RESUMO
The main objective of this work is to synthesize multifunctional nanodendritic structural molecules that can effectively encapsulate hydrophilic as well as hydrophobic therapeutic agents. Four different types of fourth-generation lysine-citric acid based dendrimer have been synthesized in this work: PE-MC-Lys-CA-PEG, TMP-MC-Lys-CA-PEG, PE-MS-Lys-CA-PEG, and TMP-MS-Lys-CA-PEG. The antibacterial drug cefotaxime (CFTX) was further conjugated to these dendrimers. The dendrimer and drug-dendrimer conjugate structures were characterized with the help of FTIR,1H-NMR, and 13C-NMR spectroscopy. Zeta sizer, AFM, and HR-TEM techniques were used to investigate the particle size, surface topography, and structural characteristics of drug-dendrimer conjugates. In vitro drug release was then investigated using dialysis method. Various kinetic drug release models were examined to evaluate the type of kinetic drug release mechanism of the formulations. Cytotoxicity study revealed that the dendrimers encapsulated with CFTX exhibited 2-3% toxicity against healthy epithelial cells, indicating their safe use. Plain dendrimers show 10-15% hemolytic toxicity against red blood cells (RBC), and the toxicity was reduced to 2-3% when CFTX was conjugated to the same dendrimers. The 3rd and 4th generation synthesized drug-dendrimer conjugates exhibit a significantly effective zone of inhibition (ZOI) against both Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, the lower concentration of 0.1 mg/mL showed more than 98% inhibition of drug-dendrimer conjugate samples against B. subtilis and more than 50% inhibition against S. aureus using 0.2 mg/mL, respectively. Moreover, samples with concentrations of 0.5 and 1.0 mg/mL exhibited more than 50% inhibition against S. typhimurium and E. coli, respectively.
Assuntos
Antibacterianos , Ácido Cítrico , Dendrímeros , Portadores de Fármacos , Liberação Controlada de Fármacos , Hemólise , Lisina , Polietilenoglicóis , Dendrímeros/química , Lisina/química , Polietilenoglicóis/química , Portadores de Fármacos/química , Antibacterianos/farmacologia , Antibacterianos/química , Ácido Cítrico/química , Humanos , Hemólise/efeitos dos fármacos , Cefotaxima/química , Cefotaxima/farmacologia , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Tamanho da Partícula , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Nanopartículas/químicaRESUMO
Antimicrobial resistance is an emerging threat to public health around the world. The study employs computational and biophysical methods to investigate the properties of cefotaxime and meropenem's binding to various beta-lactamases like TEM-1, SHV-1, KPC-2, and Amp-C. The enzyme kinetics of purified proteins revealed an increase in Michaelis constant (Km) value in the presence of meropenem and cefotaxime, indicating a decrease in enzyme affinity for nitrocefin. Proteins interact with meropenem/cefotaxime, causing quenching through complex formation. All proteins have one binding site, and binding constant (Kb) values are 104, indicating strong interaction. The study found that meropenem and cefotaxime had high fitness scores for Amp-C, KPC-2,TEM-1 and SHV-1, with binding energy ranging from -7.4 to -7.8, and hydrogen bonds between them. Molecular Dynamic simulation of protein-ligand complexes revealed cefotaxime-binding proteins have slightly lower Root Mean Square Deviation(RMSD) than meropenem-binding proteins, indicating stable association antibiotics with these proteins.
Assuntos
Cefotaxima , Meropeném , Simulação de Dinâmica Molecular , Ligação Proteica , beta-Lactamases , Meropeném/química , Meropeném/farmacologia , Meropeném/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Cefotaxima/farmacologia , beta-Lactamases/química , beta-Lactamases/metabolismo , Sítios de Ligação , Cinética , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Ligação de Hidrogênio , Tienamicinas/química , Tienamicinas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
The electrochemical behavior of cefotaxime (CTX+) was investigated at the polarized macro- and micro-interface between two immiscible electrolyte solutions (ITIES) by cyclic voltammetry and alternating current voltammetry. Miniaturization was achieved with fused silica microcapillary tubing entrapped in a polymeric casing. Scanning electron microscopy (SEM) was employed for the fabricated LLI support characterization. Voltammetric investigation of CTX+ at macro- and µ-ITIES allowed the determination of many physicochemical parameters, such as formal Galvani potential of the ion transfer reaction ([Formula: see text]), diffusion coefficients (D), formal free Gibbs energy of the ion transfer reaction (∆G'aq â org), and water-1,2-dichloroethane partition coefficient ([Formula: see text]). Additionally, based on the results obtained the analytical parameters including voltammetric sensitivity, limits of detection and the limits of quantification (in micromolar range) were calculated. The applicability of the developed procedures was verified in spiked still mineral and tap water samples.
