RESUMEN
The spread of multidrug-resistant (MDR) Klebsiella pneumoniae in the nosocomial setting represents a big challenge to infection control teams. We have recently developed a simple spectroscopic-based method with excellent accuracy, turnaround time and cost-effectiveness (Rodrigues et al. mSystems 2020) for bacterial typing. Here, we applied our method in a real clinical context to support early identification of an outbreak involving KPC-3-producing K. pneumoniae ST147 isolates. Our results further support that attenuated total reflectance Fourier transform infrared (FT-IR) spectroscopy can provide enough information to support early and adequate infection control measures and therapeutic choices in the context of nosocomial outbreaks and hospital surveillance.
Asunto(s)
Proteínas Bacterianas/genética , Infecciones por Klebsiella/tratamiento farmacológico , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/aislamiento & purificación , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Técnicas de Tipificación Bacteriana , Carbapenémicos/farmacología , Brotes de Enfermedades , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/metabolismo , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Portugal/epidemiología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
TAp73 is a key tumor suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumors with impaired p53 signaling, like neuroblastoma, TAp73 activation represents an encouraging strategy, alternative to p53 activation, to suppress tumor growth and chemoresistance. In this work, we report a new TAp73-activating agent, the 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), with potent antitumor activity. Notably, LEM2 was able to release TAp73 from its interaction with both MDM2 and mutant p53, enhancing TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutant p53-expressing tumor cells. Importantly, LEM2 displayed potent antitumor activity against patient-derived neuroblastoma cells, consistent with an activation of the TAp73 pathway. Additionally, potent synergistic effects were obtained for the combination of LEM2 with doxorubicin and cisplatin in patient-derived neuroblastoma cells. Collectively, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against neuroblastoma.
Asunto(s)
Antineoplásicos/farmacología , Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Xantonas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HT29 , Humanos , Mutación , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Transducción de Señal/efectos de los fármacos , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The growth inhibitory activity of p53 tumor suppressor is tightly regulated by interaction with two negative regulatory proteins, murine double minute 2 (MDM2) and X (MDMX), which are overexpressed in about half of all human tumors. The elucidation of crystallographic structures of MDM2/MDMX complexes with p53 has been pivotal for the identification of several classes of inhibitors of the p53-MDM2/MDMX interaction. The present review provides in silico strategies and screening approaches used in drug discovery as well as an overview of the most relevant classes of small-molecule inhibitors of the p53-MDM2/MDMX interaction, their progress in pipeline, and highlights particularities of each class of inhibitors. Most of the progress made with high-throughput screening has led to the development of inhibitors belonging to the cis-imidazoline, piperidinone, and spiro-oxindole series. However, novel potent and selective classes of inhibitors of the p53-MDM2 interaction with promising antitumor activity are emerging. Even with the discovery of the 3D structure of complex p53-MDMX, only two small molecules were reported as selective p53-MDMX antagonists, WK298 and SJ-172550. Dual inhibition of the p53-MDM2/MDMX interaction has shown to be an alternative approach since it results in full activation of the p53-dependent pathway. The knowledge of structural requirements crucial to the development of small-molecule inhibitors of the p53-MDMs interactions has enabled the identification of novel antitumor agents with improved in vivo efficacy.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular , Química Farmacéutica/métodos , Descubrimiento de Drogas , Humanos , Terapia Molecular Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Relación Estructura-Actividad Cuantitativa , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The p53 family proteins are among the most appealing targets for cancer therapy. A deeper understanding of the complex interplay that these proteins establish with murine double minute (MDM)2, MDMX, and mutant p53 could reveal new exciting therapeutic opportunities in cancer treatment. Here, we summarize the most relevant advances in the biology of p53 family protein-protein interactions (PPIs), and the latest pharmacological developments achieved from targeting these interactions. We also highlight the remarkable contributions of yeast-based assays to this research. Collectively, we emphasize promising strategies, based on the inhibition of p53 family PPIs, which have expedited anticancer drug development.
