RESUMO
Metallo-ß-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable ß-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 µM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode.
Assuntos
Tionas , Inibidores de beta-Lactamases , Humanos , Antibacterianos/farmacologia , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Carbapenêmicos/farmacologia , Ceftazidima , Ceftriaxona , Etilenos , Células HeLa , Ligantes , Meropeném , Testes de Sensibilidade Microbiana , Triazóis/química , Triazóis/farmacologia , ZincoRESUMO
In Gram-negative bacteria, the major mechanism of resistance to ß-lactam antibiotics is the production of one or several ß-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-ß-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the µM to sub-µM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.
Assuntos
Tionas/química , Inibidores de beta-Lactamases/química , beta-Lactamases/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/enzimologia , Células HeLa , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ligação Proteica , Relação Estrutura-Atividade , Tionas/metabolismo , Triazóis/química , Inibidores de beta-Lactamases/metabolismo , beta-Lactamases/metabolismoRESUMO
As shifts in the epidemiology of ß-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved ß-lactam (BL)-ß-lactamase inhibitor (BLI) combinations address widespread serine ß-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-ß-lactamases (KPC, OXA-48) or clinically important metallo-ß-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by ß-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki ) values ranging from 0.019 to 0.081 µM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 µg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.
Assuntos
Antibacterianos/farmacologia , Ácidos Borínicos/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores de beta-Lactamases/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefepima/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are increasingly prevalent and have become a major worldwide threat to human health. Carbapenem resistance is driven primarily by the acquisition of ß-lactamase enzymes, which are able to degrade carbapenem antibiotics (hence termed carbapenemases) and result in high levels of resistance and treatment failure. Clinically relevant carbapenemases include both serine ß-lactamases (SBLs; e.g., KPC-2 and OXA-48) and metallo-ß-lactamases (MBLs), such as NDM-1. MBL-producing strains are endemic within the community in many Asian countries, have successfully spread worldwide, and account for many significant CRE outbreaks. Recently approved combinations of ß-lactam antibiotics with ß-lactamase inhibitors are active only against SBL-producing pathogens. Therefore, new drugs that specifically target MBLs and which restore carbapenem efficacy against MBL-producing CRE pathogens are urgently needed. Here we report the discovery of a novel MBL inhibitor, ANT431, that can potentiate the activity of meropenem (MEM) against a broad range of MBL-producing CRE and restore its efficacy against an Escherichia coli NDM-1-producing strain in a murine thigh infection model. This is a strong starting point for a chemistry lead optimization program that could deliver a first-in-class MBL inhibitor-carbapenem combination. This would complement the existing weaponry against CRE and address an important and growing unmet medical need.
Assuntos
Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Meropeném/farmacologia , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
In the last ten years, we identified and developed a new therapeutic class of antifungal agents, the macrocyclic amidinoureas. These compounds are active against several Candida species, including clinical isolates resistant to currently available antifungal drugs. The mode of action of these molecules is still unknown. In this work, we developed an in-silico target fishing procedure to identify a possible target for this class of compounds based on shape similarity, inverse docking procedure and consensus score rank-by-rank. Chitinase enzyme emerged as possible target. To confirm this hypothesis a novel macrocyclic derivative has been produced, specifically designed to increase the inhibition of the chitinase. Biological evaluation highlights a stronger enzymatic inhibition for the new derivative, while its antifungal activity drops probably because of pharmacokinetic issues. Collectively, our data suggest that chitinase represent at least one of the main target of macrocyclic amidinoureas.
Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Trichoderma/efeitos dos fármacos , Antifúngicos/síntese química , Antifúngicos/química , Quitinases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Trichoderma/enzimologiaRESUMO
BEL-1 is an acquired class A extended-spectrum ß-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 Km 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.
