RESUMO
INTRODUCTION: Novel beta-lactam/beta-lactamase inhibitor (BIBLI) combinations are commercially available and have been used for treating carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Continuous surveillance of susceptibility profiles and resistance mechanism identification are necessary to monitor the evolution of resistance within these agents. OBJECTIVE: The purpose of this study was to evaluate the susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in CRKP isolated from patients with bloodstream infections who underwent screening for a randomized clinical trial in Brazil. METHODS: Minimum inhibitory concentrations (MICs) were determined for meropenem, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam using the gradient diffusion strip method. Carbapenemase genes were detected by multiplex real-time polymerase chain reaction. Klebsiella pneumoniae carbapenemase (KPC)-producing isolates showing resistance to any BLBLI and New Delhi Metallo-beta-lactamase (NDM)-producing isolates with susceptibility to any BLBLI isolates were further submitted for whole-genome sequencing. RESULTS: From a total of 69 CRKP isolates, 39 were positive for blaKPC, 19 for blaNDM and 11 for blaKPC and blaNDM. KPC-producing isolates demonstrated susceptibility rates above 94â¯% for all BLBLIs. Two isolates with resistance to meropenem/vaborbactam demonstrated a Gly and Asp duplication at the porin OmpK36 as well as a truncated OmpK35. All NDM-producing isolates, including KPC and NDM coproducers, demonstrated susceptibility rates to ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam of 0â¯%, 9.1-21.1â¯% and 9.1-26.3â¯%, respectively. Five NDM-producing isolates that presented susceptibility to BLBLIs also had porin alterations CONCLUSIONS: This study showed that, although high susceptibility rates to BLBLIs were found, KPC-2 isolates were able to demonstrate resistance probably as a result of porin mutations. Additionally, NDM-1 isolates showed susceptibility to BLBLIs in vitro.
Assuntos
Antibacterianos , Compostos Azabicíclicos , Enterobacteriáceas Resistentes a Carbapenêmicos , Ceftazidima , Combinação de Medicamentos , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases , beta-Lactamases , Humanos , Brasil , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Inibidores de beta-Lactamases/farmacologia , Infecções por Klebsiella/microbiologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , beta-Lactamases/genética , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Proteínas de Bactérias/genética , Meropeném/farmacologia , Imipenem/farmacologia , Bacteriemia/microbiologia , Ácidos Borônicos/farmacologia , Compostos Heterocíclicos com 1 AnelRESUMO
BACKGROUND: It has been reported that boron induces changes in the immune response, including in inflammatory processes. Recently, the effect of boric acid has been documented on the differentiation of lymphocyte clusters in mice and rats. However, the differences among boron-containing compounds (BCC) have been poorly explored. METHODS: In this study, we analyzed the effects after oral administration of boric acid (BOR), methylboronic (MET), 3-thyenylboronic (3TB), 4-hydroxymethyl-phenylboronic (4MP) and 4-methanesulfonyl-phenylboronic (4SP) acids on the populations of lymphocytes from spleen and Peyer's patch (PP) as well as on antibodies. Groups of six male BALB/c were orally treated with 4.6 mg/kg of body weight with BOR, MET, 3TB, 4MP, and 4SP/daily for 10 days or vehicle (VEH) as a control group. After euthanasia, the spleen and small intestine were dissected. We conducted flow cytometry assays to assess B, CD3+ T, CD4+ T, and CD8+ T cells. Levels of IgG and IgM in serum, and IgA in intestinal fluid samples were analyzed by enzyme immunoassay. RESULTS: In particular, we observed the effects of the administration of boronic acids on the number of lymphocytes; these changes were more notable in spleen than in PP. We found different profiles for each boron-containing compound, that is BOR induced an increase in the percentage of CD8+ T and CD19+/IgA+ cells in spleen, but a decrease in CD8+ T and B220+/CD19+ cells in PP. Meanwhile MET induced a decrease of CD4+ T in spleen, but induced an increase of CD4+ T cells and a decrease in the number of CD8+ T cells in PP. Boronic acids with an aromatic ring moiety induced changes in serum immunoglobulins levels, while 3TB acid induced a notable increase in S-IgA. CONCLUSIONS: Effects in lymphocyte populations and antibodies are different for each tested compound. These results highlight the establishment of the necessary structure-activity relationship for BCC as immunomodulatory drugs. This is relevant in the biomedical field due to their attractiveness for selecting compounds to develop therapeutic tools.