Assuntos
Antibacterianos/química , Cefotaxima/química , Eletrólitos/química , Miniaturização , Soluções/químicaRESUMO
At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.
Assuntos
Proteínas de Bactérias/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Serina-Treonina Quinases/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Bactérias Gram-Positivas/enzimologia , Repressores Lac/genética , Fosforilação , Análise de Célula ÚnicaRESUMO
Bio/chemoinformatics tools can be deployed to compare antimicrobial agents aiming to select an efficient nose-to-brain formulation targeting the meningitis disease by utilizing the differences in the main structural, topological and electronic descriptors of the drugs. Cefotaxime and ceftriaxone were compared at the formulation level (by comparing the loading in gelatin and tripalmitin matrices as bases for the formation of nanoparticulate systems), at the biopharmaceutical level (through the interaction with mucin and the P-gp efflux pumps) and at the therapeutic level (through studying the interaction with S. pneumoniae bacterial receptors). GROMACS v4.6.5 software package was used to carry-out all-atom molecular dynamics simulations. Higher affinity of ceftriaxone was observed compared to cefotaxime on the investigated biopharmaceutical and therapeutic macromolecules. Both drugs showed successful docking on mucin, P-gp efflux pump and S. pneumoniae PBP1a and 2b; but ceftriaxone showed higher affinity to the P-gp efflux pump proteins and higher docking on mucin. Ceftriaxone showed less out-of-matrix diffusion and higher entrapment on the gelatin and the tripalmitin matrices. Accordingly, Ceftriaxone gelatin nanospheres or tripalmitin solid lipid nanoparticles may pose a more feasible and efficient nose-to-brain formulation targeting the meningitis disease compared to the cefotaxime counterparts.
Assuntos
Encéfalo/metabolismo , Cefotaxima/química , Ceftriaxona/química , Quimioinformática/métodos , Antibacterianos/química , Gelatina/química , Simulação de Dinâmica Molecular , Mucinas/química , Software , Triglicerídeos/químicaRESUMO
New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for ß-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty ß-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-ß-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-ß-lactam IgE antibodies with high sensitivity (< 0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of ß-lactam antibiotics renders improved molecular recognition events when the 4-member ß-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.
Assuntos
Hipersensibilidade a Drogas/imunologia , beta-Lactamas/imunologia , Antibacterianos/imunologia , Aztreonam/química , Aztreonam/imunologia , Carbapenêmicos/imunologia , Carbapenêmicos/farmacologia , Cefotaxima/química , Cefotaxima/imunologia , Ceftriaxona/química , Ceftriaxona/imunologia , Cefuroxima/química , Cefuroxima/imunologia , Cefalosporinas/imunologia , Cefalosporinas/farmacologia , Reações Cruzadas , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Meropeném/química , Meropeném/imunologia , Monobactamas/imunologia , Monobactamas/farmacologia , Penicilinas/imunologia , Testes CutâneosRESUMO
Laboratory assays such as MIC tests assume that antibiotic molecules are stable in the chosen growth medium-but rapid degradation has been observed for antibiotics including ß-lactams under some conditions in aqueous solution. Degradation rates in bacterial growth medium are less well known. Here, we develop a 'delay time bioassay' that provides a simple way to estimate antibiotic stability in bacterial growth media, using only a plate reader and without the need to measure the antibiotic concentration directly. We use the bioassay to measure degradation half-lives of the ß-lactam antibiotics mecillinam, aztreonam and cefotaxime in widely-used bacterial growth media based on MOPS and Luria-Bertani (LB) broth. We find that mecillinam degradation can occur rapidly, with a half-life as short as 2 hours in MOPS medium at 37°C and pH 7.4, and 4-5 hours in LB, but that adjusting the pH and temperature can increase its stability to a half-life around 6 hours without excessively perturbing growth. Aztreonam and cefotaxime were found to have half-lives longer than 6 hours in MOPS medium at 37°C and pH 7.4, but still shorter than the timescale of a typical minimum inhibitory concentration (MIC) assay. Taken together, our results suggest that care is needed in interpreting MIC tests and other laboratory growth assays for ß-lactam antibiotics, since there may be significant degradation of the antibiotic during the assay.
