RESUMEN
In recent years, the rise in antimicrobial resistance (AR) in the healthcare setting as well as the environment has been recognized as a growing public health problem. The Chesapeake Bay (CB) and its upper tributaries (UT) is a large and biologically diverse estuary. This pilot study evaluated the presence of AR of gram-negative bacteria isolated from water samples collected at various sites of the Chesapeake Bay. Bacterial organisms were identified and antimicrobial susceptibility testing was performed by phenotypic and genotypic methods. Ninety-two distinctly different gram-negative bacteria were identified; Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, and Escherichia coli were most often isolated. Serratia marcescens was more frequently isolated in samples from the UT compared to the CB. Antimicrobial resistance was more frequently detected in organisms from the CB by phenotypic and genotypic methods. Antimicrobial resistance to ampicillin, imipenem, tetracycline, and chloramphenicol were the most frequently observed resistance patterns. ACT-1, CMY, and SHV genes were the most frequently detected resistance genes, with predominance in organism isolated from the CB. The results from this study emphasize the importance for further developing comprehensive surveillance programs of AR in bacterial isolates in the various environments, such as recreational and other water systems.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/aislamiento & purificación , Microbiología del Agua , Océano Atlántico , Técnicas Bacteriológicas , Enterobacteriaceae/genética , Genes Bacterianos , Técnicas de Genotipaje , Maryland , Ríos , West VirginiaRESUMEN
BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants and is associated with Crohn's disease (CD) in humans, although the latter remains controversial. In this study, we investigated the ability of MAP to adapt to anaerobic growth using the "Wayne" model of non-replicating persistence (NRP) developed for M. tuberculosis. RESULTS: All strains adapted to anaerobiosis over time in a manner similar to that seen with MTB. Susceptibility to 12 antibiotics varied widely between strains under aerobic conditions. Under anaerobic conditions, no drugs caused significant growth inhibition (>0.5 log) except metronidazole, resulting in an average decrease of ~2 logs. CONCLUSIONS: These results demonstrate that MAP is capable of adaptation to NRP similar to that observed for MTB with differential susceptibility to antibiotics under aerobic versus anaerobic conditions. Such findings have significant implications for our understanding of the pathogenesis of MAP in vivo and the treatment of CD should this organism be established as the causative agent.
RESUMEN
The etiology of Crohn's disease (CD) remains controversial. It is hypothesized that CD is the result of an abnormal immune response to the gut flora in genetically susceptible hosts. However, an infectious etiology has not been completely ruled out. Antibiotics have been utilized with some success to modify the course of the disease. Here, we report a patient with CD and pyoderma gangrenosum refractory to standard therapy, including biologics, who achieved remission with a combination of rifaximin, gentamicin and metronidazole.
RESUMEN
Despite a rising demand for anti-obesity therapeutics, few effective pharmacological options are clinically available that target the synthesis and accumulation of body fat. Moderate inhibition of mammalian glycerol-3-phosphate acyltransferase (GPAT) with 2-(alkanesulfonamido)benzoic acids has recently been described in vitro, accompanied by promising weight loss in vivo. In silico docking studies with 2-(octanesulfonamido)benzoic acid modeled into the active site of squash GPAT revealed an unoccupied volume lined with hydrophobic residues proximal to C-4 and C-5 of the benzoic acid ring. In an effort to produce more potent GPAT inhibitors, a series of 4- and 5-substituted analogs were designed, synthesized, and evaluated for inhibitory activity. In general, compounds containing hydrophobic substituents at the 4- and 5-positions, such as biphenyl and alkylphenyl hydrocarbons, exhibited an improved inhibitory activity against GPAT in vitro. The most active compound, 4-([1,1'-biphenyl]-4-carbonyl)-2-(octanesulfonamido)benzoic acid, demonstrated an IC50 of 8.5 µM and represents the best GPAT inhibitor discovered to date. Conversely, further substitution with hydroxyl or fluoro groups, led to a 3-fold decrease in activity. These results are consistent with the presence of a hydrophobic pocket and may support the binding model as a potential tool for developing more potent inhibitors.