Assuntos
Imipenem/química , Moxalactam/química , beta-Lactamases/química , beta-Lactamases/metabolismo , Antibacterianos/química , Domínio Catalítico , Ácido Cítrico/química , Cristalografia por Raios X , Dissulfetos/químicaRESUMO
ß-Lactamases are bacterial enzymes conferring resistance to ß-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of ~1400 boronic acids as potential AmpC ß-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant ß-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K i value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Ácidos Borônicos/química , Inibidores de beta-Lactamases/química , Proteínas de Bactérias/química , Sítios de Ligação , Simulação por Computador , Descoberta de Drogas , Enterobacter/enzimologia , Escherichia coli/enzimologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Serina/química , beta-Lactamases/químicaRESUMO
Metallo-ß-lactamases (MBLs) represent one of the most important and widespread mechanisms of resistance to ß-lactam antibiotics (including the life-saving carbapenems), against which no clinically useful inhibitors are currently available. We report herein a structure-based high-throughput docking (HTD) campaign on three clinically-relevant acquired MBLs (IMP-1, NDM-1 and VIM-2). The initial hit NF1810 (1) was optimized providing the broad-spectrum inhibitor 3i, which is able to potentiate the in vitro activity of cefoxitin on a VIM-2-producing E. coli strain.
Assuntos
Antibacterianos/farmacologia , Ensaios de Triagem em Larga Escala , Simulação de Acoplamento Molecular , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/químicaRESUMO
The POM-1 metallo-ß-lactamase is a subclass B3 resident enzyme produced by Pseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced in Escherichia coli BL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Pseudomonas/enzimologia , Pseudomonas/metabolismo , beta-Lactamases/metabolismo , beta-Lactamases/farmacologia , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Catálise , Cefalosporinas/farmacologia , Escherichia coli/metabolismo , Penicilinas/farmacologiaRESUMO
VIM-39, a VIM-1-like metallo-ß-lactamase variant (VIM-1 Thr33Ala His224Leu) was identified in a clinical isolate of Klebsiella pneumoniae belonging to sequence type 147. VIM-39 hydrolyzed ampicillin, cephalothin, and imipenem more efficiently than did VIM-1 and VIM-26 (a VIM-1 variant with the His224Leu substitution) because of higher turnover rates.
Assuntos
Ampicilina/metabolismo , Antibacterianos/metabolismo , Cefalotina/metabolismo , Imipenem/metabolismo , Klebsiella pneumoniae/enzimologia , Ampicilina/farmacologia , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Sequência de Bases , Biotransformação , Cefalotina/farmacologia , Expressão Gênica , Grécia , Humanos , Hidrólise , Imipenem/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Análise de Sequência de DNA , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
BACKGROUND: CTX-M-15 is the dominant type of extended-spectrum ß-lactamase in clinical isolates. This enzyme constitutes the most widespread enzymes in Tunisia. In this study, we were interested to understand the causes of the evolutionary success of CTX-M-15 in a Tunisian university hospital. METHODS: A total of of 72 cefotaxime-resistant Enterobacteriaceae were isolated from newborn patients at the hospital Taher sfar Mahdia in Tunisia and characterized their genetic support by means of molecular techniques. RESULTS: Isolates were clustered into various clonal groups, although most isolates belonged to sequence types ST39 (Klebsiella pneumoniae) and ST131 (Escherichia coli). F replicons (FIA, FIB, and FII) were the most frequently detected replicon types in our collection (91.66%). CONCLUSION: This is the first report of QnrB- and CTX-M-15-encoding large IncF-type conjugative plasmids in Tunisia.
Assuntos
Farmacorresistência Bacteriana , Enterobacteriaceae , Escherichia coli/genética , Klebsiella pneumoniae/genética , Plasmídeos/genética , beta-Lactamases/genética , Centros Médicos Acadêmicos , Cefotaxima/administração & dosagem , Infecções por Escherichia coli/microbiologia , Evolução Molecular , Variação Genética , Humanos , Recém-Nascido , Infecções por Klebsiella/microbiologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , TunísiaRESUMO
Antibiotic resistance has reached alarming levels in many clinically-relevant human pathogens, and there is an increasing clinical need for new antibiotics active on drug-resistant Gram-negative pathogens who rapidly evolve towards pandrug resistance phenotypes. Here, we report on two related classes of guanidinic compounds endowed with antibacterial activity. The two best compounds (9a and 13d) exhibited the most potent antibacterial activity with MIC values ranging 0.12-8 µg/ml with most tested pathogens, including both Gram-positive and Gram-negative bacteria. Interestingly, MIC values were not affected (1-8 µg/ml) when measured using recent clinical isolates with various antibiotic resistance determinants. The results reported herein identify guazatine derivatives as an interesting starting point for the optimization of a potentially novel class of antibacterial agents.
