RESUMEN
For the early diagnosis of cancer, leading to a better chance of full recovery, marker genes whose expression is already altered in precancerous lesions are desirable, and the tumor-suppressor gene FHIT is one candidate. The gene product, FHIT protein, has a unique dinucleoside triphosphate hydrolase (AP3Aase) activity, and in this study, we designed and synthesized a series of FHIT fluorescent probes utilizing this activity. We optimized the probe structure for high and specific reactivity with FHIT and applied the optimized probe in a screening assay for FHIT inhibitors. Screening of a compound library with this assay identified several hits. Structural development of a hit compound afforded potent FHIT inhibitors. These inhibitors induce apoptosis in FHIT-expressing cancers via caspase activation. Our results support the idea that FHIT binders, no matter whether inhibitors or agonists of AP3Aase activity, might be promising anticancer agents.
Asunto(s)
Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Estructura Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
The liposoluble tanshinones are bioactive components in Salvia miltiorrhiza and are widely investigated as anti-cancer agents, while the molecular mechanism is to be clarified. In the present study, we identified that the human fragile histidine triad (FHIT) protein is a direct binding protein of sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tanshinone IIA (TSA), with a Kd value of 268.4 ± 42.59 nM. We also found that STS inhibited the diadenosine triphosphate (Ap3A) hydrolase activity of FHIT through competing for the substrate-binding site with an IC50 value of 2.2 ± 0.05 µM. Notably, near 100 times lower binding affinities were determined between STS and other HIT proteins, including GALT, DCPS, and phosphodiesterase ENPP1, while no direct binding was detected with HINT1. Moreover, TSA, Tanshinone I (TanI), and Cryptotanshinone (CST) exhibited similar inhibitory activity as STS. Finally, we demonstrated that depletion of FHIT significantly blocked TSA's pro-apoptotic function in colorectal cancer HCT116 cells. Taken together, our study sheds new light on the molecular basis of the anti-cancer effects of the tanshinone compounds.
Asunto(s)
Abietanos/farmacología , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Apoptosis , Neoplasias Colorrectales/tratamiento farmacológico , Hidrolasas/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Salvia miltiorrhiza/química , Abietanos/química , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Células Tumorales CultivadasRESUMEN
All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the Escherichia coli Nudix hydrolase RppH, which triggers 5'-end-dependent RNA degradation by removing orthophosphate from the 5'-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5'-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5'-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5'-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , ARN/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Isomerasas de Aminoácido/metabolismo , Dominio Catalítico , Proteínas de Escherichia coli/antagonistas & inhibidores , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Magnesio/química , Modelos Moleculares , Nucleótidos/química , Nucleótidos/metabolismo , ARN/química , Especificidad por SustratoAsunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Descubrimiento de Drogas , Pirofosfatasas/metabolismo , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Humanos , Concentración 50 Inhibidora , Nucleótidos/química , Nucleótidos/metabolismo , Pirofosfatasas/antagonistas & inhibidores , Receptores Purinérgicos/química , Receptores Purinérgicos/metabolismoRESUMEN
DN604, containing a functional dicarboxylato ligand as carboplatin analogue, was significantly studied to explore its potency of antitumour activity. In vitro and in vivo experimental evidence indicated that DN604 exhibited superior antitumor activity than present platinum(II)-based agents in cervix squamous carcinoma SiHa cancer cells. Moreover, DN604 showed negligible toxic effects in vivo as confirmed as Pt accumulation and body weights of mice. Mechanistic studies have shown that DN604 suppressed CK2-mediated MRN complex to improve its antitumor efficacy by promoting DNA double-strand breaks repair. Furthermore, DN604 could inhibit Beclin1 and attenuate CK2-mediated several DSBs repair-related pathways, thus leading to cell apoptosis. Taken together, our research demonstrated that DN604 with the functional dicarboxylato ligand as the leaving group could effectively enhance chemo-sensitivity of SiHa cells to platinum-based agents via suppressing Beclin1 and CK2-mediated MRN-DSBs repair.
