RESUMEN
Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-ß-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.
Asunto(s)
Modelos Animales de Enfermedad , Leishmania mexicana , Leishmaniasis Cutánea , Ratones Endogámicos BALB C , Animales , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Ratones , Femenino , Masculino , Leishmania mexicana/efectos de los fármacos , Tubercidina/farmacología , Tubercidina/análogos & derivados , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Antiprotozoarios/administración & dosificación , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Leishmania/efectos de los fármacosRESUMEN
The emergence of new coronaviruses poses a significant threat to animal husbandry and human health. Porcine epidemic diarrhea virus (PEDV) is considered a re-emerging porcine enteric coronavirus, which causes fatal watery diarrhea in piglets. Currently, there are no effective drugs to combat PEDV. Drug repurposing screens have emerged as an attractive strategy to accelerate antiviral drug discovery and development. Here, we screened 206 natural products for antiviral activity using live PEDV infection in Vero cells and identified ten candidate antiviral agents. Among them, Tubercidin, a nucleoside analog derived from Streptomyces tubercidicus, showed promising antiviral activity against PEDV infection. Furthermore, we demonstrated that Tubercidin exhibited significant antiviral activity against both classical and variant PEDV. Time of addition assay showed that Tubercidin displayed a significant inhibitory effect on viral post-entry events but not during other periods. Molecular docking analysis indicated that Tubercidin had better docking efficiency and formed hydrophobic interactions with the active pocket of RNA-dependent RNA polymerase (RdRp) of PEDV and other nidoviruses. Additionally, Tubercidin can effectively suppress other porcine nidoviruses, such as SADS-CoV and PRRSV, demonstrating its broad-spectrum antiviral properties. In summary, our findings provide valuable evidence for the antiviral activity of Tubercidin and offer insights into the development of new strategies for the prevention and treatment of coronavirus infections.
Asunto(s)
Infecciones por Coronavirus , Coronavirus , Nidovirales , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Chlorocebus aethiops , Humanos , Animales , Porcinos , Células Vero , Tubercidina/farmacología , Tubercidina/uso terapéutico , Reposicionamiento de Medicamentos , Simulación del Acoplamiento Molecular , Virus de la Diarrea Epidémica Porcina/genética , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.
Asunto(s)
Alphainfluenzavirus , Antivirales , Betainfluenzavirus , Productos Biológicos , Inhibidores Enzimáticos , Metiltransferasas , Caperuzas de ARN , Tubercidina , Replicación Viral , Animales , Humanos , Ratones , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , Replicación Viral/efectos de los fármacos , Alphainfluenzavirus/efectos de los fármacos , Betainfluenzavirus/efectos de los fármacos , Productos Biológicos/química , Productos Biológicos/farmacología , Antivirales/química , Antivirales/farmacología , Tubercidina/análogos & derivados , Tubercidina/farmacología , Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Streptomyces/química , Simulación por Computador , Células A549RESUMEN
Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.
Asunto(s)
Senescencia Celular , Histonas , Humanos , Ratones , Animales , Histonas/genética , Senescencia Celular/genética , Tubercidina/farmacología , Epigénesis GenéticaRESUMEN
Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , SARS-CoV-2/efectos de los fármacos , Tubercidina/análogos & derivados , Línea Celular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Humanos , Pruebas de Sensibilidad Microbiana , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/genética , Tionucleósidos/farmacología , Tubercidina/farmacología , Vidarabina/análogos & derivados , Vidarabina/farmacología , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/farmacologíaRESUMEN
As a human mitotic kinase, haspin is considered as a promising target for various diseases including cancers. However, no inhibitors targeting haspin have entered clinical trials presently. 5-iTU (5-iodotubercidin) is a useful and classical chemical probe for the investigation of haspin activity, but its inhibitory mechanism remains unclear. In this study, integrated molecular dynamics (MD) of conventional MD, extended adaptive biasing force (eABF), random acceleration MD and well-tempered metadynamics were applied to investigate the thermodynamic and kinetic features of 5-iTU and three derivatives targeting haspin. To emphasize the importance of gatekeeper Phe605, two haspin mutants (F605Y and F605T) were also built. The results showed that the binding affinity of 5-iTU and haspin was highest in all wild type (WT) systems, relying on the strong halogen aromatic π interaction between 5-iTU and gatekeeper Phe605. Gatekeeper mutations, because of damage to this interaction, led to the rearrangement of water distributions at the binding site and the decrease of 5-iTU residence times. Additionally, compared with the smaller 5-fTU, 5-iTU dissociated from WT haspin with more difficulty through distinct unbinding pathways. These findings will provide crucial guidance for the design and development of novel haspin inhibitors and the rational modification of existing inhibitors.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Termodinámica , Tubercidina/análogos & derivados , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Conformación Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Tubercidina/química , Tubercidina/farmacologíaRESUMEN
Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation. These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.
