RESUMEN
Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.
Asunto(s)
Adenina/análogos & derivados , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Antibacterianos/farmacología , Bordetella pertussis/efectos de los fármacos , Profármacos/farmacología , Adenina/metabolismo , Adenina/farmacología , Toxina de Adenilato Ciclasa/metabolismo , Animales , Antibacterianos/metabolismo , Bordetella pertussis/crecimiento & desarrollo , Bordetella pertussis/patogenicidad , Células CACO-2 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Organofosfonatos/farmacología , Profármacos/metabolismoRESUMEN
A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.
Asunto(s)
Actinobacillus pleuropneumoniae/metabolismo , Toxina de Adenilato Ciclasa/metabolismo , Proteínas Bacterianas/metabolismo , Bordetella pertussis/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólisis , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Toxina de Adenilato Ciclasa/química , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Células Cultivadas , Eritrocitos/metabolismo , Proteínas Hemolisinas/antagonistas & inhibidores , Proteínas Hemolisinas/química , Hexoquinasa , Membrana Dobles de Lípidos/metabolismo , Macrófagos , Ratones , Presión Osmótica , Fosfato de Piridoxal/análogos & derivados , Colorantes de RosanilinaRESUMEN
A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC50 =9-18â nM), and one of them was more potent EF inhibitor (IC50 =12â nM), compared to adefovir diphosphate (PMEApp) with IC50 =18â nM for ACT and IC50 =36â nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC50 =490â nM compared to IC50 =150â nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics.
Asunto(s)
Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Inhibidores de Adenilato Ciclasa/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Halógenos/farmacología , Organofosfonatos/farmacología , Tiazoles/farmacología , Toxina de Adenilato Ciclasa/metabolismo , Inhibidores de Adenilato Ciclasa/síntesis química , Inhibidores de Adenilato Ciclasa/química , Antígenos Bacterianos/metabolismo , Bacillus anthracis/química , Toxinas Bacterianas/metabolismo , Bordetella pertussis/enzimología , Relación Dosis-Respuesta a Droga , Halógenos/química , Estructura Molecular , Organofosfonatos/química , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC50 as low as 37 nM) and B. anthracis edema factor (IC50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.
Asunto(s)
Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Inhibidores de Adenilato Ciclasa/farmacología , Antibacterianos/farmacología , Organofosfonatos/farmacología , Tiazoles/farmacología , Toxina de Adenilato Ciclasa/metabolismo , Inhibidores de Adenilato Ciclasa/síntesis química , Inhibidores de Adenilato Ciclasa/química , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Bacillus anthracis/efectos de los fármacos , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/enzimología , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Neuralgia/tratamiento farmacológico , Organofosfonatos/química , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC50 >10â µm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC50 values ranging from 55 to 362â nm.
Asunto(s)
Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Inhibidores de Adenilato Ciclasa/química , Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Nucleósidos/química , Organofosfonatos/química , Inhibidores de Adenilato Ciclasa/farmacología , Antígenos Bacterianos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Simulación del Acoplamiento Molecular , Nucleósidos/farmacología , Organofosfonatos/farmacología , Unión Proteica , Conformación ProteicaRESUMEN
Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussisBbronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to Bpertussis CyaA. The hemolytic activity of Bbronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, Bbronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of Bbronchiseptica CyaA based on its enzyme activity were markedly attenuated. Bparapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to Bbronchiseptica CyaA; however, Bpertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, Bpertussis CyaA impaired the barrier function of epithelial cells, whereas Bbronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/Bparapertussis and B. pertussisIMPORTANCEBordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.
Asunto(s)
Proteínas 14-3-3/metabolismo , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Infecciones por Bordetella/patología , Bordetella bronchiseptica/patogenicidad , Bordetella parapertussis/patogenicidad , Bordetella pertussis/patogenicidad , Factores de Virulencia/antagonistas & inhibidores , Toxina de Adenilato Ciclasa/metabolismo , Animales , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Eritrocitos/efectos de los fármacos , Eritrocitos/fisiología , Hemólisis , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ratas , Ovinos , Factores de Virulencia/metabolismoRESUMEN
Although CyaA has been studied for over three decades and revealed itself to be a very good prototype for developing various biotechnological applications, only a little is known about its functional dynamics and about the conformational landscape of this protein. Molecular dynamics simulations helped to clarify the view on these points in the following way. First, the model of interaction between AC and calmodulin (CaM) has evolved from an interaction centered on the surface between C-CaM hydrophobic patch and the α helix H of AC, to a more balanced view, in which the C-terminal tail of AC along with the C-CaM Calcium loops play an important role. This role has been confirmed by the reduction of the affinity of AC for calmodulin in the presence of R338, D360 and N347 mutations. In addition, enhanced sampling studies have permitted to propose a representation of the conformational space for the isolated AC. It remains to refine this representation using structural low resolution information measured on the inactive state of AC. Finally, due to a virtual screening study on another adenyl cyclase from Bacillus anthracis, weak inhibitors of AC have been discovered.