RESUMEN
Glycerol 3-phosphate acyltransferase (GPAT) isozymes are central control points for fat synthesis in mammals. Development of inhibitors of these membrane-bound enzymes could lead to an effective treatment for obesity, but is thwarted by an absence of direct structural information. Based on a highly successful study involving conformationally constrained glycerol 3-phosphate analogs functioning as potent glycerol 3-phosphate dehydrogenase inhibitors, several series of cyclic bisubstrate and transition state analogs were designed, synthesized, and tested as GPAT inhibitors. The weaker in vitro inhibitory activity of these compounds compared to a previously described benzoic acid series was then examined in docking experiments with the soluble squash chloroplast GPAT crystal structure. These in silico experiments indicate that cyclopentyl and cyclohexyl scaffolds prepared in this study may be occluded from the enzyme active site by two protein loops that sterically guard the phosphate binding region. In view of these findings, future GPAT inhibitor design will be driven toward compounds based on planar frameworks able to slide between these loops and enter the active site, resulting in improved inhibitory activity.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicerol-3-Fosfato O-Aciltransferasa/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Glicerol-3-Fosfato O-Aciltransferasa/química , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Humanos , Conformación Molecular , Estructura Molecular , Obesidad/tratamiento farmacológico , Obesidad/enzimología , Relación Estructura-ActividadRESUMEN
The incidence of obesity and other diseases associated with an increased triacylglycerol mass is growing rapidly, particularly in the United States. Glycerol 3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step of glycerolipid biosynthesis, the acylation of glycerol 3-phosphate with saturated long-chain acyl-CoAs. In an effort to produce small molecule inhibitors of this enzyme, a series of benzoic and phosphonic acids was designed and synthesized. In vitro testing of this series has led to the identification of several compounds, in particular 2-(nonylsulfonamido)benzoic acid (15g), possessing moderate GPAT inhibitory activity in an intact mitochondrial assay.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/antagonistas & inhibidores , Obesidad/tratamiento farmacológico , Organofosfonatos/síntesis química , Sulfonamidas/síntesis química , ortoaminobenzoatos/síntesis química , Acilación , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Glicéridos/biosíntesis , Glicerofosfatos/metabolismo , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Organofosfonatos/farmacología , Relación Estructura-Actividad , Sulfonamidas/farmacología , ortoaminobenzoatos/farmacologíaRESUMEN
Inhibition of brain carnitine palmitoyl-transferase-1 (CPT-1) is reported to decrease food intake and body weight in rats. Yet, the fatty acid synthase (FAS) inhibitor and CPT-1 stimulator C75 produces hypophagia and weight loss when given to rodents intracerebroventricularly (icv). Thus roles and relative contributions of altered brain CPT-1 activity and fatty acid oxidation in these phenomena remain unclarified. We administered compounds that target FAS or CPT-1 to mice by single icv bolus and examined acute and prolonged effects on feeding and body weight. C75 decreased food intake rapidly and potently at all doses (1-56 nmol) and dose dependently inhibited intake on day 1. Dose-dependent weight loss on day 1 persisted through 4 days of postinjection monitoring. The FAS inhibitor cerulenin produced dose-dependent (560 nmol) hypophagia for 1 day, weight loss for 2 days, and weight regain to vehicle control by day 3. The CPT-1 inhibitor etomoxir (32, 320 nmol) did not alter overall day 1 feeding. However, etomoxir attenuated the hypophagia produced by C75, indicating that CPT-1 stimulation is important for C75's effect. A novel compound, C89b, was characterized in vitro as a selective stimulator of CPT-1 that does not affect fatty acid synthesis. C89b (100, 320 nmol) decreased feeding in mice for 3 days and produced persistent weight loss for 6 days without producing conditioned taste aversion. Similarly, intraperitoneal administration decreased feeding and body weight without producing conditioned taste aversion. These results suggest a role for brain CPT-1 in the regulation of energy balance and implicate CPT-1 stimulation as a pharmacological approach to weight loss.
