RESUMEN
Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-ß (TGF-ß) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFß-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.
Asunto(s)
Hipertensión Pulmonar , Músculo Liso Vascular , Inhibidor 1 de Activador Plasminogénico , Remodelación Vascular , Animales , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ratones , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Transducción de Señal , Masculino , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proliferación Celular , Ratones Noqueados , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Apoptosis , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Hipertrofia Ventricular Derecha/metabolismo , Hipertrofia Ventricular Derecha/patología , Hipertrofia Ventricular Derecha/fisiopatología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Inhibidor 2 de Activador Plasminogénico/metabolismo , Inhibidor 2 de Activador Plasminogénico/genéticaRESUMEN
Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.
Asunto(s)
Canales Iónicos Sensibles al Ácido , Amilorida , Ratas , Animales , Canales Iónicos Sensibles al Ácido/farmacología , Canales Iónicos Sensibles al Ácido/fisiología , Amilorida/farmacología , NeuronasRESUMEN
The K+-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N,N-hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure-property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies.
Asunto(s)
Amilorida/análogos & derivados , Amilorida/farmacología , Antineoplásicos/farmacología , Amilorida/farmacocinética , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Células CACO-2 , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Microsomas Hepáticos/efectos de los fármacos , Estructura Molecular , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.
Asunto(s)
Amilorida/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Intercambiador 1 de Sodio-Hidrógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Amilorida/síntesis química , Amilorida/química , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Diuréticos/síntesis química , Diuréticos/química , Diuréticos/farmacología , Femenino , Humanos , Modelos Moleculares , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Intercambiador 1 de Sodio-Hidrógeno/antagonistas & inhibidores , Relación Estructura-Actividad , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
OBJECTIVES: The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D ('DiEthylAmin-Cephalosporin-3'-Diazeniumdiolate') has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. METHODS: ß-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. RESULTS: DEA-C3D was confirmed to selectively release NO in response to contact with bacterial ß-lactamase. Despite lacking direct, cephalosporin/ß-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. CONCLUSIONS: DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.
Asunto(s)
Biopelículas/efectos de los fármacos , Cefalosporinas/farmacología , Fibrosis Quística/microbiología , Donantes de Óxido Nítrico/farmacología , Profármacos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Adolescente , Antibacterianos/farmacología , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Adulto JovenRESUMEN
Fatty acids are a structurally diverse category of lipids with a myriad of biochemical functions, which includes their role as building blocks of more complex lipids (e.g., glycerophospholipids and triacylglycerols). Increasingly, the analysis of fatty acids is undertaken using liquid chromatography-mass spectrometry (LC-MS), due to its versatility in the detection of lipids across a wide range of concentrations and diversity of molecular structures and masses. Previous work has shown that fixed-charge pyridinium derivatives are effective in enhancing the detection of fatty acids in LC-MS workflows. Herein, we describe the development of two novel pyridinium fixed-charged derivatization reagents that incorporate a photolabile aryl iodide that is selectively activated by laser irradiation inside the mass spectrometer. Photodissociation mass spectra of fatty acids conjugated to 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) and 1-(4-(aminomethyl)-3-iodophenyl)pyridin-1-ium (3-I-AMPP+) derivatives reveal structurally diagnostic product ions. These spectra feature radical-directed dissociation of the carbon-carbon bonds within the fatty acyl chain, enabling structural assignments of fatty acids and discrimination of isomers that differ in site(s) of unsaturation, methyl branching or cyclopropanation. These derivatives are shown to be suitable for hyphenated LC-MS methods, and their predictable photodissociation behavior allows de novo identification of unusual fatty acids within a biological context.
Asunto(s)
Ácidos Grasos/química , Procesos Fotoquímicos , Cromatografía Liquida , Yodo/química , Espectrometría de Masas , Compuestos de Piridinio/químicaRESUMEN
Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.