Asunto(s)
Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Beclina-1/metabolismo , Carboplatino/análogos & derivados , Quinasa de la Caseína II/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteína Homóloga de MRE11/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias del Cuello Uterino/tratamiento farmacológico , Animales , Apoptosis , Beclina-1/genética , Biomarcadores de Tumor , Carboplatino/farmacología , Ciclo Celular , Proliferación Celular , Reparación del ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases.
Asunto(s)
Ácido Anhídrido Hidrolasas/biosíntesis , Aldehído Deshidrogenasa Mitocondrial/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Herbicidas/toxicidad , Neostriado/enzimología , Células-Madre Neurales/enzimología , Síndromes de Neurotoxicidad/enzimología , Oxadiazoles/toxicidad , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Aldehído Deshidrogenasa Mitocondrial/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Ensayo Cometa , Humanos , Neostriado/citología , Neostriado/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Síndromes de Neurotoxicidad/patología , Estrés Oxidativo/efectos de los fármacosRESUMEN
The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap3 A) are important factors in cancer development and progression. Fhit has Ap3 A hydrolase activity and cleaves Ap3 A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit-mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhitâ Ap3 A complex might exert its growth-suppressive function remain to be discovered. Small-molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor-suppressive functions. Here we describe the development of a high-throughput screen for the identification of such small-molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC50 values in the nanomolar range.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de Neoplasias/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Concentración 50 Inhibidora , Proteínas de Neoplasias/antagonistas & inhibidores , Unión Proteica , Quinolonas/química , Quinolonas/metabolismo , Quinolonas/toxicidad , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/toxicidadRESUMEN
Messenger RNAs are decorated by a cap structure, which is essential for their translation into proteins. Many viruses have developed strategies in order to cap their mRNAs. The cap is either synthetized by a subset of viral or cellular enzymes, or stolen from capped cellular mRNAs by viral endonucleases ('cap-snatching'). Reverse genetic studies provide evidence that inhibition of viral enzymes belonging to the capping pathway leads to inhibition of virus replication. The replication defect results from reduced protein synthesis as well as from detection of incompletely capped RNAs by cellular innate immunity sensors. Thus, it is now admitted that capping enzymes are validated antiviral targets, as their inhibition will support an antiviral response in addition to the attenuation of viral mRNA translation. In this review, we describe the different viral enzymes involved in mRNA capping together with relevant inhibitors, and their biochemical features useful in inhibitor discovery.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hidrolasas/metabolismo , Caperuzas de ARN/metabolismo , ARN Viral/metabolismo , Transferasas/metabolismo , Proteínas Virales/metabolismo , Virus/enzimología , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Antivirales/química , Antivirales/metabolismo , Replicación del ADN/efectos de los fármacos , Endonucleasas/antagonistas & inhibidores , Endonucleasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Humanos , Hidrolasas/antagonistas & inhibidores , Modelos Moleculares , Caperuzas de ARN/química , ARN Mensajero/metabolismo , ARN Viral/química , Transferasas/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología , Virus/efectos de los fármacos , Virus/genéticaRESUMEN
Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5'-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5'-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5'-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro with IC50s of 5-8 µM and show antifungal activity with MICs ranging from 3.12 to 100 µg ml-1 against S. cerevisiae.
Asunto(s)
Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Actinobacteria/metabolismo , Antifúngicos/farmacología , Inhibidores Enzimáticos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Antifúngicos/administración & dosificación , Antifúngicos/aislamiento & purificación , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/aislamiento & purificación , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/enzimologíaRESUMEN
UNLABELLED: Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals-including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics-that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. IMPORTANCE: The stark differences between the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus in pathogenic protozoa, fungi, and viruses and those of their metazoan hosts highlight RTPase as a target for anti-infective drug discovery. Protozoan, fungal, and DNA virus RTPases belong to the triphosphate tunnel metalloenzyme family. This study shows that a protozoan RTPase, TbCet1 from Trypanosoma brucei, is essential for growth of the parasite in culture and identifies, via in vitro screening of chemical libraries, several classes of potent small-molecule inhibitors of TbCet1 phosphohydrolase activity.