Asunto(s)
Peso Corporal/fisiología , Carnitina O-Palmitoiltransferasa/metabolismo , Ingestión de Alimentos/fisiología , Hipotálamo/enzimología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Animales , Peso Corporal/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Inhibidores de la Síntesis de Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Femenino , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Embarazo , RatasRESUMEN
PURPOSE: Fatty acid synthase (FAS) is overexpressed in many human cancers and is considered to be a promising target for therapy. However, in vitro use of previous generations of FAS inhibitors has been limited by severe, but reversible, anorexia in treated animals, which is thought to be related to a parallel stimulation of fatty acid oxidation by these agents. This study investigated pharmacologic inhibition of FAS using C93, a rationally designed molecule that inhibits FAS activity without affecting fatty acid oxidation in preclinical models of lung cancer. EXPERIMENTAL DESIGN: Activity of C93 on FAS and fatty acid oxidation was evaluated in cultured non-small cell lung cancer (NSCLC) cells. Antineoplastic activity of the compound, given orally or by i.p. injection, was evaluated in s.c. and orthotopic NSCLC xenografts. RESULTS: Our experiments confirm that C93 effectively inhibits FAS without stimulating fatty acid oxidation in lung cancer cells. More importantly, C93 significantly inhibits the growth of both s.c. and orthotopic xenograft tumors from human NSCLC cell lines without causing anorexia and weight loss in the treated animals. CONCLUSIONS: We conclude that inhibition of FAS can be achieved without parallel stimulation of fatty acid oxidation and that inhibition of tumor growth in vivo can be achieved without anorexia and weight loss. Thus, this therapeutic strategy holds promise for clinical treatment of cancers, including non-small cell lung cancer, the leading cause of cancer mortality in the United States and Europe.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Ácido Graso Sintasas/antagonistas & inhibidores , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Administración Oral , Animales , Anorexia/inducido químicamente , Antineoplásicos/toxicidad , Línea Celular Tumoral , Ácido Graso Sintasas/biosíntesis , Ácido Graso Sintasas/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/toxicidad , Ácidos Grasos/metabolismo , Humanos , Ratones , Ratones Desnudos , Oxidación-Reducción/efectos de los fármacos , Pérdida de Peso/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fatty acid synthase (FAS), the enzyme responsible for the de novo synthesis of fatty acids, is highly expressed in ovarian cancers and most common human carcinomas. Inhibition of FAS and activation of AMP-activated protein kinase (AMPK) have been shown to be cytotoxic to human cancer cells in vitro and in vivo. In this report, we explore the cytotoxic mechanism of action of FAS inhibition and show that C93, a synthetic FAS inhibitor, increases the AMP/ATP ratio, activating AMPK in SKOV3 human ovarian cancer cells, which leads to cytotoxicity. As a physiologic consequence of AMPK activation, acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis, was phosphorylated and inhibited whereas glucose oxidation was increased. Despite these attempts to conserve energy, the AMP/ATP ratio increased with worsening cellular redox status. Pretreatment of SKOV3 cells with compound C, an AMPK inhibitor, substantially rescued the cells from C93 cytotoxicity, indicating its dependence on AMPK activation. 5-(Tetradecyloxy)-2-furoic acid, an ACC inhibitor, did not activate AMPK despite inhibiting fatty acid synthesis pathway activity and was not significantly cytotoxic to SKOV3 cells. This indicates that substrate accumulation from FAS inhibition triggering AMPK activation, not end-product depletion of fatty acids, is likely responsible for AMPK activation. C93 also exhibited significant antitumor activity and apoptosis against SKOV3 xenografts in athymic mice without significant weight loss or cytotoxicity to proliferating cellular compartments such as bone marrow, gastrointestinal tract, or skin. Thus, pharmacologic FAS inhibition selectively activates AMPK in ovarian cancer cells, inducing cytotoxicity while sparing most normal human tissues from the pleiotropic effects of AMPK activation.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activación Enzimática , Ácidos Grasos/metabolismo , Femenino , Furanos/farmacología , Glucosa/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , NAD/metabolismo , Neoplasias Ováricas/metabolismo , Oxidación-Reducción , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fatty acid synthase (FAS) catalyzes the synthesis of palmitate from the sequential condensation of an acetyl primer with two carbon units added from malonyl-CoA. Inhibition of the beta-ketoacyl synthase domain of mammalian FAS leads to selective cytotoxicity to various cancer cell lines in vitro and in vivo. Also, inhibitors of FAS can cause reduced food intake and body weight in mice. Naturally occurring thiolactomycin (TLM) was used as a template to develop a new class of type I FAS inhibitors. Using a flexible synthesis, families of TLM structural analogues were obtained that possess selective FAS activity and display anticancer and weight loss effects. Compounds 13a and 13d inhibit pure FAS (ZR-75-1 breast cancer, IC(50) = Asunto(s)
Fármacos Antiobesidad/síntesis química
, Antineoplásicos/síntesis química
, Ácido Graso Sintasas/antagonistas & inhibidores
, Tiofenos/síntesis química
, Animales
, Fármacos Antiobesidad/química
, Fármacos Antiobesidad/farmacología
, Antineoplásicos/química
, Antineoplásicos/farmacología
, Peso Corporal/efectos de los fármacos
, Línea Celular Tumoral
, Ensayos de Selección de Medicamentos Antitumorales
, Ácido Graso Sintasas/química
, Humanos
, Ratones
, Ratones Endogámicos BALB C
, Modelos Moleculares
, Estereoisomerismo
, Relación Estructura-Actividad
, Tiofenos/química
, Tiofenos/farmacología
RESUMEN
C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the cytotoxic response. The continued ability of TOFA to rescue cancer cells from C75 cytotoxicity implies a proapoptotic role for malonyl-CoA independent of CPT-1 that selectively targets cancer cells as they progress into S phase.