Asunto(s)
Antineoplásicos/síntesis química , Citotoxinas/química , Profármacos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Alcoholes/química , Aminas/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Citotoxinas/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Floxuridina/química , Humanos , Indoles/química , Indoles/farmacología , Estructura Molecular , Profármacos/farmacología , Prueba de Estudio Conceptual , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/química , Pirroles/farmacología , Relación Estructura-Actividad , Hipoxia TumoralRESUMEN
The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Kiâ¯=â¯2.4⯵M), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.
Asunto(s)
Amilorida/análogos & derivados , Amilorida/uso terapéutico , Diuréticos/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Amilorida/farmacología , Diuréticos/farmacología , Humanos , Relación Estructura-ActividadRESUMEN
The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.
Asunto(s)
Ácidos Grasos/química , Ácidos Grasos/síntesis química , Espectrometría de Masas , Técnicas de Química Sintética , Ésteres/química , Humanos , Glándulas Tarsales/química , EstereoisomerismoRESUMEN
Bacterial sliding clamps bind to DNA and act as protein-protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding clamps via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of ß sliding clamps from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli clamps and similar patterns of binding to linear clamp-binding motif peptides. The results suggest that linear motif-sliding clamp interactions are well conserved and an antibiotic targeting the sliding clamp should have broad-spectrum activity against Gram-negative pathogens.
Asunto(s)
Acinetobacter baumannii/genética , ADN Bacteriano/química , Enterobacter cloacae/genética , Pseudomonas aeruginosa/genética , Algoritmos , Secuencias de Aminoácidos/genética , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Cristalografía por Rayos X , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64⯵g/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.
Asunto(s)
Antibacterianos/química , Pirazoles/química , Tiocarbamatos/química , Tiocarbamatos/farmacología , Antibacterianos/síntesis química , Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pirazoles/síntesis química , Pirazoles/farmacología , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Tiocarbamatos/síntesis químicaRESUMEN
Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en-MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en-MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Indoles/farmacología , Azul de Metileno/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Indoles/química , Azul de Metileno/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Relación Estructura-ActividadRESUMEN
PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking ß-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.
Asunto(s)
Compuestos Azo/farmacología , Biopelículas/efectos de los fármacos , Cefalosporinas/farmacología , Haemophilus influenzae/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Profármacos/farmacología , Antibacterianos/farmacología , Azitromicina/farmacología , Cromatografía Liquida , Farmacorresistencia Bacteriana , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Óxidos de Nitrógeno/metabolismo , Proteómica , beta-Lactamasas/metabolismoRESUMEN
Non-typeable Haemophilus influenzae (NTHi) is the most common pathogen in primary ciliary dyskinesia (PCD) patients. We hypothesised that abnormal ciliary motility and low airway nitric oxide (NO) levels on airway epithelial cells from PCD patients might be permissive for NTHi colonisation and biofilm development.We used a primary epithelial cell co-culture model to investigate NTHi infection. Primary airway epithelial cells from PCD and non-PCD patients were differentiated to ciliation using an air-liquid interface culture and then co-cultured with NTHi.NTHi adherence was greater on PCD epithelial cells compared to non-PCD cells (p<0.05) and the distribution of NTHi on PCD epithelium showed more aggregated NTHi in biofilms (p<0.001). Apart from defective ciliary motility, PCD cells did not significantly differ from non-PCD epithelial cells in the degree of ciliation and epithelial integrity or in cytokine, LL-37 and NO production. Treatment of PCD epithelia using exogenous NO and antibiotic significantly reduced NTHi viability in biofilms compared with antibiotic treatment alone.Impaired ciliary function was the primary defect in PCD airway epithelium underlying susceptibility to NTHi biofilm development compared with non-PCD epithelium. Although NO responses were similar, use of targeted NO with antibiotics enhanced killing of NTHi in biofilms, suggesting a novel therapeutic approach.