Assuntos
Antibacterianos/farmacologia , Guanidinas/química , Resistência Microbiana a Medicamentos , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Class D ß-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae. Carbapenemase activity is an unusual feature among class D ß-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands ß5 and ß6 (the ß5-ß6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D ß-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the ß5-ß6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10-derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted ß5-ß6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the ß5-ß6 loop in the carbapenemase activity of class D ß-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.
Assuntos
Carbapenêmicos/metabolismo , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Homologia de Sequência de Aminoácidos , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
Although ß-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of ß-lactamase enzymes that are not affected by currently marketed ß-lactam/ß-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-ß-lactam ß-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D ß-lactamases. Here we describe the structures of two clinically important ß-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum ß-lactamase and the class C Pseudomonas aeruginosa AmpC ß-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.
Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Pseudomonas aeruginosa/efeitos dos fármacos , Inibidores de beta-Lactamases , beta-Lactamases/química , Acilação , Antibacterianos/metabolismo , Compostos Azabicíclicos/química , Compostos Azabicíclicos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Pseudomonas aeruginosa/enzimologia , Relação Estrutura-Atividade , Difração de Raios X , beta-Lactamases/metabolismo , beta-Lactamas/química , beta-Lactamas/metabolismo , beta-Lactamas/farmacologiaRESUMO
The diffusion of antibiotic-resistant, Gram-negative, opportunistic pathogens, an increasingly important global public health issue, causes a significant socioeconomic burden. Acinetobacter baumannii isolates, despite causing a lower number of infections than Enterobacterales, often show multidrug-resistant phenotypes. Carbapenem resistance is also rather common, prompting the WHO to include carbapenem-resistant A. baumannii as a "critical priority" for the discovery and development of new antibacterial agents. In a previous work, we identified several series of compounds showing either direct-acting or synergistic activity against relevant Gram-negative species, including A. baumannii. Among these, two pyrazole compounds, despite being devoid of any direct-acting activity, showed remarkable synergistic activity in the presence of a subinhibitory concentration of colistin on K. pneumoniae and A. baumannii and served as a starting point for the synthesis of new analogues. In this work, a new series of 47 pyrazole compounds was synthesized. Some compounds showed significant direct-acting antibacterial activity on Gram-positive organisms. Furthermore, an evaluation of their activity as potential antibiotic adjuvants allowed for the identification of two highly active compounds on MDR Acinetobacter baumannii, including colistin-resistant isolates. This work confirms the interest in pyrazole amides as a starting point for the optimization of synergistic antibacterial compounds active on antibiotic-resistant, Gram-negative pathogens.
RESUMO
Metallo-ß-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, these compounds were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we synthesized and characterized compounds where the hydrazone-like bond of the Schiff base analogues was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but showed a significantly better activity on VIM-type enzymes, with Ki values in the µM to sub-µM range. The resolution of the crystallographic structure of VIM-2 in complex with one of the best inhibitors yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the ß-lactam susceptibility of VIM-type-producing E. coli laboratory strains and also of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cancer cells at high concentration, thus showing promising safety.
Assuntos
Tionas , Inibidores de beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli , Humanos , Testes de Sensibilidade Microbiana , Tionas/farmacologia , Triazóis/química , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
A very rare variant of mammary Paget disease (MPD) is the pigmented MPD, first described in 1956. It is very difficult to distinguish this variant from melanoma both clinically and dermoscopically. The diagnosis is confirmed by histopathology and immunohistochemistry. Correct diagnosis is crucial for surgical treatment, which is different for these two diseases. We report the case of a 92-year-old woman, who presented an asymptomatic pigmented lesion of the right nipple and areola. The lesion was arisen for about 6 months and was suspected for melanoma because of clinical and dersmoscopic characteristics. Incisional biopsy revealed tumor cells, that proliferate in the major mammary ducts, and tumor cells in the overlying epidermis of the nipple, thus diagnosing pigmented mammary Paget disease. The patient underwent radical mastectomy.