Assuntos
Ácidos Bóricos , Nódulos Linfáticos Agregados , Animais , Boro/farmacologia , Ácidos Borônicos/farmacologia , Linfócitos T CD8-Positivos , Imunidade , Imunoglobulina A , Agentes de Imunomodulação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RatosRESUMO
AmpC is a type of ß-lactamase enzyme produced by bacteria; these enzymes are classified in Class C and Group 1, and these confer resistance to cephamycin. Enterobacterales producing AmpC are reported worldwide and have great clinical importance due to therapeutic restriction and epidemiological importance once the easy dissemination by plasmidic genes to other bacteria is a real threat. These genes are naturally found in some enterobacteria as Enterobacter cloacae, Morganella morganii, and Citrobacter freundii, but other species have demonstrated similar resistance phenotype of AmpC production. Genes carried in plasmids have been described in these species conferring resistance to cefoxitin and causing therapeutic failure in some bacterial infections. This work detected and described five clinical strains of Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae that presented plasmid ampC (pAmpC) isolated from the north of Portugal collected in 2009. AmpC production was confirmed by inhibition of the enzyme by cloxacillin and boronic acid in agar diffusion tests. Also, PCR (polymerase chain reaction) was performed for the detection of gene universal to AmpC, blaampC, and others to AmpC group: blaACC, blaCIT, blaCMY, blaDHA, and blaEBC. The conjugation in liquid medium for 24 h was realized to determine if gene is localized in chromosome or plasmid. The isolates and their conjugants showed phenotypic characteristics and blaCMY and blaCIT were detected by PCR corroborating the AmpC characteristics observed in these bacteria. Confirmation of transfer of plasmid containing genes encoding AmpC is of high epidemiological relevance to the hospital studied and demonstrated the importance of AmpC surveillance and studies in hospital and community environments in order to choose the appropriate therapy for bacterial infections.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , Enterobacteriaceae/genética , Plasmídeos/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Ácidos Borônicos/farmacologia , Cloxacilina/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/classificação , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Portugal/epidemiologia , beta-Lactamases/metabolismoRESUMO
The emergent Zika virus (ZIKV) infection has become a threat to global health due to its association with severe neurological abnormalities, namely Guillain-Barré Syndrome (GBS) in adults and Congenital Zika virus Syndrome (CZS) in neonates. Many studies are nowadays being conducted to find an effective antiviral drug against ZIKV. In particular, NS2B-NS3 protease is an attractive drug target due to its essential function in viral replication, although a drug is not yet commercially available. In this context, we present here a comparative structural study, based on quantum chemistry calculations, to analyze the intermolecular binding energies between the crystallographic structure of NS2B-NS3 protease and dipeptide boronic acid (cn-716) and aldehyde (acyl-KR-aldehyde) peptidomimetic inhibitors, by using the molecular fractionation with conjugate caps (MFCC) scheme within the density functional theory (DFT) formalism. Most intermolecular interactions in cn-716/NS2B-NS3 (acyl-KR-aldehyde/NS2B-NS3) are due to the amino acid residues Asp83*, His51, Asp129, Ser81*, Gly133, Ala132, Tyr161, Asn152 and Asp75 (Asp83*, Asp129, His51, Asn152, Tyr161, Tyr130, Gly153, Gly151, Asp75, Pro131, and Gly82). Additionally, we have considered missense mutation analysis of these residues to evaluate the destabilization and the increase of the flexibility of the protease, showing that mutation of the residues Tyr161 and Tyr130 causes more impact. Our simulations are a valuable tool for a better understanding of the binding mechanism of recognized inhibitors of NS2B-NS3 protease, and can lead to the rational design and development of novel anti-Zika drugs with improved efficiency.
Assuntos
Aldeídos/farmacologia , Antivirais/farmacologia , Ácidos Borônicos/farmacologia , Dipeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Zika virus/efeitos dos fármacos , Aldeídos/química , Antivirais/química , Ácidos Borônicos/química , Teoria da Densidade Funcional , Dipeptídeos/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Peptidomiméticos/antagonistas & inibidores , Peptidomiméticos/metabolismo , Inibidores de Proteases/química , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Zika virus/metabolismoRESUMO
The P2X7 receptor (P2X7R) is an ion channel which is activated by interactions with the extracellular ATP molecules. The molecular complex P2X7R/ATP induces conformational changes in the protein subunits, opening a pore in the ion channel macromolecular structure. Currently, the P2X7R has been studied as a potential therapeutic target of anti-inflammatory drugs. Based on this, a series of eight boronic acids (NO) analogs were evaluated on the biologic effect of this pharmacophoric group on the human and murine P2X7R. The boronic acids derivatives NO-01 and NO-12 inhibited in vitro human and murine P2X7R function. These analogs compounds showed effect better than compound BBG and similar to inhibitor A740003 for inhibiting dye uptake, in vitro IL-1ß release and ATP-induced paw edema in vivo. In both, in vitro and in vivo assays the compound NO-01 showed to be the hit compound in the present series of the arylboronic acids analogs. The molecular docking suggests that the NO derivatives bind into the upper body domain of the P2X7 pore and that the main intermolecular interaction with the two most active NO derivatives occur with the residues Phe 95, 103 and 293 by hydrophobic interactions and with Leu97, Gln98 and Ser101 by hydrogen bonds.. These results indicate that the boronic acid derivative NO-01 shows the lead compound characteristics to be used as a scaffold structure to the development of new P2X7R inhibitors with anti-inflammatory action.