Asunto(s)
Células Epiteliales/microbiología , Infecciones por Haemophilus/fisiopatología , Síndrome de Kartagener/microbiología , Óxido Nítrico/farmacología , Adolescente , Adulto , Antibacterianos/farmacología , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Estudios de Casos y Controles , Niño , Preescolar , Citocinas/metabolismo , Femenino , Haemophilus influenzae/patogenicidad , Haemophilus influenzae/fisiología , Humanos , Síndrome de Kartagener/fisiopatología , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Adulto JovenRESUMEN
Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3'-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with ß-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following ß-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (ß-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks ß-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of ß-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the ß-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Compuestos Azo/farmacología , Biopelículas/efectos de los fármacos , Cefalosporinas/farmacología , Donantes de Óxido Nítrico/farmacología , Profármacos/farmacología , Streptococcus pneumoniae/efectos de los fármacos , Amoxicilina/farmacología , Antiinflamatorios no Esteroideos/química , Azitromicina/farmacología , Compuestos Azo/química , Cefalosporinas/química , Óxido Nítrico/análisis , Donantes de Óxido Nítrico/química , Penicilinasa/química , Plancton/microbiología , Profármacos/químicaRESUMEN
RATIONALE: (O-acyl)-hydroxy fatty acids (OAHFAs) are a recently discovered class of endogenous lipids, generating significant interest for their correlation with enhanced glucose tolerance. Structural variants that differ in the position of the ester linkage have been described, including the ω-OAHFA sub-class, that plays a key role in stabilizing the human tear film. Developing analytical tools for rapid and unambiguous structural elucidation of OAHFAs is essential to understanding their diverse physiological functions. METHODS: Commercially available and synthesized OAHFA standards were dissolved in chloroform and subsequently diluted into methanol with 1.5 mM ammonium acetate. Negative ion collision-induced dissociation (CID) MSn spectra were acquired using chip-based nano-electrospray ionization (Advion TriVersa NanoMate) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). RESULTS: Major product ions observed during CID of [OAHFA - H]- ions readily identify the constituent fatty acid and hydroxy fatty acid; however, isomers are not easily distinguished. Interrogation of the hydroxy fatty acid and dehydrated hydroxy fatty acid product ions by MSn and ion-molecule reactions yielded diagnostic ions that readily pinpoint hydroxylation position and, thus, the OAHFA ester location. Conversely, these ions are characteristically absent in the MS3 spectra of ω-OAHFAs. Unimolecular dissociation mechanisms are proposed, which are shown to be consistent with prior isotopic labelling experiments. CONCLUSIONS: A mechanistic rationale is provided to explain the unimolecular dissociation of [OAHFA - H]- ions in an ion trap mass spectrometer, thus enabling near-complete de novo structural elucidation of OAHFAs in shotgun lipidomics workflows, even if synthetic standards are unavailable for comparison. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Ésteres/química , Ácidos Grasos/química , Humanos , Isomerismo , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Lágrimas/químicaRESUMEN
Sunitinib (Sutent®) is a receptor tyrosine kinase (RTK) and angiogenesis inhibitor approved for the treatment of renal cell carcinomas, gastrointestinal stromal tumours and pancreatic neuroendocrine tumours. A key structural motif retained throughout medicinal chemistry efforts during sunitinib's development was the indoline-2-one group. In the search for new anti-angiogenic scaffolds, we previously reported that non-indoline-2-one-based derivatives of semaxanib (SU5416, a structurally simpler sunitinib predecessor that underwent Phase III trials) are active as angiogenesis inhibitors, indicating that the group is not essential for activity. This Letter describes the synthesis and structure-activity relationships of another class of non-indoline-2-one angiogenesis inhibitors related to sunitinib/semaxanib; the 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones. A focussed library of 19 analogues was prepared using a simple novel process, wherein commercially available substituted arylacetic acids activated with an amide coupling reagent (HBTU) were reacted with the potassium salt of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde in one-pot. Screening of the library using a cell-based endothelial tube formation assay identified 6 compounds with anti-angiogenesis activity. Two of the compounds were advanced to the more physiologically relevant rat aortic ring assay, where they showed similar inhibitory effects to semaxanib at 10µg/mL, confirming that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones represent a new class of angiogenesis inhibitors.