RESUMO
Expression levels of p27(kip1) , a negative regulator of the G1 phase of the cell cycle, and 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, were assessed by immunostaining in a series of renal cell carcinomas (RCCs) and their prognostic significance was evaluated. Expression of p27(kip1) as well as of the α-subunit of the dystroglycan (DG) complex, previously reported to be altered in RCC, was also evaluated by western blot analysis. Nuclear expression of p27(kip1) was reduced in a significant fraction of tumors and low p27(kip1) staining correlated with higher tumor grade (P < 0.01). Recurrence and death from clear cell RCCs were significantly more frequent in p27(kip1) -low expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor groups for both disease-free (P = 0.011) and overall (P = 0.002) survival. Low nuclear expression of p27(kip1) as well as loss of α-DG were confirmed to be independent prognostic parameters at a multivariate analysis and the simultaneous loss of both molecules defined a "high-risk" group of patients with increased risk of recurrence (RR = 28.7; P = 0.01) and death (RR = 12.9; P = 0.03). No significant correlation with clinical or pathological parameters was found for 8-OHdG staining. Western blot analyses suggested a post-translational mechanism for the loss of α-DG expression and demonstrated that cytoplasmic dislocation of the protein contributes to the loss of active nuclear p27(kip1) . Loss of nuclear p27(kip1) is a frequent event in human RCCs and is a powerful predictor of poor outcome which, in combination with low DG expression, could help to identify high-risk patients with clear cell RCC.
Assuntos
Carcinoma de Células Renais/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Distroglicanas/metabolismo , Neoplasias Renais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma de Células Renais/patologia , Núcleo Celular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Avaliação de Resultados em Cuidados de Saúde , Valor Preditivo dos Testes , Prognóstico , Adulto JovemRESUMO
Aeromonas enteropelogenes (formerly A. tructi) was described to be an ampicillin-susceptible and cephalothin-resistant Aeromonas species, which suggests the production of a cephalosporinase. Strain ATCC 49803 was susceptible to amoxicillin, cefotaxime, and imipenem but resistant to cefazolin (MICs of 2, 0.032, 0.125, and >256 microg/ml, respectively) and produced an inducible beta-lactamase. Cefotaxime-resistant mutants (MIC, 32 microg/ml) that showed constitutive beta-lactamase production could be selected in vitro. The gene coding for the cephalosporinase of A. enteropelogenes ATCC 49803 was cloned, and its biochemical properties were investigated. Escherichia coli transformants showing resistance to various beta-lactams carried a 3.5-kb plasmid insert whose sequence revealed a 1,146-bp open reading frame (ORF) encoding a class C beta-lactamase, named TRU-1, showing the highest identity scores with A. punctata CAV-1 (75%), A. salmonicida AmpC (75%), and A. hydrophila CepH (71%). The bla(TRU-1) locus includes open reading frames (ORFs) showing significant homology with genes found in the genomes of other Aeromonas species, although it exhibits a different organization, as reflected by the presence of additional ORFs located downstream of the beta-lactamase gene in the A. hydrophila and A. salmonicida genomes. Specific PCR assays were negative for cphA-like and bla(OXA-12)-like genes in three A. enteropelogenes ATCC strains. Purified TRU-1 showed a broad substrate profile, efficiently hydrolyzing benzylpenicillin, cephalothin, cefoxitin, and, although with significantly lower turnover rates, oxyiminocephalosporins. Cephaloridine and cefepime were poorly recognized by the enzyme, as reflected by the high K(m) values observed with these substrates. Thus far, A. enteropelogenes represents the only known example of an Aeromonas species that produces only one beta-lactamase belonging to molecular class C.
Assuntos
Aeromonas/enzimologia , Aeromonas/genética , beta-Lactamases/genética , Aeromonas/efeitos dos fármacos , Aeromonas/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Humanos , Cinética , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transformação Genética , beta-Lactamases/classificação , beta-Lactamases/metabolismoRESUMO
A Pseudomonas aeruginosa isolate recovered in Belgium produced a novel extended-spectrum ss-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The bla(BEL-2) gene was identified from a P. aeruginosa isolate clonally related to another bla(BEL-1)-positive isolate.