RESUMEN
Drug-resistant Neisseria gonorrhoeae represents a major threat to public health; without new effective antibiotics, untreatable gonococcal infections loom as a real possibility. In a previous drug-repurposing study, we reported that salicylic acid had good potency against azithromycin-resistant N. gonorrhoeae. We now report that the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution, but that select substituted naphthyl analogs (3b, 3o and 3p) have superior activity to salicylic acid itself. Furthermore, these compounds retained potency against multiple ceftriaxone- and azithromycin-resistant strains, exhibited rapid bactericidal activity against N. gonorrhoeae, and showed high tolerability to mammalian cells (CC50 > 128 µg/mL). Promisingly, these compounds also show very weak growth inhibition of commensal vaginal bacteria.
Asunto(s)
Gonorrea , Neisseria gonorrhoeae , Animales , Femenino , Ácido Salicílico/farmacología , Azitromicina , Gonorrea/tratamiento farmacológico , Bacterias , MamíferosRESUMEN
Neisseria gonorrhoeae has been classified by the U.S. Centers for Disease Control and Prevention as an urgent threat due to the rapid development of antibiotic resistance to currently available antibiotics. Therefore, there is an urgent need to find new antibiotics to treat gonococcal infections. In our previous study, the gold-containing drug auranofin demonstrated potent in vitro activity against clinical isolates of N. gonorrhoeae, including multidrug-resistant strains. Therefore, the aim of this study was to investigate the in vivo activity of auranofin against N. gonorrhoeae using a murine model of vaginal infection. A significant reduction in N. gonorrhoeae recovered from the vagina was observed for infected mice treated with auranofin compared to the vehicle over the course of treatment. Relative to the vehicle, after three and five days of treatment with auranofin, a 1.04 (91%) and 1.40 (96%) average log10-reduction of recovered N. gonorrhoeae was observed. In conclusion, auranofin has the potential to be further investigated as a novel, safe anti-gonococcal agent to help meet the urgent need for new antimicrobial agents for N. gonorrhoeae infection.
Asunto(s)
Gonorrea , Infecciones del Sistema Genital , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Auranofina/farmacología , Auranofina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Oro/uso terapéutico , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Humanos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae , Infecciones del Sistema Genital/tratamiento farmacológicoRESUMEN
Gonococcal infections represent an urgent public health threat worldwide due to the increasing incidence of infections that has been accompanied by an increase in bacterial resistance to most antibiotics. This has resulted in a dwindling number of effective treatment options. Undoubtedly, there is a critical need to develop new, effective anti-gonococcal agents. In an effort to discover new anti-gonococcal therapeutics, we previously identified acetazolamide, a carbonic anhydrase inhibitor, as a novel inhibitor of Neisseria gonorrhoeae. Acetazolamide exhibited potent anti-gonococcal activity in vitro as it inhibited growth of strains of N. gonorrhoeae at concentrations that ranged from 0.5 to 4 µg/mL. The aim of this study was to investigate the in vivo efficacy of acetazolamide in a mouse model of N. gonorrhoeae genital tract infection. Compared to vehicle-treated mice, acetazolamide significantly reduced the gonococcal burden by 90% in the vagina of infected mice after three days of treatment. These results indicate that acetazolamide warrants further investigation as a promising treatment option to supplement the limited pipeline of anti-gonococcal therapeutics.
Asunto(s)
Gonorrea , Acetazolamida/farmacología , Acetazolamida/uso terapéutico , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Ratones , Neisseria gonorrhoeaeRESUMEN
Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide's Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.