Asunto(s)
Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Indoles/química , Indoles/farmacología , Pirroles/química , Pirroles/farmacología , Inhibidores de la Angiogénesis/síntesis química , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Indoles/síntesis química , Metilación , Modelos Moleculares , Neovascularización Fisiológica/efectos de los fármacos , Pirroles/síntesis química , Ratas , SunitinibRESUMEN
The emergence of multidrug-resistant bacterial strains has heightened the need for new antimicrobial agents based on novel chemical scaffolds that are able to circumvent current modes of resistance. We recently developed a whole-animal drug-screening methodology in pursuit of this goal and now report the discovery of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC) as a novel antibacterial effective against resistant nosocomial pathogens. The minimum inhibitory concentrations (MIC) of PSPC against Staphylococcus aureus and Enterococcus faecium were 4 µg/mL and 8 µg/mL, respectively, whereas the MICs were higher against the Gram-negative bacteria Klebsiella pneumoniae (64 µg/mL), Acinetobacter baumannii (32 µg/mL), Pseudomonas aeruginosa (>64 µg/mL), and Enterobacter spp. (>64 µg/mL). However, co-treatment of PSPC with the efflux pump inhibitor phenylalanine arginyl ß-naphthylamide (PAßN) or with sub-inhibitory concentrations of the lipopeptide antibiotic polymyxin B reduced the MICs of PSPC against the Gram-negative strains by >4-fold. A sulfide analog of PSPC (PSPC-1S) showed no antibacterial activity, whereas the sulfoxide analog (PSPC-6S) showed identical activity as PSPC across all strains, confirming structure-dependent activity for PSPC and suggesting a target-based mechanism of action. PSPC displayed dose dependent toxicity to both Caenorhabditis elegans and HEK-293 mammalian cells, culminating with a survival rate of 16% (100 µg/mL) and 8.5% (64 µg/mL), respectively, at the maximum tested concentration. However, PSPC did not result in hemolysis of erythrocytes, even at a concentration of 64 µg/mL. Together these results support PSPC as a new chemotype suitable for further development of new antibiotics against Gram-positive and Gram-negative bacteria.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pirazinas/farmacología , Animales , Caenorhabditis elegans , Dipéptidos/farmacología , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Polimixina B/farmacología , Pirazinas/sangre , Ovinos , Vancomicina/farmacologíaRESUMEN
Berberine-INF55 hybrids are a promising class of antibacterials that combine berberine and the NorA multidrug resistance pump inhibitor INF55 (5-nitro-2-phenylindole) together in one molecule via a chemically stable linkage. Previous studies demonstrated the potential of these compounds for countering efflux-mediated antibacterial drug resistance but they didn't establish whether the compounds function as originally intended, i.e. with the berberine moiety providing antibacterial activity and the attached INF55 component independently blocking multidrug resistance pumps, thereby enhancing the activity of berberine by reducing its efflux. We hypothesised that if the proposed mechanism is correct, then hybrids carrying more potent INF55 pump inhibitor structures should show enhanced antibacterial effects relative to those bearing weaker inhibitors. Two INF55 analogues showing graded reductions in NorA inhibitory activity compared with INF55 were identified and their corresponding berberine-INF55 hybrids carrying equivalent INF55 inhibitor structures synthesised. Multiple assays comparing the antibacterial effects of the hybrids and their corresponding berberine-INF55 analogue combinations showed that the three hybrids all show very similar activities, leading us to conclude that the antibacterial mechanism(s) of berberine-INF55 hybrids is different from berberine-INF55 combinations.