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Nature Communications Nov 2022Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding...
Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from Stenotrophomonas maltophilia. Using time-resolved serial synchrotron crystallography, we show the time course of β-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs.
Topics: beta-Lactams; Moxalactam; beta-Lactamases; Crystallography, X-Ray
PubMed: 36450742
DOI: 10.1038/s41467-022-35029-3 -
The Western Journal of Medicine Mar 1984These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from transcriptions, they...
These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from transcriptions, they are prepared by Drs Homer A. Boushey, Associate Professor of Medicine, and David G. Warnock, Associate Professor of Medicine, under the direction of Dr Lloyd H. Smith, Jr, Professor of Medicine and Chairman of the Department of Medicine. Requests for reprints should be sent to the Department of Medicine, University of California, San Francisco, School of Medicine, San Francisco, CA 94143.
Topics: Bacterial Infections; Cephalosporins; Humans; Meningitis; Moxalactam; Penicillins
PubMed: 6710984
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy Mar 2023In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during...
In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during β-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all -containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population . Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII -containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the gene were detected. A single copy of was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of ( and ) were presented in pJCL-3, and three copies of were presented in pJCL-4. The -harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure . Experimental evolution assays revealed that multicopy-containing cells were increased in the original single-copy -harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by β-lactam antibiotics other than ceftazidime-avibactam. Notably, gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.
Topics: Humans; Ceftazidime; Klebsiella pneumoniae; Meropenem; Klebsiella; Moxalactam; Klebsiella Infections; Anti-Bacterial Agents; Azabicyclo Compounds; beta-Lactamases; Bacterial Proteins; Drug Combinations; Microbial Sensitivity Tests; Cefiderocol
PubMed: 36794957
DOI: 10.1128/aac.01279-22 -
Antimicrobial Agents and Chemotherapy Dec 2016BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs...
BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower K values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.
Topics: Anti-Bacterial Agents; Catalytic Domain; Citric Acid; Crystallography, X-Ray; Disulfides; Imipenem; Moxalactam; beta-Lactamases
PubMed: 27671060
DOI: 10.1128/AAC.00936-16 -
Antimicrobial Agents and Chemotherapy Sep 1980The pharmacokinetics of cefotaxime and moxalactam were compared in six healthy male volunteers aftr the administration of 1-g doses intravenously. Penetration of the... (Comparative Study)
Comparative Study
The pharmacokinetics of cefotaxime and moxalactam were compared in six healthy male volunteers aftr the administration of 1-g doses intravenously. Penetration of the compounds into tissue fluid was studied in cantharides-induced blisters. Both serum and tissue fluid levels of moxalactam were higher than those of cefotaxime. The elimination half-life of cefotaxime was 1.2 h, and that of moxalactam was 2.85 h. On the average, 50.5% of cefotaxime and 87.5% of moxalactam were recovered in the urine in 24 h.
Topics: Adult; Blister; Body Fluids; Cefotaxime; Cephalosporins; Cephamycins; Escherichia coli; Humans; Kinetics; Male; Moxalactam
PubMed: 6252833
DOI: 10.1128/AAC.18.3.369 -
Antimicrobial Agents and Chemotherapy Jul 1982We measured the serum concentrations of moxalactam in 10 children receiving antibiotic prophylaxis for surgery and in 18 children treated for documented or suspected...
We measured the serum concentrations of moxalactam in 10 children receiving antibiotic prophylaxis for surgery and in 18 children treated for documented or suspected infections. Moxalactam was administered intravenously at a dose of 50 mg/kg every 8 h. After the first dose in 28 patients, mean moxalactam concentrations 5 min, 30 min, 2 h, and 8 h after infusion were 257, 177, 82.2, and 17.5 micrograms/ml, respectively. The mean half-life (T 1/2) was 2.44 h (range: 0.55 to 7.96 h). The mean distribution volume (VD) was 0.30 liters per kg. No significant accumulation was observed with multiple doses. Prophylactic and therapeutic groups had similar serum levels, T 1/2, and VD. The five infants less than 1 year of age had a lower mean 30-min level (P less than 0.01), larger VD (P less than 0.001) and longer T 1/2 (P less than 0.025) than the children older than 1 year. A dose of 50 mg/kg produced 30-min levels in excess of 64 microgram/ml in all patients studied, but 8-h trough levels were below the minimal inhibitory concentrations breakpoint of 17 mg/ml in 32% of patients.