Assuntos
Anti-Inflamatórios , Ácidos Borônicos , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X7/metabolismo , Acetamidas/química , Acetamidas/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Receptores Purinérgicos P2X7/genéticaRESUMO
It has been reported that boron induces changes in carbohydrate and lipid metabolism, body weight and inflammatory processes. This is relevant to the biomedical field due to the requirement for developing therapeutic tools with potential application in metabolic disorders affecting humankind. However, most of the reported data from both humans and animals were obtained after boron was administered as borax or boric acid. In this work, we determined the effects of boric, cyclohexylboronic (CHB) and phenylboronic (PBA) acids (10 mg/kg of body weight/daily for two weeks) on the body weight, metabolism and inflammatory markers in the blood of control, fat-feeding and experimental diabetic rats. In particular, we observed the effects of the administration of these compounds on glycaemia and cholesterol, triglyceride, insulin, IL-6 and C-reactive protein levels, as well as visceral fat and body weight. We found different profiles for each boron-containing compound: boric acid induced decreasing body weight, insulin and IL-6 levels; CHB administration induced an increase in body weight and cholesterol but decreased IL-6 levels; and PBA administration induced a decrease in visceral fat and glucose and insulin levels. These results can improve the understanding of boron as a metabolic regulator and help develop new potential strategies to use compounds with this trace element for therapeutic purposes.
Assuntos
Peso Corporal/efeitos dos fármacos , Ácidos Bóricos/farmacologia , Ácidos Borônicos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Boro , Inflamação/sangue , Inflamação/metabolismo , Insulina/sangue , Masculino , RatosRESUMO
The activity of meropenem/vaborbactam was evaluated against 11 559 Enterobacteriaceae isolates, including 330 carbapenem-resistant phenotypes (CRE) and carbapenemase genotypes collected worldwide during 2015. Antimicrobial susceptibility testing for meropenem/vaborbactam (inhibitor at 8 mg/L) and comparators was performed by the reference broth microdilution method. CRE isolates were screened for the presence of genes encoding carbapenemases, and 292 (88.5%) of the CRE isolates carried these resistance genes. A total of 209 isolates (63.3% of the CRE; 1.8% of the overall Enterobacteriaceae population) carried blaKPC, including genes encoding KPC-2 (90 isolates), KPC-3 (117 isolates) and KPC-17 (2 isolates). Overall, meropenem/vaborbactam (vaborbactam at 8 mg/L) inhibited 99.3% of all Enterobacteriaceae isolates at the US-FDA susceptibility breakpoint of ≤4/8 mg/L. Meropenem alone inhibited 96.9% of the isolates at the current CLSI susceptibility breakpoint of ≤2 mg/L. Susceptibility rates for comparator antimicrobial agents tested against Enterobacteriaceae isolates ranged from 82.1-98.2% applying the CLSI breakpoints. Against CRE isolates, meropenem/vaborbactam displayed MIC50/90 values at 0.5/32 mg/L, whereas meropenem MIC50/90 values were 16/>32 mg/L. Meropenem/vaborbactam was very active against KPC-producers, and 99.5% of these isolates were inhibited by ≤4/8 mg/L. The single resistant isolate was shown to harbour an outer membrane porin alteration. Meropenem/vaborbactam had limited activity against MBL-producing isolates (including 49 NDM-, 1 IMP-64- and 2 VIM-producers) and/or oxacillinases (47 OXA-48/-232) that were detected mainly in European countries. Meropenem/vaborbactam was active against contemporary CRE and wild-type Enterobacteriaceae collected worldwide and this combination demonstrated enhanced activity compared with meropenem and most comparator agents against CRE and KPC-producers.