Assuntos
Andinocilina/química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Cefotaxima/química , Andinocilina/farmacologia , Antibacterianos/farmacologia , Bioensaio , Cefotaxima/farmacologia , Meios de Cultura , Estabilidade de Medicamentos , Meia-Vida , Testes de Sensibilidade Microbiana , Fatores de TempoRESUMO
OBJECTIVES: Due to the expanded bacterial genetic tolerance to antibiotics through different mechanisms, infectious diseases of MDR bacteria are difficult for treatment. Consequently, we synthesized drug conjugated nanoparticles to dissolve this problem. Moreover, the present study aims to display the cell death status treated with cefotaxime-CS-AgNPs and also, apoptosis pathways of human RPE-1 normal cells and human MCF-7 breast cancer cells. METHODS: Here, we demonstrate the possibility to synthesize AgNPs and conjugate them with cefotaxime to survey the probability of cefotaxime-CS-AgNPs as an antimicrobial agent against cefotaxime-resistant strains E. coli and MRSA. RESULTS: TEM showed the size of AgNPs, CS-AgNPs and cefotaxime-CS-AgNPs ranged from 7.42 to 18.3 nm, 8.05-23.89 nm and 8.48-25.3 nm, respectively, with a spherical shape. The cefotaxime-CS-AgNPs enhanced the high antimicrobial properties compared to AgNPs or pure antibiotic. The MIC of Cefotaxime-CS-AgNPs ranged from 3 µg/mL to 8 µg/mL against tested E. coli and MRSA bacteria. Consequently, the highest reduction in the MIC of cefotaxime-CS-AgNPs was noted against tested strains ranging from 22% to 96%. Comparing cefotaime-CS-AgNPs to AgNPs we showed that cefotaime-CS-AgNPs have no cytotoxic effect on normal cells at even 12 µg/mL for 24 hrs. The IC50 for the AgNPs and cefotaxime-CS-AgNPs was 12 µg/mL for human RPE-1 normal cells and human MCF-7 breast cancer cell lines. The pro-apoptotic genes p53, p21, and Bax of cancer cell lines significantly upregulated followed by downregulated by anti-apoptotic gene Bcl-2 after 48 hrs at 24 µg/mL, and this concentration represents the most effective dose. CONCLUSION: Results enhanced the conjugating utility in old unresponsive cefotaxime to AgNPs to restore its efficiency against previous strains and demonstrated potential therapeutic applications of cefotaxime-CS-AgNPs. Moreover, this research gives remarkable insights for designing nanoscale delivery and curative systems that have a pronounced cytotoxic activity on cancer cells and are safe to normal cells.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Cefotaxima/química , Cefotaxima/farmacologia , Nanopartículas Metálicas/química , Antibacterianos/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Prata/química , Prata/farmacologia , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels into chemically complex natural products is particularly important for techniques such as NMR, IR, mass spectrometry, imaging, and kinetic isotope effects. Toward this goal, we developed a biosynthetic strategy for the site-specific incorporation of 13C labels into the canonical ß-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin C. This was achieved through sulfur-replacement with 1-13C-l-cysteine, resulting in high isotope incorporations and milligram-scale yields. Using 13C NMR and isotope-edited IR difference spectroscopy, we illustrate how these molecules can be used to interrogate interactions with their protein targets, e.g., TEM-1 ß-lactamase. This method provides a feasible route to isotopically labeled penicillin and cephalosporin precursors for future biophysical studies.