Asunto(s)
Acetazolamida/farmacología , Antibacterianos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Etoxzolamida/farmacología , Neisseria gonorrhoeae/efectos de los fármacos , Acetazolamida/síntesis química , Acetazolamida/química , Antibacterianos/síntesis química , Antibacterianos/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Etoxzolamida/síntesis química , Etoxzolamida/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Neisseria gonorrhoeae/enzimología , Relación Estructura-Actividad , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Vancomycin-resistant enterococci (VRE) are a serious public health threat and a leading cause of healthcare-associated infections. Bacterial resistance to antibiotics recommended for the treatment of enterococcal infections complicates the management of these infections. Hence, there is a critical need for the discovery of new anti-VRE agents. We previously reported carbonic anhydrase inhibitors (CAIs) as new potent VRE inhibitors. In the present study, the activity of the CAI, dorzolamide was evaluated against VRE both in vitro and in vivo. Dorzolamide exhibited potent activity against a panel of clinical VRE isolates, with minimum inhibitory concentration (MIC) values ranging from 1 µg/mL to 8 µg/mL. A killing kinetics experiment determined that dorzolamide exhibited a bacteriostatic effect against VRE, which was similar to the drug of choice (linezolid). Dorzolamide interacted synergistically with gentamicin against four strains of VRE, and exhibited an additive interaction with gentamicin against six VRE strains, reducing gentamicin's MIC by several folds. Moreover, dorzolamide outperformed linezolid in an in vivo VRE colonization reduction mouse model. Dorzolamide significantly reduced the VRE burden in fecal samples of mice by 2.9-log10 (99.9%) and 3.86-log10 (99.99%) after 3 and 5 days of treatment, respectively. Furthermore, dorzolamide reduced the VRE count in the cecal (1.74-log10 (98.2%) reduction) and ileal contents (1.5-log10 (96.3%)) of mice, which was superior to linezolid. Collectively, these results indicate that dorzolamide represents a promising treatment option that warrants consideration as a supplement to current therapeutics used for VRE infections.
RESUMEN
Neisseria gonorrhoeae is an urgent threat to public health in the United States and around the world. Many of the current classes of antibiotics to treat N. gonorrhoeae infection are quickly becoming obsolete due to increased rates of resistance. Thus, there is a critical need for alternative antimicrobial targets and new chemical entities. Our team has repurposed the FDA-approved carbonic anhydrase inhibitor scaffold of acetazolamide to target N. gonorrhoeae and the bacteria's essential carbonic anhydrase, NgCA. This study established both structure-activity and structure-property relationships that contribute to both antimicrobial activity and NgCA activity. This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 µg/mL equating to an 8- to 16-fold improvement in antigonococcal activity compared to acetazolamide. These analogues were determined to be bacteriostatic against the pathogen and likely on-target against NgCA. Additionally, they did not exhibit any detrimental effects in cellular toxicity assays against both a human endocervical (End1/E6E7) cell line or colorectal adenocarcinoma cell line (Caco-2) at concentrations up to 128 µg/mL. Taken together, this study presents a class of antigonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models.
Asunto(s)
Inhibidores de Anhidrasa Carbónica , Neisseria gonorrhoeae , Acetazolamida/farmacología , Células CACO-2 , Inhibidores de Anhidrasa Carbónica/farmacología , Humanos , Relación Estructura-ActividadRESUMEN
The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-ß) expression in lungs, together with an increased expression of TGF-ß related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin ß3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Apoptosis/fisiología , Líquido del Lavado Bronquioalveolar/citología , Granuloma/inducido químicamente , Granuloma/metabolismo , Nanotubos de Carbono/efectos adversos , Fagocitosis/fisiología , Animales , Lavado Broncoalveolar/métodos , Modelos Animales de Enfermedad , Enfermedad Granulomatosa Crónica/metabolismo , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/metabolismo , Fibrosis Pulmonar/metabolismo , Sarcoidosis Pulmonar/metabolismoRESUMEN