Asunto(s)
Ciclo Celular , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Colorantes Fluorescentes , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Fase G2/efectos de los fármacos , Humanos , Interfase/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis/efectos de los fármacos , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Fase S/efectos de los fármacos , Tubercidina/análogos & derivados , Tubercidina/farmacología , Proteína p53 Supresora de Tumor/genética , Ubiquitinación , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in patients with refractory epilepsy. Centrally-mediated respiratory dysfunction has been identified as one of the principal mechanisms responsible for SUDEP. Seizures generate a surge in adenosine release. Elevated adenosine levels suppress breathing. Insufficient metabolic clearance of a seizure-induced adenosine surge might be a precipitating factor in SUDEP. In order to deliver targeted therapies to prevent SUDEP, reliable biomarkers must be identified to enable prompt intervention. Because of the integral role of the phrenic nerve in breathing, we hypothesized that suppression of phrenic nerve activity could be utilized as predictive biomarker for imminent SUDEP. We used a rat model of kainic acid-induced seizures in combination with pharmacological suppression of metabolic adenosine clearance to trigger seizure-induced death in tracheostomized rats. Recordings of EEG, blood pressure, and phrenic nerve activity were made concomitant to the seizure. We found suppression of phrenic nerve burst frequency to 58.9% of baseline (p < 0.001, one-way ANOVA) which preceded seizure-induced death; importantly, irregularities of phrenic nerve activity were partly reversible by the adenosine receptor antagonist caffeine. Suppression of phrenic nerve activity may be a useful biomarker for imminent SUDEP. The ability to reliably detect the onset of SUDEP may be instrumental in the timely administration of potentially lifesaving interventions.
Asunto(s)
Adenosina Quinasa/antagonistas & inhibidores , Nervio Frénico/enzimología , Nervio Frénico/fisiopatología , Convulsiones/enzimología , Convulsiones/fisiopatología , Muerte Súbita e Inesperada en la Epilepsia , Adenosina Quinasa/metabolismo , Animales , Ácido Kaínico/toxicidad , Masculino , Nervio Frénico/efectos de los fármacos , Valor Predictivo de las Pruebas , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Tubercidina/análogos & derivados , Tubercidina/farmacologíaRESUMEN
Previously considered a neglected flavivirus, Zika virus has recently emerged as a public health concern due to its ability to spread rapidly and cause severe neurological disorders, such as microcephaly in newborn babies from infected mothers, and Guillain-Barré syndrome in adults. Despite extensive efforts towards the identification of effective therapies, specific antivirals are still not available. As part of ongoing medicinal chemistry studies to identify new antiviral agents, we screened against Zika virus replication in vitro in a targeted internal library of small-molecule agents, comprising both nucleoside and non-nucleoside agents. Among the compounds evaluated, novel aryloxyphosphoramidate prodrugs of the nucleosides 2'-C-methyl-adenosine, 2-CMA, and 7-deaza-2'C-methyl-adenosine, 7-DMA, were found to significantly inhibit the virus-induced cytopathic effect in multiple relevant cell lines. In addition, one of these prodrugs exhibits a synergistic antiviral effect against Zika virus when applied in combination with an indirect antiviral agent, a l-dideoxy bicyclic pyrimidine nucleoside analogue, which potently inhibits vaccinia and measles viruses in vitro by targeting a host pathway. Our findings provide a solid basis for further development of an antiviral therapy for Zika virus infections, possibly exploiting a dual approach combining two different agents, one targeting the viral polymerase (direct-acting antiviral), the second targeting a host-directed autophagy mechanism.