Topics: Adolescent; Bacterial Infections; Cephalosporins; Cephamycins; Child; Child, Preschool; Humans; Infant; Infant, Newborn; Kinetics; Microbial Sensitivity Tests; Moxalactam; Time Factors
PubMed: 6214997
DOI: 10.1128/AAC.22.1.47 -
International Journal of Environmental... Oct 2019Cephalosporins that contain the N-methylthiotetrazole side chain (NMTT-cephalosporin) have been reported to be associated with coagulation-related adverse events;... (Meta-Analysis)
Meta-Analysis
Cephalosporins that contain the N-methylthiotetrazole side chain (NMTT-cephalosporin) have been reported to be associated with coagulation-related adverse events; however, a comprehensive evaluation regarding the association is lacking. A systematic review and meta-analysis were conducted to assess the safety profile of NMTT-cephalosporins with respect to hypoprothrombinemia and bleeding. The MEDLINE, Embase, Cochrane, and RISS databases were systematically searched for clinical studies up to October 2018. The association between NMTT-cephalosporins and hypoprothrombinemia was estimated using an odds ratio (OR) with a 95% confidence interval (CI). A total of 15 studies on cefamandole, cefoperazone, cefotetan, cefmetazole, and moxalactam were identified and included in the meta-analysis. Hypoprothrombinemia (OR 1.676, 95% CI 1.275-2.203) and prothrombin time (PT) prolongation (OR 2.050, 95% CI 1.398-3.005) were significantly associated with NMTT-cephalosporins, whereas bleeding was not (OR 1.359, 95% CI 0.920-2.009). Subgroup analyses revealed that cefoperazone (OR 2.506, 95% CI 1.293-4.860), cefamandole (OR 3.247, 95% CI 1.083-9.733), and moxalactam (OR 3.367, 95% CI 1.725-6.572) were significantly associated with hypoprothrombinemia. An Antimicrobial Stewardship Program led by a multidisciplinary team could play a critical role in monitoring cephalosporin-related hypoprothrombinemia or PT prolongation in patients with underlying clinical conditions at risk for bleeding. The multidisciplinary team could also assist in communicating the potential safety concerns regarding NMTT-cephalosporin use with healthcare professionals to decrease the risk of adverse events.
Topics: Anti-Bacterial Agents; Cephalosporins; Humans; Hypoprothrombinemias; Male
PubMed: 31623191
DOI: 10.3390/ijerph16203937 -
Antimicrobial Agents and Chemotherapy Apr 1981Moxalactam, a new beta-lactam antibiotic with a wide in vitro spectrum of activity, was compared with cefazolin after intravenous and intramuscular administration of 1.0... (Clinical Trial)
Clinical Trial
Moxalactam, a new beta-lactam antibiotic with a wide in vitro spectrum of activity, was compared with cefazolin after intravenous and intramuscular administration of 1.0 g in a double-blind crossover design in 21 adult male subjects with normal renal function. Serum samples were obtained at 0.5, 1, 2, 3, 4, 6, 8, and 12 h, and urine was collected at 0 to 2, 2 to 4, 4 to 6, 6 to 8, and 8 to 12 h after dosing. Intravenous kinetics were described by a linear two-compartment model. For moxalactam, the drug clearance and volume of distribution were larger (115.2 versus 75.9 ml/min per 70 kg, P = 0.001, and 0.44 versus 0.19 liter/kg, P less than 0.001, respectively), and the t1/2beta was longer (3.47 versus 2.18 h, P = 0.01), with correspondingly smaller area under the curve (151 versus 236 h x mg/ml, P = 0.003) and lower serum concentration at 30 min (62 versus 106 micrograms/ml, P = 0.003) than cefazolin. Intramuscular kinetics were similar and were well described by a single-compartment model. Urinary recovery was essentially identical for both drugs: 55 to 75% in 8 h. Consistent departures from the two-compartment model for moxalactam (not noted for cefazolin) suggested enterohepatic recirculation of moxalactam. Both drugs were well tolerated, and no adverse reactions were noted.
Topics: Adult; Cefazolin; Cephalosporins; Cephamycins; Humans; Injections, Intramuscular; Injections, Intravenous; Kinetics; Male; Moxalactam
PubMed: 6454387
DOI: 10.1128/AAC.19.4.613 -
Protein Science : a Publication of the... Mar 2020Emergence of Enterobacteriaceae harboring metallo-β-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of...
Structural and biochemical analysis of the metallo-β-lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di-metal scaffold for catalytic activity.
Emergence of Enterobacteriaceae harboring metallo-β-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a. We determined several crystal structures of L1 complexes with three different classes of β-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn , Cd , and Cu ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn ions, however, the protein does not bind the substrate moxalactam. When two Zn ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-β-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. STATEMENT: The L1 metallo-β-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several β-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.
Topics: Anti-Bacterial Agents; Binding Sites; Biocatalysis; Lactams; Metals, Heavy; Microbial Sensitivity Tests; Models, Molecular; Stenotrophomonas maltophilia; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 31846104
DOI: 10.1002/pro.3804 -
Journal of Medicinal Chemistry Nov 2019Small-molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are...
Small-molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are more susceptible to inhibition than others. A possible reason for this apparent selectivity is that aggregation-based inhibition, as a stoichiometric process, is sensitive to protein concentration, which varies across assays. Alternatively, local protein unfolding by aggregates may lead to selectivity since stability varies among proteins. To deconvolute these effects, we used differentially stable point mutants of a single protein, TEM-1 β-lactamase. Broadly, destabilized mutants had higher affinities for and were more potently inhibited by aggregates versus more stable variants. The addition of the irreversible inhibitor moxalactam destabilized several mutants, and these typically bound tighter to a colloidal particle, while the only mutant it stabilized bound weaker. These results suggest that less-stable enzymes are more easily sequestered and inhibited by colloidal aggregates.
Topics: Enzyme Inhibitors; Mutation; Protein Stability; Thermodynamics; beta-Lactamases
PubMed: 31589047
DOI: 10.1021/acs.jmedchem.9b01019