Human exposure to carbon nanotubes (CNT) has been associated with the development of pulmonary sarcoid-like granulomatous disease. Our previous studies demonstrated that multi-walled carbon nanotubes (MWCNT) induced chronic pulmonary granulomatous inflammation in mice. Granuloma formation was accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) and disrupted intracellular lipid homeostasis in alveolar macrophages. Others have shown that PPARγ activation increases mitochondrial fatty acid oxidation (FAO) to reduce free fatty acid accumulation. Hence, we hypothesized that the disrupted lipid metabolism suppresses mitochondrial FAO. To test our hypothesis, C57BL/6 J mice were instilled by an oropharyngeal route with 100 µg MWCNT freshly suspended in 35 % Infasurf. Control sham mice received vehicle alone. Sixty days following instillation, mitochondrial FAO was measured in permeabilized bronchoalveolar lavage (BAL) cells. MWCNT instillation reduced the mitochondrial oxygen consumption rate of BAL cells in the presence of palmitoyl-carnitine as mitochondrial fuel. MWCNT also reduced mRNA expression of mitochondrial genes regulating FAO, carnitine palmitoyl transferase-1 (CPT1), carnitine palmitoyl transferase-2 (CPT2), hydroxyacyl-CoA dehydrogenase subunit beta (HADHB), and PPARγ coactivator 1 alpha (PPARGC1A). Importantly, both oxidative stress and apoptosis in alveolar macrophages and lung tissues of MWCNT-instilled mice were increased. Because macrophage PPARγ expression has been reported to be controlled by miR-27b which is known to induce oxidative stress and apoptosis, we measured the expression of miR-27b. Results indicated elevated levels in alveolar macrophages from MWCNT-instilled mice compared to controls. Given that inhibition of FAO and apoptosis are linked to M1 and M2 macrophage activation, respectively, the expression of both M1 and M2 key indicator genes were measured. Interestingly, results showed that both M1 and M2 phenotypes of alveolar macrophages were activated in MWCNT-instilled mice. In conclusion, alveolar macrophages of MWCNT-instilled mice had increased miR-27b expression, which may reduce the expression of PPARγ resulting in attenuation of FAO. This reduction in FAO may lead to activation of M1 macrophages. The upregulation of miR-27b may also induce apoptosis, which in turn can cause M2 activation of alveolar macrophages. These observations indicate a possible role of miR-27b in impaired mitochondrial function in the chronic activation of alveolar macrophages by MWCNT and the development of chronic pulmonary granulomatous inflammation.
Asunto(s)
Enfermedad Granulomatosa Crónica/inducido químicamente , Enfermedades Pulmonares/inducido químicamente , Macrófagos Alveolares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Animales , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/patología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiologíaRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Artemisinin derivatives, including the dihydroartemisinin (DHA) monomers, are widely used as clinical agents for the treatment of malaria. Numerous studies demonstrate that these molecules also display antineoplastic activity with minimal toxicity. Of interest, dimeric DHA molecules are more active than their monomeric counterparts. Our previous data showed that the DHA dimer, NSC735847, was a potent inducer of death in different cancer cell types. However, the mechanism of action and activity of NSC735847 in colon cancer cells was not explored. The present study investigated the anticancer activity of NSC735847 and four structurally similar analog in human tumorigenic (HT-29 and HCT-116) and non-tumorigenic (FHC) colon cell lines. NSC735847 was more cytotoxic toward tumorigenic than non-tumorigenic colonocytes. In addition, NSC735847 exhibited greater cytotoxicity and tumor selectivity than the NSC735847 derivatives. To gain insight into mechanisms of NSC735847 activity, the requirement for endoplasmic reticulum (ER) stress and oxidative stress was tested. The data show that ER stress played a key role in the cytotoxicity of NSC735847 while oxidative stress had little impact on cell fate. In addition, it was observed that the cytotoxic activity of NSC735847 required the presence of heme, but not iron. The activity of NSC735847 was then compared to clinically utilized CRC therapeutics. NSC735847 was cytotoxic toward colon tumor cells at lower concentrations than oxaliplatin (OX). In addition, cell death was achieved at lower concentrations in colon cancer cells that were co-treated with folinic acid (Fol), 5-FU (F), and NSC735847 (FolFNSC), than Fol, F, and OX (FolFOX). The selective activity of NSC735847 and its ability to induce cytotoxicity at low concentrations suggest that NSC735847 may be an alternative for oxaliplatin in the FolFOX regimen for patients who are unable to tolerate its adverse effects.