Asunto(s)
Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Ácido Anhídrido Hidrolasas/genética , Antioxidantes/química , Antioxidantes/farmacología , Apirasa/metabolismo , Sitios de Unión , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacología , Dominio Catalítico , Cinamatos/química , Cinamatos/farmacología , Depsidos/química , Depsidos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Ácido Gálico/farmacología , Glucósidos/química , Glucósidos/farmacología , Concentración 50 Inhibidora , Proteínas Protozoarias/genética , Quercetina/análogos & derivados , Quercetina/química , Quercetina/farmacología , Ácido Quínico/análogos & derivados , Ácido Quínico/química , Ácido Quínico/farmacología , Interferencia de ARN , Bibliotecas de Moléculas Pequeñas/química , Tripanocidas/química , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/crecimiento & desarrollo , Ácido RosmarínicoRESUMEN
Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Antimaláricos/química , Antimaláricos/farmacología , Sistemas de Liberación de Medicamentos , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Dominios Proteicos , Proteínas Protozoarias/antagonistas & inhibidoresRESUMEN
Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarinâ A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarinâ A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarinâ A binds a dimeric PfAp3Aase and prevents the formation of the enzymeâ substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarinâ A in Mycobacterium tuberculosis. Thus, cyclomarinâ A is a rare example of a natural product with two distinct and specific modes of action.
Asunto(s)
Productos Biológicos/química , Oligopéptidos/química , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Endopeptidasa Clp/antagonistas & inhibidores , Endopeptidasa Clp/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
FHIT (Fragile Histidin Triad) is a tumor suppressor gene involved in regulating cell death during DNA damage conditions. The exact mechanism of DNA damage-induced FHIT signaling is not well understood. It is known that p38 kinase and CHK2 kinase are being activated during stress-induced conditions and DNA damage, resulting in cell death. Since both CHK2 and FHIT are being influenced by DNA damage, we have evaluated the interplay of p38, CHK2 and FHIT in response to etoposide-induced cell death. DNA damage was induced by etoposide in MCF-7 cells and viability was examined using MTT. FHIT expression was blocked using siRNA. Protein expression was measured using western blotting. Our results indicated that etoposide induced cytotoxicity in MCF-7. Block of FHIT expression, completely reversed etoposide cytotoxicity. Besides, etoposide induced p38 and CHK2 phosphorylation and reduced FHIT expression in a time-dependent manner. The time-course study indicated that CHK2 had been phosphorylated prior to p38 activation. Knockdown of FHIT expression reduced CHK2 phosphorylation but had no significant effect on p38 activation. Inhibition of p38 kinase and CHK2 prevented etoposide induced alteration in FHIT expression. Furthermore, p38 inhibitors augmented etoposide-induced CHK2 phosphorylation. These results indicate that etoposide lowers FHIT expression and induces cell death via p38 and CHK2 phosphorylation. These results demonstrate a time dependent complex crosstalk of FHIT, p38 and CHK2 pathways in response to etoposide. Moreover, our findings suggest signaling interaction for these pathways which can be targeted for manipulating cell proliferation.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Antineoplásicos Fitogénicos/farmacología , Proliferación Celular/efectos de los fármacos , Etopósido/farmacología , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/genética , Quinasa de Punto de Control 2 , Daño del ADN , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Células MCF-7 , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Inorganic polyphosphate (poly-P), guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) are ubiquitous in bacteria. These molecules play a variety of important physiological roles associated with stress resistance, persistence, and virulence. In the bacterial pathogen Mycobacterium tuberculosis, the identities of the proteins responsible for the metabolism of polyphosphate and (p)ppGpp remain to be fully established. M. tuberculosis encodes two PPX-GppA homologues, Rv0496 (MTB-PPX1) and Rv1026, which share significant sequence similarity with bacterial exopolyphosphatase (PPX) and guanosine pentaphosphate 5'-phosphohydrolase (GPP) proteins. Here we delineate the respective biochemical activities of the Rv0496 and Rv1026 proteins and benchmark these against the activities of the PPX and GPP proteins from Escherichia coli. We demonstrate that Rv0496 functions as an exopolyphosphatase, showing a distinct preference for relatively short-chain poly-P substrates. In contrast, Rv1026 has no detectable exopolyphosphatase activities. Analogous to the E. coli PPX and GPP enzymes, the exopolyphosphatase activities of Rv0496 are inhibited by pppGpp and, to a lesser extent, by ppGpp alarmones, which are produced during the bacterial stringent response. However, neither Rv0496 nor Rv1026 have the ability to hydrolyze pppGpp to ppGpp; a reaction catalyzed by E. coli PPX and GPP. Both the Rv0496 and Rv1026 proteins have modest ATPase and to a lesser extent ADPase activities. pppGpp alarmones inhibit the ATPase activities of Rv1026 and, to a lesser extent, the ATPase activities of Rv0496. We conclude that PPX-GppA family proteins may not possess all the catalytic activities implied by their name and may play distinct biochemical roles involved in polyphosphate and (p)ppGpp metabolic pathways.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Mycobacterium tuberculosis/enzimología , Homología de Secuencia de Aminoácido , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Sistema Libre de Células/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , GTP Fosfohidrolasas/metabolismo , Guanosina Tetrafosfato/farmacología , Hidrólisis/efectos de los fármacos , Cinética , Mycobacterium tuberculosis/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.