Asunto(s)
Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Saccharomyces cerevisiae , Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , Neoplasias/tratamiento farmacológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
AIM: Chalcones are naturally occurring compounds with recognized anticancer activity. It was recently shown that the O-prenyl derivative (2) of 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (1) had a remarkably increased cytotoxicity against human tumour cells compared to its precursor. With this study, we aimed to investigate the molecular mechanism underlying the improved tumour cytotoxicity of prenylchalcone 2. MAIN METHODS: The impact of chalcones 1 and 2 on p53-MDM2 interaction was investigated using yeast growth-inhibitory and p53 transactivation assays. Their tumour growth-inhibitory effects were assessed on human colon adenocarcinoma HCT116 cell lines with wild-type p53 and its p53-null derivative, followed by analysis of cell cycle and apoptosis. In tumour cells, the activation of a mitochondrial pathway was checked by analysis of reactive oxygen species generation, Bax mitochondrial translocation and cytochrome c release. Additionally, the up-regulation of p53 transcriptional activity was investigated through Western blot analysis of p53 target expression levels, and the disruption of the p53-MDM2 interaction was confirmed by co-immunoprecipitation. KEY FINDINGS: The potent cell tumour growth-inhibitory activity of prenylchalcone 2 was associated with the activation of a p53 pathway involving cell cycle arrest and a mitochondria-dependent apoptosis. Furthermore, a correlation between the distinct cytotoxicity of chalcones 1 and 2 and their ability to disrupt the p53-MDM2 interaction was established. SIGNIFICANCE: This work shows that prenylation is a determinant factor for the enhancement of chalcones tumour cytotoxicity by improving their ability to disrupt the p53-MDM2 interaction. Prenylchalcone 2 represents a starting basis for the design of new p53-MDM2 interaction inhibitors with improved antitumor properties.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Chalconas/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Proteínas Proto-Oncogénicas c-mdm2/genética , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Inactivation of the p53 tumor suppressor protein by interaction with murine double minute (MDM) proteins, MDM2 and MDMX, is a common event in human tumors expressing wild-type p53. In these tumors, the simultaneous inhibition of these interactions with MDMs, for a full p53 reactivation, represents a promising anticancer strategy. Herein, we report the identification of a dual inhibitor of the p53 interaction with MDM2 and MDMX, the (S)-tryptophanol derivative OXAZ-1, from the screening of a small library of enantiopure tryptophanol-derived oxazolopiperidone lactams, using a yeast-based assay. With human colon adenocarcinoma HCT116 cell lines expressing wild-type p53 (HCT116 p53(+/+)) and its p53-null isogenic derivative (HCT116 p53(-/-)), it was shown that OXAZ-1 induced a p53-dependent tumor growth-inhibitory effect. In fact, OXAZ-1 induced p53 stabilization, up-regulated p53 transcription targets, such as MDM2, MDMX, p21, Puma and Bax, and led to PARP cleavage, in p53(+/+), but not in p53(-/-), HCT116 cells. In addition, similar tumor cytotoxic effects were observed for OXAZ-1 against MDMX-overexpressing breast adenocarcinoma MCF-7 tumor cells, commonly described as highly resistant to MDM2-only inhibitors. In HCT116 p53(+/+) cells, the disruption of the p53 interaction with MDMs by OXAZ-1 was further confirmed by co-immunoprecipitation. It was also shown that OXAZ-1 potently triggered a p53-dependent mitochondria-mediated apoptosis, characterized by reactive oxygen species generation, mitochondrial membrane potential dissipation, Bax translocation to mitochondria, and cytochrome c release, and exhibited a p53-dependent synergistic effect with conventional chemotherapeutic drugs. Collectively, in this work, a novel selective activator of the p53 pathway is reported with promising antitumor properties to be explored either alone or combined with conventional chemotherapeutic drugs. Moreover, OXAZ-1 may represent a promising starting scaffold to search for new dual inhibitors of the p53-MDMs interaction.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Nucleares/metabolismo , Oxazoles/farmacología , Piperidonas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Triptófano/análogos & derivados , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Células HCT116 , Humanos , Células MCF-7 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estructura Molecular , Proteínas Nucleares/genética , Oxazoles/síntesis química , Oxazoles/química , Piperidonas/síntesis química , Piperidonas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Triptófano/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53-MDM2 interaction inhibitors.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Isoindoles/química , Isoindoles/farmacología , Oxazoles/química , Oxazoles/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Simulación por Computador , Computadores Moleculares , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Saccharomyces cerevisiae/efectos de los fármacos , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions.