Assuntos
Antibacterianos/metabolismo , Isótopos de Carbono/química , beta-Lactamas/metabolismo , Antibacterianos/química , Sítios de Ligação , Cefotaxima/química , Cefotaxima/metabolismo , Cefalosporinas/metabolismo , Descoberta de Drogas , Resistência Microbiana a Medicamentos , Penicilina G/química , Penicilina G/metabolismo , Penicillium/metabolismo , Conformação Proteica , beta-Lactamases/metabolismo , beta-Lactamas/químicaRESUMO
Due to the potential threatening of antibiotics in aqueous environment, a novel electro-oxidation (EO) - electro-Fenton (EF) -persulfate (PS) system with the addition of peroxydisulfate and Fe2+ was installed for the degradation of cefotaxime. Ti/CNT/SnO2-Sb-Er with an ultra-high oxygen evolution potential (2.15 V) and enhanced electrocatalytic surface area was adopted as anode. The OH production and electrode stability test demonstrated great improvement in the electrochemical performances. Ni@NCNT cathode was tested with higher H2O2 generation by the presence of nitrogen functionalities due to the acceleration of electron transfer of O2 reduction. Experiment results indicated CNT and ErO2 modification increased the molecular and TOC removal of cefotaxime. Coupling processes of EO-EF and EO-PS both resulted in shorter electrolysis time for complete cefotaxime removal, however, the mineralization ability of EO-PS process was lower than EO-EF, which might result from the immediate vanishing of PS. Thus, a further improved treatment EO-EF-PS system achieved an 81.6% TOC removal towards 50 mg L-1 cefotaxime after 4 h electrolysis, under the optimal working condition Fe2+ = PS = 1 mM. The influence of current density and initial concentration on the performance of all processes was assessed. Methanol and tert-butanol were added in the system as OH and SO4- scavengers, which illustrating the mechanism of EO-EF-PS oxidizing process was the result of the two free radicals. Major intermediates were deduced and the degradation pathway of cefotaxime was analyzed. This research provides a potential coupling process with high antibiotic removal efficiency and effective materials for practical uses.
Assuntos
Cefotaxima/química , Poluentes Químicos da Água/química , Antibacterianos/química , Eletrodos , Eletrólise/métodos , Peróxido de Hidrogênio/química , Oxirredução , Titânio , Águas Residuárias/química , Água , Purificação da Água/métodosRESUMO
Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are a diverse family of enzymes that are rapidly becoming the predominant cause of bacterial resistance against ß-lactam antibiotics in many regions of the world. OXA-48, an atypical member of CHDLs, is one of the most frequently observed in the clinic and exhibits a unique substrate profile. We applied X-ray crystallography to OXA-48 complexes with multiple ß-lactam antibiotics to elucidate this enzyme's carbapenemase activity and its preference of imipenem over meropenem and other substrates such as cefotaxime. In particular, we obtained acyl-enzyme complexes of OXA-48 with imipenem, meropenem, faropenem, cefotaxime, and cefoxitin, and a product complex with imipenem. Importantly, the product complex captures a key reaction milestone with the newly generated carboxylate group still in the oxyanion hole, and represents the first such complex with a wild-type serine ß-lactamase. A potential hydrogen bond is observed between the two carboxylate groups from the product and the carbamylated Lys73, representing the stage immediately after the breakage of the acyl-enzyme bond where the product carboxylate would be neutral. The placement of the product carboxylate also illustrates the approximate transient location of the deacylation water that has long eluded structural characterization in class D ß-lactamases. Additionally, comparing the product complex with the acyl-enzyme intermediates provides new insights into the various mechanisms by which specific side chain groups hinder the access of the deacylation water to the acyl-enzyme linkage, especially in meropenem. Taken together, these data offer valuable information on the substrate specificity of OXA-48 and the catalytic mechanism of CHDLs.
Assuntos
Modelos Moleculares , beta-Lactamases/química , beta-Lactamases/metabolismo , Domínio Catalítico , Cefotaxima/química , Cefotaxima/metabolismo , Cristalografia por Raios X , Hidrólise , Imipenem/química , Imipenem/metabolismo , Meropeném/química , Meropeném/metabolismo , Especificidade por SubstratoRESUMO
In recent years dendrimers have fascinated the investigators towards targeted drug delivery because of their versatile framework and exhibit immense potentiality in entrapping drug moieties through host-guest interactions and serve as a promising vector in biological applications. The current investigation is focused on developing pegylated citric acid cefotaxime dendrimers through the divergent method and its characterization through spectroscopic, microscopic, thermal and microscopic techniques. Among the spectroscopic techniques, 1H NMR and 13C NMR elucidated the key functional groups at various chemical shifts while ESI-MS pointed out the molecular weight of cefotaxime sodium in various generations. Similarly, FTIR, DSC, and AFM investigations detailed that the generations are devoid of incompatibilities, structural deformities and can be opted for targeted drug delivery. The drug entrapment studies and in-vitro drug release studies highlight CFTX G5 containing 92.4% entrapment efficacy and 83.8% drug release in 48â¯h and specifies a sustain release characteristics. In connection to the above, the in-vivo studies reveal a potent antibacterial activity against various gram-positive and gram-negative microorganisms with a decreased hemolysis and cytotoxicity effects and reflect a high margin of safety regarding pegylated CFTX dendrimers. Further, the antibacterial activities are supported through confocal microscopy that clarified the cellular uptake of dendritic molecules and their internalization.