Assuntos
Antibacterianos/farmacologia , Ácidos Borônicos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Infecções por Enterobacteriaceae/epidemiologia , Tienamicinas/farmacologia , Resistência beta-Lactâmica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterobacteriáceas Resistentes a Carbapenêmicos/classificação , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/microbiologia , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Expressão Gênica , Genótipo , Humanos , Meropeném , Testes de Sensibilidade Microbiana , Mutação , América do Norte/epidemiologia , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , Porinas/genética , Porinas/metabolismo , América do Sul/epidemiologia , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
LASSBio-1524 was designed as inhibitor of the IKK-ß (kappa ß kinase inhibitor) enzyme, which participates in the activation of the nuclear factor κB (NF-κB) canonical pathway, and its three N-acylhydrazone new analogues, LASSBio-1760, LASSBio-1763 and LASSBio-1764 are now being tested on their anti-inflammatory potential. The activity of these compounds was evaluated with the subcutaneous air pouch induced by carrageenan and by subsequent measurement of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive oxygen species (ROS). In the acute inflammation model, the oral pretreatment with doses from 0.3 to 30 mg/kg of N-acylhydrazone derivatives was able to significantly reduce leukocyte migration to the cavity. Pretreatment with LASSBio-1524 and its analogues also decreased NO, TNF-α and ROS biosynthesis an events closely involved with NF-kB pathway. The tetrahydronaphthyl-N-acylhydrazone derivative LASSBio-1764 was the most promising compound from this series, surpassing even LASSBio-1524. Additionally, none of the compounds demonstrated myelotoxicity or cytotoxicity. Cell viability was assayed and these compounds demonstrated to be safe at different concentrations. Western blot analysis demonstrated that LASSBio-1524 and LASSBio-1760 inhibited NF-κB expression in RAW 264.7 cell lineage. Our data indicate that the tested compounds have anti-inflammatory activity, which may be related to inhibition of leukocyte migration, reducing the production of NO, TNF-α and ROS. LASSBio-1524 and LASSBio-1760, in addition to these features, also reduced p65 nuclear expression assessed by western blot in RAW 264.7 murine cells.
Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Borônicos/farmacologia , Descoberta de Drogas , Hidrazonas/farmacologia , Inflamação/tratamento farmacológico , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/química , Carragenina/toxicidade , Hidrazonas/administração & dosagem , Hidrazonas/química , Quinase I-kappa B/antagonistas & inibidores , Inflamação/induzido quimicamente , Inflamação/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Leishmania arginase is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme initiates de novo polyamine biosynthesis by catalyzing the hydrolysis of L-arginine to generate L-ornithine and urea. The product L-ornithine subsequently undergoes decarboxylation to yield putrescine, which in turn is utilized for spermidine biosynthesis. Polyamines such as spermidine are essential for the growth and survival of the parasite, so inhibition of enzymes in the polyamine-biosynthetic pathway comprises an effective strategy for treating parasitic infections. To this end, two X-ray crystal structures of L. mexicana arginase complexed with α,α-disubstituted boronic amino-acid inhibitors based on the molecular scaffold of 2-(S)-amino-6-boronohexanoic acid are now reported. Structural comparisons with human and parasitic arginase complexes reveal interesting differences in the binding modes of the additional α-substituents, i.e. the D side chains, of these inhibitors. Subtle differences in the three-dimensional contours of the outer active-site rims among arginases from different species lead to different conformations of the D side chains and thus different inhibitor-affinity trends. The structures suggest that it is possible to maintain affinity while fine-tuning intermolecular interactions of the D side chain of α,α-disubstituted boronic amino-acid inhibitors in the search for isozyme-specific and species-specific arginase inhibitors.
Assuntos
Aminoácidos/química , Arginase/química , Ácidos Borônicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania mexicana/enzimologia , Animais , Arginase/antagonistas & inibidores , Ácidos Borônicos/química , Modelos MolecularesRESUMO
BACKGROUND AND PURPOSE: The Kaposi sarcoma (KS)-associated herpesvirus GPCR (vGPCR) is a key molecule in the pathogenesis of KS, where it increases NF-κB gene expression and activates the NF-κB pathway. We investigated whether the less calcemic vitamin D analogue TX 527 inhibited the proliferation of endothelial cells transformed by vGPCR by modulation of the NF-κB pathway. EXPERIMENTAL APPROACH: Endothelial cells transformed by vGPCR (SVEC-vGPCR) were treated with TX 527. Proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and cell cycle by flow cytometry. mRNA and protein levels were measured by real-time quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot analysis respectively. KEY RESULTS: TX 527, similar to bortezomib (0.5 nM), a proteasome inhibitor that inhibits the activation of NF-κB, reduced proliferation and induced G0/G1 cell cycle arrest in SVEC-vGPCR. TX 527 like 1α,25(OH)2 D3 , biological active form of vitamin D, decreased the activity of NF-κB comparable with the effect of bortezomib. Time-response studies showed that TX 527 significantly decreased NF-κB and increased IκBα mRNA and protein levels. The increase of IκBα was accompanied by a reduction in p65/NF-κB translocation to the nucleus. These responses were abolished when vitamin D receptor (VDR) expression was suppressed by stable transfection of shRNA against VDR. In parallel with NF-κB inhibition, there was a down-regulation of inflammatory genes such as IL-6, CCL2/MCP and CCL20/MIP3α. CONCLUSIONS AND IMPLICATIONS: These results suggest that the anti-proliferative effects of the vitamin D analogue TX 527 in SVEC-vGPCR occur by modulation of the NF-κB pathway and are VDR dependent.