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Ácido Anhídrido Hidrolasas/metabolismo , Alanina-Deshidrogenasa/metabolismo , Biocatálisis , Escherichia coli K12/enzimología , Proteínas de Escherichia coli/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/genética , Alanina/genética , Alanina/metabolismo , Transporte Biológico , Escherichia coli K12/genética , Escherichia coli K12/crecimiento & desarrollo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Genes Bacterianos/genética , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Modelos Moleculares , Operón/genética , Fenotipo , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
Partially purified endopolyphosphatase from cytosol of the yeast Saccharomyces cerevisiae with inactivated genes PPX1 and PPN1 encoding exopolyphosphatases was obtained with ion-exchange and affinity chromatography. The enzyme activity was estimated by decrease of polyphosphate chain length determined by PAGE. The enzyme cleaved inorganic polyphosphate without the release of orthophosphate (P(i)) and was inhibited by heparin and insensitive to fluoride. Mg2+, Mn2+, and Co2+ (1.5 mM) stimulated the activity, and Ca2+ was ineffective. The molecular mass of the endopolyphosphatase determined by gel filtration was of ~20 kDa.
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Ácido Anhídrido Hidrolasas/química , Saccharomyces cerevisiae/enzimología , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Calcio/química , Cobalto/química , Pruebas de Enzimas , Heparina/química , Magnesio/química , Manganeso/química , Peso Molecular , Polifosfatos/químicaRESUMEN
Novel inhibitors of lupin diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolase have been identified by in silico screening of a large virtual chemical library. Compounds were ranked on the basis of a consensus from six scoring functions. From the top 100 ranked compounds six were selected and initially screened for inhibitory activity using a single concentration isothermal titration calorimetry assay. Two of these compounds that showed excellent solubility properties were further analyzed, but only one [NSC51531; 2-((8-hydroxy-4-(4-methyl-2-sulfoanilino)-9,10-dioxo-9,10-dihydro-1-anthracenyl)amino)-5-methylbenzenesulfonic acid] exhibited competitive inhibition with a K(i) of 1 microM. A structural analogue of this compound also exhibited competitive inhibition with a comparable K(i) of 2.9 microM. (1)H, (15)N NMR spectroscopy was used to map the binding site of NSC51531 on lupin Ap(4)A hydrolase and demonstrated that the compound bound specifically in the substrate-binding site, consistent with the competitive inhibition results. Binding of NSC51531 to the human form of Ap(4)A hydrolase is nonspecific, suggesting that this compound may represent a useful lead in the design of specific inhibitors of the plant-like form of Ap(4)A hydrolases.