Assuntos
Cefotaxima/química , Ácido Cítrico/química , Dendrímeros/química , Nanoestruturas/química , Células A549 , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Cefotaxima/metabolismo , Cefotaxima/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Meia-Vida , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Nanoestruturas/toxicidade , Polietilenoglicóis/químicaRESUMO
The emergence of antibiotic resistant Gram-negative bacteria has become a serious global health issue. In this study, we have employed the intrinsically resistant opportunistic pathogen Stenotrophomonas maltophilia as a model to study the mechanisms involved in the acquisition of mutation-driven resistance to antibiotics. To this aim, laboratory experimental evolution studies, followed by whole-genome sequencing, were performed in the presence of the third-generation cephalosporin ceftazidime. Using this approach, we determined that exposure to increasing concentrations of ceftazidime selects high-level resistance in S. maltophilia through a novel mechanism: amino acid substitutions in SmeH, the transporter protein of the SmeGH RND efflux pump. The recreation of these mutants in a wild-type background demonstrated that, in addition to ceftazidime, the existence of these substitutions provides bacteria with cross-resistance to other beta-lactam drugs. This acquired resistance does not impose relevant fitness costs when bacteria grow in the absence of antibiotics. Structural prediction of both amino acid residues points that the observed resistance phenotype could be driven by changes in substrate access and recognition.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Ceftazidima/farmacologia , Stenotrophomonas maltophilia/efeitos dos fármacos , Resistência beta-Lactâmica/genética , Substituição de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cefotaxima/química , Cefotaxima/farmacologia , Cefoxitina/química , Cefoxitina/farmacologia , Ceftazidima/química , Expressão Gênica , Genes MDR , Aptidão Genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Estrutura Secundária de Proteína , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Sequenciamento Completo do GenomaRESUMO
The MALDI Biotyper Selective Testing of Antibiotic Resistance-ß-Lactamase (MBT STAR-BL) assay, which analyzes bacterial induced hydrolysis of cefotaxime using MALDI-TOF MS, correctly identified 100.0% of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae as positive and 94.7% of non-ESBL producers as negative in 80 strains tested.
Assuntos
Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/isolamento & purificação , Klebsiella pneumoniae/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Resistência beta-Lactâmica , Cefotaxima/química , Hospitais Universitários , Japão , Programas de Rastreamento/métodosRESUMO
Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.
Assuntos
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropeném/química , Zinco/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Domínio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropeném/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Zinco/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
The CTX-M ß-lactamases have emerged as the most widespread extended-spectrum ß-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 ß-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation.
Assuntos
Resistência às Cefalosporinas/genética , beta-Lactamases/genética , Substituição de Aminoácidos , Cefotaxima/química , Ceftazidima/química , Cristalografia por Raios X , Estabilidade Enzimática , Epistasia Genética , Escherichia coli/genética , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Conformação Proteica , beta-Lactamases/químicaRESUMO
CTX-M-15 type ß-lactamases are a class of enzymes which hydrolyzes cefotaxime and aztreonam (a monobactam) antibiotics. The emergence of CTX-M-15 producing Enterobacteriaceae member is a major threat to public health. The objective of the study was to check the potency of aztreonam and cefotaxime in combination against ß-lactamase producing strains and to monitor the mechanism behind their interaction. FICI results showed the synergistic effect of aztreonam-cefotaxime against CTX-M-15 producing strain. The expressed and purified CTX-M-15 protein was used as the source of enzyme. Kinetic studies confirmed that the catalytic efficiency of the CTX-M-15 enzyme was decreased to about 78% when it was treated with aztreonam then with cefotaxime in 5 and 10 times molar ratio, respectively, in comparison to the studies where efficiency was enhanced by 33% when cefotaxime was taken alone. Fluorescence study showed that aztreonam binding with CTX-M-15 was an endothermic and spontaneous process with Ka of the order of 104â¯M-1. CD spectroscopic study showed conformational changes upon aztreonam/aztreonam-cefotaxime binding with CTX-M-15. The study concludes that aztreonam in combination with cefotaxime synergistically inhibits CTX-M-15 efficiency significantly. Hence the combination of a monobactam and cephalosporin can be used as the potential therapeutic candidates against ß-lactamase producing CTX-M-15 strains.