Asunto(s)
Autofagia , Saccharomyces cerevisiae/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetatos/farmacología , Actinas/genética , Actinas/metabolismo , Proteínas de Ciclo Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Imidazoles/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/farmacología , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
INTRODUCTION: Chronic kidney disease (CKD) and smoking are public health problems. OBJECTIVE: To assess smoking as a risk factor for progression of CKD. METHODS: We conducted a systematic review in Medline, LILACS, SciELO, Google Scholar, Embase and Trials.gov with articles published until February/2013. Were included: cohort, clinical trials and case-control. Performed in humans, aged ≥ 18 years with smoking as a risk factor for progression of CKD. We excluded studies that reported no smoking and CKD in the title or had proposed to reduce smoking. RESULTS: Among 94 citations, 12 articles were selected. Of these, six were multicenter conducted in developed countries, four were randomized. Males predominated 51-76%. There was associated with smoking progression in 11 studies. It was found that the consumption ≥ 15 packs/ year increases the risk of progression of CKD. CONCLUSION: Smoking is a risk factor for progression of CKD.
Asunto(s)
Insuficiencia Renal Crónica/epidemiología , Insuficiencia Renal Crónica/etiología , Fumar/efectos adversos , Progresión de la Enfermedad , Humanos , Factores de RiesgoRESUMEN
Introdução: A doença renal crônica (DRC) e o tabagismo são problemas de saúde pública. Objetivo: Analisar o tabagismo como fator risco para a progressão da DRC. Métodos: Realizou-se uma revisão sistemática nas bases Medline, LILACS, SciELO, Google Acadêmico, Trials.gov e Embase com artigos publicados até fevereiro de 2013. Incluíram-se estudos: tipo coorte, ensaios clínicos e caso-controle. Realizados em seres humanos com idade ≥ 18 anos tendo tabagismo como fator de risco para progressão da DRC. Excluíram-se estudos que não referiam tabagismo e DRC no título ou tinham proposta de combate ao fumo. Resultados: Das 94 citações, 12 artigos foram selecionados. Destes, seis eram multicêntricos realizados em países desenvolvidos e quatro foram aleatorizados. Predominou o sexo masculino 51%-76%. Houve progressão associada ao tabagismo em 11 estudos. Identificou-se que o consumo ≥ 15 maços/ ano aumenta o risco de progressão da DRC. Conclusão: Tabagismo é fator de risco para progressão da DRC. .
Introduction: Chronic kidney disease (CKD) and smoking are public health problems. Objective: To assess smoking as a risk factor for progression of CKD. Methods: We conducted a systematic review in Medline, LILACS, SciELO, Google Scholar, Embase and Trials.gov with articles published until February/2013. Were included: cohort, clinical trials and case-control. Performed in humans, aged ≥ 18 years with smoking as a risk factor for progression of CKD. We excluded studies that reported no smoking and CKD in the title or had proposed to reduce smoking. Results: Among 94 citations, 12 articles were selected. Of these, six were multicenter conducted in developed countries, four were randomized. Males predominated 51-76%. There was associated with smoking progression in 11 studies. It was found that the consumption ≥ 15 packs/ year increases the risk of progression of CKD. Conclusion: Smoking is a risk factor for progression of CKD. .
Asunto(s)
Femenino , Humanos , Neoplasias de la Mama/genética , /genética , Amplificación de Genes , Proteínas de Neoplasias , Fosfoproteínas Fosfatasas/genética , Apoptosis/genética , Neoplasias de la Mama/etiología , Transformación Celular Neoplásica/genética , Oncogenes/genéticaRESUMEN
INTRODUCTION: Obesity is an important cause of cardiovascular disease, especially coronary artery disease. Severely obese patients are particularly prone to this risk. The coronary artery calcium (CAC) score is a strong predictor of coronary heart disease and provides incremental information beyond traditional risk factors. We sought to determine the prevalence of abnormally high CAC scores in the preoperative setting among patients undergoing bariatric surgery and to establish risk predictors for higher scores. METHODS: We performed an observational study of 202 patients free of known coronary artery disease who were referred for bariatric surgery. In each patient, the presence of CAC was detected with computed tomography, and coronary risk variables were either measured or determined via questionnaire. RESULTS: CAC was found in 14.4% of the overall population (26% of male and 10.5% of female patients). Participants with altered CAC scores were older (mean age, 46.8 years). The variables positively associated with an altered CAC score were older age, male sex, type 2 diabetes, hypertension, and hypercholesterolemia. Multivariate-adjusted analysis showed that age (OR, 1.11; 95% CI, 1.06-1.17; p = 0.001), male sex (OR, 4.17; 95% CI, 1.52-11.47; p = 0.006), and hypercholesterolemia (OR, 6.21; 95% CI, 1.81-21.29; p = 0.004) were most closely related to the presence of CAC. CONCLUSION: Obese patients in the preoperative bariatric surgery setting have a high prevalence of abnormal CAC scores. Traditional risk factors play a important role in this scenario.
RESUMEN
Yeast has proven to be an efficient model system for functional and pharmacological studies of the p53 tumour suppressor protein. In this work, the human p53-MDMX regulatory pathway was reconstituted in yeast. Additionally, by using the known inhibitor of p53-MDMX interaction, SJ-172550, the efficacy of a simplified yeast-based screening assay to search for inhibitors of p53-MDMX interaction is demonstrated for the first time. Moreover, further insights on p53 transcriptional activity in yeast are provided. In particular, it is shown that the reported wild-type (wt) p53-induced yeast growth inhibition and cell cycle arrest is associated with actin depolarization and with an increase of actin mRNA and protein expression levels. The increase of actin protein levels was not observed with the p53 R273H mutant (a loss of function p53 mutation hotspot) and was further intensified with the toxic p53 V122A mutant (reported to exhibit higher transcriptional activity than wt p53 for selected p53 target sequences). Moreover, it is shown that the wt p53-induced actin protein levels are modulated by natural (MDM2 and MDMX) and chemical (pifithrin-α, nutlin-3a and SJ-172550) regulators of p53 activity. Furthermore, wt p53 could stimulate transcription from a minimal promoter containing a fragment of the ACT1 upstream sequence. Thus, ACT1 is proposed as a putative endogenous p53 target gene. This finding may open the way for the development of simpler yeast p53 transactivation assays, not based on artificial reporter constructs, for the analysis of the impact of mutants, cofactors and small molecules on p53 transcriptional activity.
Asunto(s)
Actinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetatos/farmacología , Actinas/genética , Western Blotting , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Luciferasas/metabolismo , Mutación/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Pirazoles/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The regulation of protein kinase C (PKC) isoforms by ceramide is still controversial. In this work, the yeast Saccharomyces cerevisiae was used as a model to elucidate the effect of ceramide on the activity of mammalian PKC isoforms. For that, isc1Δ cells, with a deletion in the pathway for ceramide production by hydrolysis of complex sphingolipids, individually expressing mammalian PKCα, δ and ζ were used. Contrary to PKCα and ζ, expression of PKCδ in isc1Δ cells exhibited a similar phenotype to that observed with wild-type yeast cells expressing PKCδ treated with a PKC activator, as phorbol 12-myristate 13-acetate (PMA), specifically a growth inhibition associated with a G2/M cell cycle arrest. Interestingly, in isc1Δ yeast cells expressing PKCδ this phenotype was completely abrogated in the presence of exogenous ceramide. Moreover, using a yeast-based assay previously developed for the screening of PKC inhibitors, it was also shown that, like the known PKC inhibitor NPC 15437, ceramide reduced the PMA-induced growth inhibition, supporting an inhibitory effect of ceramide on PKCδ. Altogether, these results may indicate that ceramide distinctly interfere with the activity of PKCα, δ and ζ. Most importantly, they showed that ceramide is an inhibitor of PKCδ.
Asunto(s)
Ceramidas/metabolismo , Proteína Quinasa C/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
α-Mangostin (1) and gambogic acid (2) are natural products with potent cytotoxic activity against several human tumor cells. However, their molecular mechanisms of action remain controversial. In this work, using yeast-based assays, it was shown that both xanthones are potential inhibitors of the p53-MDM2 interaction. This activity on p53-MDM2 interaction was confirmed by a gene reporter assay in a human tumor cell. Additionally, computational docking studies supported the potential of these xanthones to bind to MDM2 and therefore act as putative MDM2 inhibitors. Altogether, this work provides a new insight concerning the molecular basis of activity for these compounds.