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Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Lupinus/enzimología , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/genética , Animales , Calorimetría , Dominio Catalítico , Simulación por Computador , Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Conformación ProteicaRESUMEN
Dinucleoside polyphosphates (Np(n)N's; where N and N' are nucleosides and n = 3-6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of Np(n)N' analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap(4)A hydrolase [bis(5'-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41] from E. coli and of two asymmetrically acting Ap(4)A hydrolases [bis(5'-nucleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow-leaved lupin. The six chemically synthesized analogs were: ApCH(2)OpOCH(2)pA (1), ApOCH(2)pCH(2)OpA (2), ApOpCH(2)OpOpA (3), ApCH(2)OpOpOCH(2)pA (4), ApOCH(2)pOpCH(2)OpA (5) and ApOpOCH(2)pCH(2)OpOpA (6). The eukaryotic asymmetrical Ap(4)A hydrolases degrade two compounds, 3 and 5, as anticipated in their design. Analog 3 was cleaved to AMP (pA) and beta,gamma-methyleneoxy-ATP (pOCH(2)pOpA), whereas hydrolysis of analog 5 gave two molecules of alpha,beta-oxymethylene ADP (pCH(2)OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having K(i) values within the range of the K(m) for Ap(4)A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap(4)A hydrolase.
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Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Fosfatos de Dinucleósidos/farmacología , Inhibidores Enzimáticos/farmacología , Oxígeno/química , Cromatografía Líquida de Alta Presión , Fosfatos de Dinucleósidos/química , Inhibidores Enzimáticos/química , HidrólisisRESUMEN
The present work evaluated polyphosphate (poly P) metabolism in nuclear and mitochondrial fractions during Rhipicephalus microplus embryogenesis. Nuclear poly P decreased and activity of exopolyphosphatase (PPX - polyphosphate-phosphohydrolases; EC 3.6.1.11) increased after embryo cellularization until the end of embryogenesis. The utilization of mitochondrial poly P content occurred between embryo cellularization and segmentation stages. Increasing amounts of total RNA extracted from eggs progressively enhanced nuclear PPX activity, whereas it exerted no effect on mitochondrial PPX activity. The decline in total poly P content after the 7th day of embryogenesis does not reflect the free P(i) increase and the total poly P chain length decrease after embryo cellularization. The Km(app) utilizing poly P(3), poly P(15) and poly P(65) as substrate was almost the same for the nuclear fraction (around 1muM), while the affinity for substrate in mitochondrial fraction was around 10 times higher for poly P(3) (Km(app) = 0.2muM) than for poly P(15) (Km(app) = 2.8muM) and poly P(65) (Km(app) = 3.6muM). PPX activity was stimulated by a factor of two by Mg2+ and Co2+ in the nuclear fraction and only by Mg2+ in the mitochondrial fraction. Heparin (20microg/mL) inhibited nuclear and mitochondrial PPX activity in about 90 and 95% respectively. Together, these data are consistent with the existence of two different PPX isoforms operating in the nuclei and mitochondria of the hard tick R. microplus with distinct metal dependence, inhibitor and activator sensitivities. The data also shed new light on poly P biochemistry during arthropod embryogenesis, opening new routes for future comparative studies on the physiological roles of different poly P pools distributed over cell compartments.
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Ácido Anhídrido Hidrolasas/metabolismo , Núcleo Celular/enzimología , Mitocondrias/enzimología , Rhipicephalus/enzimología , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Animales , Fraccionamiento Celular , Embrión no Mamífero/enzimología , Heparina/farmacología , Rhipicephalus/embriologíaRESUMEN
Fhit, the product of tumor suppressor fragile histidine triad (FHIT) gene, exhibits antitumor activity of still largely unknown cellular background. However, it is believed that Fhit-Ap(3)A or Fhit-AMP complex might act as a second class messenger in cellular signal transduction pathway involved in cell proliferation and apoptosis. We demonstrate here for the first time that the photosensitizer, protoporphyrin IX (which is a natural precursor of heme) binds to Fhit protein and its mutants in the active site in vitro. Furthermore, PpIX inhibits the enzymatic activity of Fhit. Simultaneously, PpIX shows lower binding capacity to mutant Fhit-H96N of highly reduced hydrolase activity. In cell-based assay PpIX induced HeLa cell death in Fhit and Fhit-H96N-dependent manner which was measured by means of MTT assay. Moreover, HeLa cells stably expressing Fhit or mutant Fhit-H96N were more susceptible to protoporphyrin IX-mediated photodynamic therapy (2J/cm(2)) than parental cells.