Assuntos
Aztreonam/química , Proteínas de Bactérias , Cefotaxima/química , Enterobacteriaceae/enzimologia , beta-Lactamases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , CatáliseRESUMO
Assessment of incompatibilities between active pharmaceutical ingredient and pharmaceutical excipients is an important part of preformulation studies. The objective of the work was to assess the effect of moisture content of chitin calcium silicate of two size ranges (two specific surface areas) on the rate of degradation of cefotaxime sodium. The surface area of the excipient was determined using adsorption method. The effect of moisture content of a given size range on the stability of the drug was determined at 40°C in the solid state. The moisture content was determined at the beginning and the end of the kinetic study using TGA. The degradation in solution was studied for comparison. Increasing the moisture content of the excipient of size range 63-180 µm (surface area 7.2 m2/g) from 3.88 to 8.06% increased the rate of degradation of the drug more than two times (from 0.0317 to 0.0718 h-1). While an opposite trend was observed for the excipient of size range < 63 µm (surface area 55.4 m2/g). The rate of degradation at moisture content < 3% was 0.4547 h-1, almost two times higher than that (0.2594 h-1) at moisture content of 8.54%, and the degradation in solid state at both moisture contents was higher than that in solution (0.0871 h-1). In conclusion, the rate of degradation in solid should be studied taking into consideration the specific surface area and moisture content of the excipient at the storage condition and it may be higher than that in solution.
Assuntos
Compostos de Cálcio/metabolismo , Cefotaxima/metabolismo , Quitina/metabolismo , Silicatos/metabolismo , Água/metabolismo , Compostos de Cálcio/química , Cefotaxima/química , Quitina/química , Estabilidade de Medicamentos , Excipientes/química , Excipientes/metabolismo , Cinética , Silicatos/química , Água/químicaRESUMO
Cefotaxime is third generation antibiotic with known therapeutic efficacy against bacterial infections including cerebral abscesses and bacterial meningitis. The ß-lactam group of drugs are considered safest antibiotics. Many antibiotics directly interact with DNA and alter their expression profile. Thus, it is necessary to understand the binding mode and its relevance to drug activity and toxicity. There is considerably a remarkable focus on deciphering the binding mechanism of these therapeutic agents as DNA is one of the major target for wide range of drugs. Cefotaxime has been extensively studied for its pharmacological properties while its binding mode to DNA has not been explicated so far. In this study, we have unveiled the binding mechanism of cefotaxime to DNA by using various biophysical, thermodynamic and in silico techniques. UV-vis spectroscopy confirmed the formation cefotaxime-DNA complex along with a brief idea about the extent of interaction. Fluorescence spectroscopy yielded the values of various binding constants and explained mode of fluorescence quenching to be static. CD spectroscopy, thermal denaturation, KI quenching and viscosity measurement explained that cefotaxime is groove binder. Measuring the effect of ions on cefotaxime-DNA complex ensured that it does not bind to DNA electrostatically. Dye displacement experiments finally confirmed that cefotaxime binds to the minor groove of DNA. ITC gave the thermodynamic profile of this binding in which negative value of Gibb's free energy change revealed that the process is spontaneous. Molecular modelling finally strengthened our experimental results that cefotaxime was located in curved contour of minor groove of DNA. The findings support on safety of drug and may have a little interference on normal biological functions.
Assuntos
Antibacterianos/química , Cefotaxima/química , DNA/química , Simulação de Acoplamento Molecular/métodos , Conformação de Ácido Nucleico , Antibacterianos/toxicidade , Calorimetria/métodos , Cefotaxima/toxicidade , Simulação por Computador , DNA/efeitos dos fármacos , Espectrometria de Fluorescência/instrumentação , Espectrofotometria Ultravioleta/instrumentação , Espectrofotometria Ultravioleta/métodos , TermodinâmicaRESUMO
OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced â¼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of ß-lactam drugs.