Asunto(s)
Clusiaceae/química , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Xantonas/aislamiento & purificación , Xantonas/farmacología , Western Blotting , Frutas/química , Humanos , Estructura Molecular , Resinas de Plantas/química , Xantonas/química , Levaduras/metabolismoRESUMEN
The virtual screening of a library of xanthone derivatives led us to the identification of potential novel MDM2 ligands. The activity of these compounds as inhibitors of p53-MDM2 interaction was investigated using a yeast phenotypic assay, herein developed for the initial screening. Using this approach, in association with a yeast p53 transactivation assay, the pyranoxanthone (3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one) (1) was identified as a putative small-molecule inhibitor of p53-MDM2 interaction. The activity of the pyranoxanthone 1 as inhibitor of p53-MDM2 interaction was further investigated in human tumor cells with wild-type p53 and overexpressed MDM2. Notably, the pyranoxanthone 1 mimicked the activity of known p53 activators, leading to p53 stabilization and activation of p53-dependent transcriptional activity. Additionally, it led to increased protein levels of p21 and Bax, and to caspase-7 cleavage. By computational docking studies, it was predicted that, like nutlin-3a, a known small-molecule inhibitor of p53-MDM2 interaction, pyranoxanthone 1 binds to the p53-binding site of MDM2. Overall, in this work, a novel small-molecule inhibitor of p53-MDM2 interaction with a xanthone scaffold was identified for the first time. Besides its potential use as molecular probe and possible lead to develop anticancer agents, the pyranoxanthone 1 will pave the way for the structure-based design of a new class of p53-MDM2 inhibitors.
Asunto(s)
Antineoplásicos/química , Benzopiranos/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Xantenos/química , Xantonas/química , Antineoplásicos/farmacología , Benzopiranos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/genética , Xantenos/farmacología , Xantonas/farmacologíaRESUMEN
As a model organism Saccharomyces cerevisiae has greatly contributed to our understanding of many fundamental aspects of cellular biology in higher eukaryotes. More recently, engineered yeast models developed to study endogenous or heterologous proteins that lay at the root of a given disease have become powerful tools for unraveling the molecular basis of complex human diseases like neurodegeneration. Additionally, with the possibility of performing target-directed large-scale screenings, yeast models have emerged as promising first-line approaches in the discovery process of novel therapeutic opportunities against these pathologies. In this paper, several yeast models that have contributed to the uncovering of the etiology and pathogenesis of several neurodegenerative diseases are described, including the most common forms of neurodegeneration worldwide, Alzheimer's, Parkinson's, and Huntington's diseases. Moreover, the potential input of these cell systems in the development of more effective therapies in neurodegeneration, through the identification of genetic and chemical suppressors, is also addressed.
Asunto(s)
Investigación Biomédica , Modelos Biológicos , Enfermedades Neurodegenerativas/patología , Levaduras/metabolismo , Animales , Humanos , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismoRESUMEN
Despite great advances in understanding the molecular etiology of cancer and neurodegeneration, therapeutic strategies against these diseases are still largely lacking. Hence, acceleration of the discovery of new therapeutic agents against these pathologies is of enormous interest. This review is focused on the role of multi-faceted and expanding yeast cell-based systems in the search for new drugs and therapeutic targets in cancer and neurodegeneration. Though the obvious limitations of using a microorganism to address human diseases, when used in the early phase and with complementary mammalian systems, it can have a tremendous impact in the discovery of new therapeutic opportunities. In this review, many evidence are provided demonstrating the valuable contribution of yeast in this area. Additionally, several yeast target-based drug screening approaches based on a readily screenable phenotype on genomic technologies increasingly oriented towards genetic and chemical high-throughput analysis are addressed. Altogether, with this review, we intend not only to recognize previous successes and ongoing work in this area, but also to point out new opportunities that may be of interest for yeast as a model organism and as a powerful system in the discovery of new lead compounds that have the potential to become novel drugs in cancer and neurodegeneration.
Asunto(s)
Diseño de Fármacos , Modelos Biológicos , Levaduras/metabolismo , Animales , Descubrimiento de Drogas , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/fisiopatologíaRESUMEN
A cardiomiopatia periparto (CMPP) é uma das causas comuns de cardiomiopatia secundária de etiologia desconhecida. Caracteriza-se pela presença de insuficiência cardíaca congestiva (ICC) materna, no último mês de gestação ou até cinco meses pós-parto, com disfunção ventricular sistólica esquerda, na ausência de outras causas de insuficiência cardíaca e em mulheres previamente saudáveis. A terapêutica médica consiste no bloqueio neuro-hormonal, suporte inotrópico, redução da pré e pós-carga cardíaca e anticoagulação. O transplante cardíaco está reservado aos casos graves e refratários à terapêutica medicamentosa. O prognóstico é variável: aproximadamente 50-60% das pacientes recuperam completamente a função cardíaca, na maioria das vezes dentro dos primeiros seis meses. Relata-se um caso de mulher negra, 37 anos, multípara, pré-natal sem intercorrências, sem comorbidades prévias ou uso de drogas, que desenvolveu um quadro de ICC, de instalação súbita, com disfunção ventricular comprovada ao ecocardiograma 15 dias após parto normal. Houve suspeição clínica de miocárdio não-compactado (MNC) que levou a dúvida quanto ao diagnóstico de CMPP, posto ser esta um diagnóstico de exclusão. A presença de MNC foi afastada pela ressonância nuclear magnética cardíaca.