Asunto(s)
Toxina de Adenilato Ciclasa/química , Modelos Moleculares , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Toxina de Adenilato Ciclasa/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Dominio CatalíticoRESUMEN
Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B.â pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145â µM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.
Asunto(s)
Adenina/análogos & derivados , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Bordetella pertussis/metabolismo , Organofosfonatos/química , Profármacos/química , Adenina/síntesis química , Adenina/química , Adenina/toxicidad , Toxina de Adenilato Ciclasa/metabolismo , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Semivida , Humanos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Organofosfonatos/síntesis química , Organofosfonatos/toxicidad , Profármacos/farmacocinética , Profármacos/toxicidad , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas WistarRESUMEN
Whooping cough is a very important medical problem that requires novel approaches for treatment. The disease is caused by Bordetella pertussis, with the calmodulin (CaM)-activated adenylyl cyclase (AC) toxin (also known as CyaA) being a major virulence factor. Hence, CyaA inhibitors could constitute novel therapeutics, but it has been difficult to develop potent drugs with high selectivity over mammalian membranous ACs (mACs). Recent studies have shown that bis-anthraniloyl-substituted nucleoside 5'-triphosphates are potent and selective CyaA inhibitors. In addition, the interaction of CyaA with CaM is very different from the interaction of membranous mAC1 with CaM. Accordingly, compounds that interfere with the CyaA-CaM interaction may constitute a novel class of drugs against whooping cough.
Asunto(s)
Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Antibacterianos/farmacología , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/enzimología , Inhibidores Enzimáticos/farmacología , Toxina de Adenilato Ciclasa/química , Antibacterianos/química , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformación Proteica , Ribonucleótidos/química , Ribonucleótidos/farmacologíaRESUMEN
Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-alpha, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.
Asunto(s)
Toxina de Adenilato Ciclasa/química , Toxina de Adenilato Ciclasa/fisiología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/fisiología , Bordetella pertussis/enzimología , Bordetella pertussis/inmunología , Inmunidad Activa , Inmunidad Innata , Acilación , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Toxina de Adenilato Ciclasa/aislamiento & purificación , Adyuvantes Inmunológicos/antagonistas & inhibidores , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Antígeno CD11b/fisiología , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/inmunología , Diferenciación Celular/inmunología , Línea Celular , Células Cultivadas , AMP Cíclico/química , Citocinas/metabolismo , Pruebas Inmunológicas de Citotoxicidad , Proteínas de Unión al ADN/fisiología , Células Dendríticas/citología , Células Dendríticas/inmunología , Activación Enzimática/inmunología , Femenino , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Oligodesoxirribonucleótidos/química , Receptores de Superficie Celular/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 9RESUMEN
Edema factor (EF) and CyaA are adenylyl cyclase toxins secreted by pathogenic bacteria that cause anthrax and whooping cough, respectively. Using the structure of the catalytic site of EF, we screened a data base of commercially available, small molecular weight chemicals for those that could specifically inhibit adenylyl cyclase activity of EF. From 24 compounds tested, we have identified one quinazoline compound, ethyl 5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, that specifically inhibits adenylyl cyclase activity of EF and CyaA with approximately 20 microm Ki. This compound neither affects the activity of host resident adenylyl cyclases type I, II, and V nor exhibits promiscuous inhibition. The compound is a competitive inhibitor, consistent with the prediction that it binds to the adenine portion of the ATP binding site on EF. EF is activated by the host calcium sensor, calmodulin. Surface plasmon resonance spectroscopic analysis shows that this compound does not affect the binding of calmodulin to EF. This compound is dissimilar from a previously described, non-nucleoside inhibitor of host adenylyl cyclase. It may serve as a lead to design antitoxins to address the role of adenylyl cyclase toxins in bacterial pathogenesis and to fight against anthrax and whooping cough.