RESUMEN
The phosphodiesterase-3 inhibitor, cilostazol has been recently shown to protect against chemically induced colitis in animal models. However, whether cyclic adenosine monophosphate (cAMP) contributes to the anti-inflammatory activity of cilostazol in colitis is still unknown. In the current study, we investigated the role of cAMP/silent information regulator-1 (SIRT-1) pathway in the protective effect of cilostazol using rat model of acetic acid-induced colitis. Upregulation of SIRT1 activity and expression has been recently shown to protect against chemically induced colitis. Our results demonstrated that cilostazol alleviated the histopathological changes associated with acetic acid-induced colitis. Interestingly, pre-administration of cilostazol increased cAMP concentration and SIRT1 expression in colonic mucosa to levels similar to that observed in control animals without induction of colitis. In addition, cilostazol inhibited the SIRT1 targets; NF-κB, Akt and MAPK inflammatory pathways as demonstrated by suppression of acetic acid-induced upregulation of NF-κB activity, p-AKT levels and the expression of p38 MAPK. NF-κB activity and the levels of p-AKT, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) were similar in rats pretreated with cilostazol prior to induction of colitis and the control rats without colitis. Furthermore, cilostazol reduced acetic acid-induced oxidative stress and apoptosis. In conclusion, the protective effect of cilostazol against acetic acid-induced colitis may be attributed to activation of SIRT1 expression by cAMP. SIRT1 is suggested to contribute to cilostazol-induced suppression of NF-κB, Akt and MAPK inflammatory pathways, oxidative stress and apoptosis.
Asunto(s)
Antiinflamatorios/farmacología , Cilostazol/farmacología , Colitis/prevención & control , Colon/efectos de los fármacos , AMP Cíclico/metabolismo , Sirtuina 1/metabolismo , Ácido Acético , Animales , Colitis/inducido químicamente , Colitis/enzimología , Colitis/patología , Colon/enzimología , Colon/patología , Citoprotección , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Transducción de Señal , Sirtuina 1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The combined incidence of melanoma and non-melanoma skin cancer (NMSC) is greater than the incidence of all other malignancies in the US. Previously, we demonstrated that the endocannabinoid, arachidonoyl-ethanolamide (AEA), was a potent inducer of apoptosis in NMSC. The metabolism of AEA to the prostaglandin, PGD2-EA, was a prerequisite for AEA cytotoxicity. However, the mechanism of PGD2-EA cell death has not been clearly defined. In the present study, we report that PGD2-EA causes apoptosis in melanoma and NMSC cells. Mass spectrometry analysis revealed that PGD2-EA was dehydrated to three J-series prostaglandins; PGJ2-EA, Δ12PGJ2-EA, and 15deoxy,Δ12,14 PGJ2-EA. PGD2-EA inhibited the antioxidant activity of glutathione and thioredoxin which then caused oxidative stress. This increase in oxidative stress was accompanied by the activation of endoplasmic reticulum (ER) stress and apoptosis. The effect of PGD2-EA was independent of DP1, DP2, and PPARγ receptors suggesting that PGD2-EA cytotoxicity was mediated by its metabolic product, 15dPGJ2-EA.
Asunto(s)
Apoptosis/efectos de los fármacos , Prostaglandina D2/química , Prostaglandina D2/farmacología , Neoplasias Cutáneas/patología , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glutatión/metabolismo , Melanoma/patología , Ratones , Estrés Oxidativo/efectos de los fármacos , Tiorredoxinas/metabolismoRESUMEN
Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837-0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucellaâ FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortusâ ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortusâ ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host.
Asunto(s)
Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Brucelosis/microbiología , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Factores de Virulencia/metabolismo , Animales , Brucella abortus/genética , Brucella abortus/crecimiento & desarrollo , Brucelosis/patología , Células Cultivadas , Medios de Cultivo/química , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Radioisótopos de Hierro/metabolismo , Marcaje Isotópico , Macrófagos/inmunología , Macrófagos/microbiología , Proteínas de Transporte de Membrana/genética , Ratones , Factores de Virulencia/genéticaRESUMEN
The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants.