RESUMEN
Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.
Asunto(s)
Inhibidores de Fosfodiesterasa 4 , Esquistosomiasis , Animales , Humanos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Schistosoma mansoni , Benzamidas/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Esquistosomiasis/tratamiento farmacológico , Nucleótidos CíclicosRESUMEN
Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/efectos de los fármacos , Inhibidores de Fosfodiesterasa/uso terapéutico , Proteínas Protozoarias/efectos de los fármacos , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Relación Estructura-ActividadRESUMEN
Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.
Asunto(s)
Inhibidores de Fosfodiesterasa/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Relación Estructura-ActividadRESUMEN
In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Inhibidores de Fosfodiesterasa/análisis , Inhibidores de Fosfodiesterasa/química , Proteínas Protozoarias/química , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Resonancia por Plasmón de Superficie , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Sitios de Unión , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Humanos , Unión Proteica , Proteínas Protozoarias/metabolismo , Especificidad por SustratoRESUMEN
In this work we describe the optimization of a lead compound based on the quinazoline template to give a new series of potent pyrido[3,2-d]pyrimidines as histamine H4 receptor antagonists. The pyrido[3,2-d]pyrimidine ligands have significantly reduced hERG binding compared to clinical stage compound PF-3893787 while showing good affinities at the human and rodent histamine receptors. The receptor residence time of several of these new compounds was determined for the human H4R and compared with JNJ7777120 and PF-3893787. The pyrido[3,2-d]pyrimidines showed residence times lower than JNJ7777120 but comparable to the residence time of PF-3893787. Overall, the pyrido[3,2-d]pyrimidines show an excellent in vitro profile that warrants their further investigation in relevant models of human disease.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Antagonistas de los Receptores Histamínicos/química , Piridinas/química , Pirimidinas/química , Pirrolidinas/química , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Línea Celular , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/química , Semivida , Antagonistas de los Receptores Histamínicos/síntesis química , Antagonistas de los Receptores Histamínicos/farmacocinética , Humanos , Indoles/química , Indoles/farmacocinética , Cinética , Ratones , Piperazinas/química , Piperazinas/farmacocinética , Unión Proteica , Piridinas/síntesis química , Piridinas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Pirrolidinas/farmacocinética , Ratas , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H4 , Relación Estructura-ActividadRESUMEN
Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei, is one of the neglected tropical diseases with a continuing need for new medication. We here describe the discovery of 5-phenylpyrazolopyrimidinone analogs as a novel series of phenotypic antitrypanosomal agents. The most potent compound, 30 (NPD-2975), has an in vitro IC50 of 70 nM against T. b. brucei with no apparent toxicity against human MRC-5 lung fibroblasts. Showing good physicochemical properties, low toxicity potential, acceptable metabolic stability, and other pharmacokinetic features, 30 was further evaluated in an acute mouse model of T. b. brucei infection. After oral dosing at 50 mg/kg twice per day for five consecutive days, all infected mice were cured. Given its good drug-like properties and high in vivo antitrypanosomal potential, the 5-phenylpyrazolopyrimidinone analog 30 represents a promising lead for future drug development to treat HAT.
Asunto(s)
Tripanocidas , Trypanosoma brucei brucei , Tripanosomiasis Africana , Ratones , Humanos , Animales , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Descubrimiento de Drogas , Desarrollo de MedicamentosRESUMEN
The histamine H(4) receptor is a G protein-coupled receptor that has attracted much interest for its role in inflammatory and immunomodulatory functions. In our search for new H(4)R ligands, a low affinity isoquinoline fragment was optimized to 7-(furan-2-yl)-4-(piperazin-1-yl)quinazolin-2-amine (VUF11489), as a new H(4)R antagonist. Analysis of its binding kinetics at the human H(4)R showed this compound to have a very different dissociative half-life in comparison with reference antagonist JNJ7777120.
Asunto(s)
Antagonistas de los Receptores Histamínicos/síntesis química , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Histamínicos/química , Animales , Disponibilidad Biológica , Química Farmacéutica/métodos , Diseño de Fármacos , Antagonistas de los Receptores Histamínicos/farmacología , Humanos , Hipersensibilidad/tratamiento farmacológico , Concentración 50 Inhibidora , Cinética , Ligandos , Ratones , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Ratas , Receptores Histamínicos H4 , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Several members of the 3',5'-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 µM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.
RESUMEN
Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.
Asunto(s)
Inhibidores de Fosfodiesterasa/farmacología , Ftalazinas/farmacología , Tripanocidas/farmacología , Animales , Cristalografía por Rayos X , Estabilidad de Medicamentos , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Ftalazinas/síntesis química , Ftalazinas/metabolismo , Unión Proteica , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/metabolismo , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Aim: Due to the urgent need for effective drugs to treat schistosomiasis that act through a known molecular mechanism of action, we focused on a target-based approach with the aim to discover inhibitors of a cyclic nucleotide phosphodiesterase from Schistosoma mansoni (SmPDE4A). Materials & methods: To discover new inhibitors of SmPDE4A homology models of the enzyme structure were constructed based on known human and protozoan homologs. The best two models were selected for subsequent virtual screening of our in-house chemical library. Results & conclusion: A total of 25 library compounds were selected for experimental confirmation as SmPDE4A inhibitors and after dose-response experiments, three top hits were identified. The results presented validate the virtual screening approach to identify new inhibitors for clinically relevant phosphodiesterases.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Descubrimiento de Drogas , Inhibidores de Fosfodiesterasa 4/farmacología , Schistosoma mansoni/enzimología , Esquistosomiasis/tratamiento farmacológico , Animales , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Inhibidores de Fosfodiesterasa 4/química , Esquistosomiasis/metabolismo , Relación Estructura-ActividadRESUMEN
Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.
Asunto(s)
Condrocitos/citología , Proteínas , Alelos , Animales , Northern Blotting , Diferenciación Celular , Línea Celular , Condrocitos/metabolismo , Colágeno/metabolismo , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Transgénicos , Modelos Genéticos , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Fenotipo , Fosfoproteínas/metabolismo , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Retroviridae/genética , Factores de TiempoRESUMEN
Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Ciclina A/metabolismo , Reparación del ADN , ADN/metabolismo , ADN/efectos de la radiación , Regulación de la Expresión Génica , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Quinasas CDC2-CDC28/genética , Células Cultivadas , Ciclina A/genética , Ciclina A1 , Quinasa 2 Dependiente de la Ciclina , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Rayos gamma , Células Madre Hematopoyéticas , Humanos , Autoantígeno Ku , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Regiones Promotoras Genéticas , Distribución Aleatoria , Recombinación Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Rayos UltravioletaRESUMEN
In virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the Emu and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. Emu-pp-E2F2 mice start to develop cytokeratin- and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4(+)CD8(+) double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, Emu-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells.
Asunto(s)
Ratones Transgénicos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias de la Tiroides/genética , Transactivadores/genética , Factores de Edad , Animales , Northern Blotting , Southern Blotting , Western Blotting , Ciclo Celular , División Celular , Transformación Celular Neoplásica , Factor de Transcripción E2F2 , Células Epiteliales/metabolismo , Citometría de Flujo , Hiperplasia/metabolismo , Inmunohistoquímica , Ratones , Mutación , Regiones Promotoras Genéticas , Timoma/genética , Timoma/metabolismo , TransgenesRESUMEN
In higher eukaryotes, the cyclins constitute a family of proteins involved in progression through the cell cycle. The cyclin A1 gene (Ccna1) is expressed during meiosis and is required for spermatogenesis. Targeted disruption of the Ccna1 gene with a LacZ reporter gene has allowed us to study the expression pattern of this gene in more detail. We have confirmed expression in mouse pre-meiotic spermatocytes and also detected expression in the accessory olfactory bulb, hippocampus and amygdala of the adult brain. We have also found that the amount of cyclin A1 protein influences the fertility of male mice and its action is modulated by genetic background. On an outbred genetic background (129S6/SvEv x MF1), Ccna1 (tm1Col) -/- animals are sterile due to spermatogenic arrest prior to the first meiotic division while Ccna1 (tm1Col) +/- mice show reduced sperm production and fertility. This is even more pronounced on an inbred genetic background (129S6/SvEv) where Ccna1 (tm1Col) +/- male mice are sterile due to a severe reduction in the total number of sperm.