La miocardiopatía periparto (MCPP) es una causa frecuente de miocardiopatía secundaria de etiología desconocida. Se caracteriza por la presencia de insuficiencia cardíaca congestiva (ICC) en la madre en el último mes del embarazo o hasta cinco meses después del parto, con disfunción sistólica del ventrículo izquierdo en ausencia de otras causas de insuficiencia cardíaca en mujeres previamente sanas. El tratamiento médico consiste en el bloqueo neurohormonal, soporte inotrópico, la reducción de la pre y post-carga cardíaca y anticoagulación. El trasplante cardíaco está reservado para casos graves y refractarios al tratamiento farmacológico. El pronóstico es variable: aproximadamente el 50-60% de las pacientes recuperan completamente la función cardíaca, en la mayoría de los casos en los primeros seis meses. Se presenta el caso clínico de una mujer de raza negra de 37 años, multípara, prenatal sin complicaciones, sin comorbilidades previas o uso de drogas, que desarrollo ICC, de aparición súbita, con disfunción ventricular demostrada en el ecocardiograma 15 días después de un parto normal. No había sospecha clínica de miocardiopatía no compactada (MNC) que dio lugar a duda sobre el diagnóstico de MCPP, ya que éste es un diagnóstico de exclusión. La presencia de MNC fue descartada por resonancia magnética nuclear cardíaca.
Peripartum cardiomyopathy (PPCM) is a common cause of secondary cardiomyopathy of unknown etiology. It is characterized by the presence of congestive heart failure (CHF) in the mother in the last month of pregnancy or until five months after birth with left ventricular systolic dysfunction in the absence of other causes of heart failure in previously healthy women. Medical treatment consists of neurohormonal blockade, inotropic support, reduced pre-and post-cardiac load and anticoagulation. Heart transplantation is reserved for severe cases refractory to medical therapy. The prognosis is variable: approximately 50-60% of patients recover full cardiac function, in most cases in the first six months. We report the case of a black woman of 37 years, multiparous, prenatal without complications, comorbidities or previous drug use that developed CHF, sudden onset, demonstrated ventricular dysfunction on echocardiography 15 days after childbirth normal. There was no clinical suspicion of non-compaction cardiomyopathy (NCC) which gave rise to doubt about the diagnosis of PPCM, since this is a diagnosis of exclusion. The presence of NCC was ruled out by cardiac magnetic resonance imaging.
RESUMEN
Fundamentos: Na avaliação da doença arterial coronariana, o teste ergométrico deve ser interpretado através da análise de dados clínicos, hemodinâmicos eeletrocardiográficos, mas na prática clínica algumas dessas informações não são adequadamente identificadas.Objetivo: Analisar a prática de realização do teste ergométrico para avaliação da doença ateroscleróticacoronariana em relação às recomendações consensuais. Métodos: Estudo de série de casos, transversal,observacional e descritivo. Entre maio e junho 2011 analisaram-se os resultados dos testes ergométricos de 166 pacientes submetidos a cineangiocoronariografiasconsecutivas. Resultados: População predominante masculina (60,8%)submetida a teste ergométrico e cineangiocoronariografia, com elevada frequência de fatores de risco cardiovascular. Observada baixa frequência de precordialgia típica(6,4%), com maioria (80,8%) dos testes sem relatos de precordialgia. Teste ergométrico máximo em 17,5% daamostra e submáximo na maioria (60,8%); sugestivo de isquemia em 63,9%, não sugestivo ou inconclusivo nos demais. A cineangiocoronariografia identificou aterosclerose significativa em 60,2% (100/166) dos pacientes. A sensibilidade do teste de esforço foi 65% (IC95% 54,8-74,2), especificidade 37,8% (IC95% 26,2-50,6), valor preditivo positivo 61,3% (IC95% 51,3-70,6) e valor preditivo negativo do teste ergométrico em relação ao padrão-ouro foi 41,6% (IC95% 29,0-55,1). A acurácia foide apenas 54,2% (IC95% 43,6-66,6) dos exames de esforço. Informações importantes para análise adequada do teste ergométrico não foram encontradas na maioria dos exames.