,8-dihydrobiopterin and normal mode analyses based on ab initio quantum mechanic methods, we have positively identified some of the Raman bands in the enzyme-bound inhibitor, particularly the important N5=C6 stretch mode. The spectrum of the enzyme-bound inhibitor shows that the pK(a) of N5 is not significantly increased in the complex. This result suggests that N5 of 7,8-dihydroneopterin is not protonated before the bond cleavage of 7,8-dihydroneopterin during the DHNA-catalyzed reaction as has been suggested. Our results also show that the N5=C6 stretch mode of 7, 8-dihydrobiopterin shifts 19 cm(-)(1) upon binding to DHNA. Various possibilities on how the enzyme can bring about such large frequency change of the N5=C6 stretch mode are discussed.

Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon.

Neutral endopeptidase is a mammalian type II integral membrane zinc-containing endopeptidase, which degrades and inactivates a number of bioactive peptides. The range of substrates cleaved by neutral endopeptidase in vitro includes the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor. Due to the physiological importance of neutral endopeptidase in the modulation of nociceptive and pressor responses there is considerable interest in inhibitors of this enzyme as novel analgesics and anti-hypertensive agents. Here we describe the crystal structure of the extracellular domain (residues 52-749) of human NEP complexed with the generic metalloproteinase inhibitor phosphoramidon at 2.1 A resolution. The structure reveals two multiply connected folding domains which embrace a large central cavity containing the active site. The inhibitor is bound to one side of this cavity and its binding mode provides a detailed understanding of the ligand-binding and specificity determinants.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus.

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow single well defined crystals in the microbatch system which belonged to the space group C2 and diffracted isotropically to approximately 2 A resolution.

The structure and function of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Haemophilus influenzae.

The gene encoding the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase of Haemophilus influenzae has been cloned and expressed in Escherichia coli. A complex of the purified protein with a substrate analog has been crystallized and its structure solved by multiple anomalous dispersion using phase information obtained from a single crystal of selenomethione-labeled protein. The enzyme folds into a four-stranded antiparallel beta-sheet flanked on one side by two alpha-helices and on the other by three consecutive alpha-helices, giving a novel beta1alpha1beta2beta3alpha2beta4alpha3alpha4alpha5 polypeptide topology. The three-dimensional structure of a binary complex has been refined at 2.1 A resolution. The location of the substrate analog and a sulfate ion gives important insight into the molecular mechanism of the enzyme.

Crystal structure and reaction mechanism of 7,8-dihydroneopterin aldolase from Staphylococcus aureus.

Dihydroneopterin aldolase catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin during the de novo synthesis of folic acid from guanosine triphosphate. The gene encoding the dihydroneopterin aldolase from S. aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and its X-ray structure determined at 1.65 A resolution. The protein forms an octamer of 110,000 Mr molecular weight. Four molecules assemble into a ring, and two rings come together to give a cylinder with a hole of at least 13 A diameter. The structure of the binary complex with the product 6-hydroxymethyl-7,8-dihydropterin has defined the location of the active site. The structural information and results of site directed mutagenesis allow an enzyme reaction mechanism to be proposed.

Structure and function of the dihydropteroate synthase from Staphylococcus aureus.

The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of "TIM-barrel" proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmpr DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmpr staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 or 33 isolates highly resistant to Tmp (MICs, > or = 512 micrograms/ml) contained dfrA sequences, whereas among the Tmpr (MICs, < or = 256 micrograms/ml) and Tmps isolates only the Staphylococcus epidermidis isolates (both Tmpr and Tmps) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmpr DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 +/- 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.

Increased solubility of trimethoprim-resistant type S1 DHFR from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES.

The production of the trimethoprim-resistant type S1 dihydrofolate reductase (DHFR) from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES is described. The simultaneous overproduction of the chaperonins with DHFR results in an increased solubility of the enzyme. We compare the time course of production of active type S1 DHFR by measuring enzyme activity in cells overproducing or not overproducing the chaperonins. Although co-overproduction of the chaperonins reduces the total production level of type S1 DHFR, the amount of soluble and active DHFR is increased several-fold in comparison with cells producing only DHFR. Thus, the higher concentrations of GroES and GroEL in cells overproducing the chaperonins partially protect DHFR from aggregation, resulting in higher concentrations of soluble and active DHFR in the cell. Furthermore, we also demonstrate that the chaperonins can improve in vitro refolding yields of type S1 DHFR. These results suggest that it is possible to purify suitable amounts of trimethoprim-resistant type S1 DHFR for X-ray crystallographic studies.

Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread and several Tmp-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from Gram-negative bacteria. In staphylococci, however, only one Tmpr DHFR (type S1 DHFR) has been found so far, and this is located on transposon Tn4003. To help understand the mechanism of resistance, we are interested in determining the 3-D structure of the recombinant enzyme produced in Escherichia coli. However, the production level of the type S1 DHFR was very low and > 95% of the total recombinant protein accumulated in inclusion bodies. Furthermore, as a result of an internal start of translation, a truncated derivative of the enzyme that copurified with the full-length enzyme was produced. We were able to increase the expression level 20-fold by changing 18 N-terminal codons and to eliminate the internal start of translation. In addition, through molecular modelling and subsequent site-directed mutagenesis to replace two amino acids, we constructed a biochemically similar but soluble derivative of the type S1 DHFR that, after production in E.coli, resulted in a 264-fold increase in DHFR activity. The highly overproduced enzyme was purified to homogeneity, characterized biochemically and crystallized.

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly overexpressed in Escherichia coli, with most of the recombinant protein occurring in a soluble and an active form.