Asunto(s)
Antivirales/farmacología , Nucleósidos/farmacología , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacología , Antivirales/química , Autofagia/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Nucleósidos/análogos & derivados , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/farmacología , Replicación Viral/efectos de los fármacos , Virus Zika/patogenicidad , Infección por el Virus Zika/virologíaRESUMEN
3-Deazadenosine (3-DA) is a general methylation inhibitor that depletes S-adenosylmethionine, a methyl donor, by blocking S-adenosylhomocysteine hydrolase (SAHH). In this study, we investigated the inhibitory activity and molecular mechanisms of 3-DA in inflammatory responses. 3-DA suppressed the secretion of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide-treated RAW264.7 cells and phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells. It also reduced mRNA expression of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, interleukin-1ß (IL-1 ß), and IL-6, indicating that 3-DA has anti-inflammatory properties in murine and human macrophages. Moreover, 3-DA strongly blocked AP-1 and NF-κB luciferase activity under PMA-, MyD88-, and TRIF-stimulated conditions and decreased the translocation of c-Jun, c-Fos, p65, and p50 into the nucleus. In addition, the p-ERK level in AP-1 signaling and the p-IκBα level in NF-kB signaling were diminished by 3-DA treatment. Interestingly, 3-DA did not alter the phosphorylation of MEK1/2, an ERK modulator, or IKKα/ß, an IκBα regulator. Instead, 3-DA prevented MEK1/2 and IKKα/ß from combining with ERK and IκBα, respectively, and directly suppressed MEK1/2 and IKKα/ß kinase activity. These results indicate that MEK1/2 and IKKα/ß are direct targets of 3-DA. In addition, suppression of SAHH by siRNA or treatment with adenosine dialdehyde, another SAHH inhibitor, showed inhibitory patterns against p-ERK and IκBα similar to those of 3-DA. Taken together, this study demonstrates that 3-DA inhibits AP-1 and NF-κB signaling by directly blocking MEK1/2 and IKKα/ß or indirectly mediating SAHH, resulting in anti-inflammatory activity.
Asunto(s)
Adenosilhomocisteinasa/antagonistas & inhibidores , Mediadores de Inflamación/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , FN-kappa B/antagonistas & inhibidores , Factor de Transcripción AP-1/antagonistas & inhibidores , Tubercidina/farmacología , Adenosilhomocisteinasa/metabolismo , Animales , Antiinflamatorios/farmacología , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Ratones , FN-kappa B/metabolismo , Células RAW 264.7 , Factor de Transcripción AP-1/metabolismo , Células U937RESUMEN
N6methyladenosine (m6A) RNA modification maintained by N6methyltransferases and demethylases is involved in multiple biological functions. Methyltransferase like 3 (METTL3) is a major N6methyltransferase. However, the role of METTL3 and its installed m6A modification in colorectal tumorigenesis remains to be fully elucidated. METTL3 is highly expressed as indicated in colorectal cancer samples in the TCGA and Oncomine databases, implying its potential role in colon tumorigenesis. SW480 cell line with stable METTL3 knockout (METTL3KO) was generated using CRISPR/Cas9 and were confirmed by the loss of METTL3 expression and suppression of m6A modification. The proliferation of METTL3KO cells was significantly inhibited compared with that of control cells. METTL3KO decreased the decay rate of suppressor of cytokine signaling 2 (SOCS2) RNA, resulting in elevated SOCS2 protein expression. m6ARNA immunoprecipitationqPCR (MeRIPqPCR) revealed that SOCS2 mRNA was targeted by METTL3 for m6A modification. Similar to METTL3KO SW480 cells, SW480 cells treated with 3deazaadenosine, an RNA methylation inhibitor, exhibited elevated SOCS2 protein expression. Increased levels of SOCS2 in METTL3KO SW480 cells were associated with decreased expression of leucinerich repeatcontaining G proteincoupled receptor 5 (LGR5), contributing to the inhibition of cell proliferation. The underlying associations among METTL3, SOCS2, and LGR5 were further confirmed in SW480 cells transfected with siMETTL3 and in tumor samples from patients with colorectal cancer. Taken together, our data demonstrate that an increased level of METTL3 may maintain the tumorigenicity of colon cancer cells by suppressing SOCS2.
Asunto(s)
Carcinogénesis/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica/genética , Metiltransferasas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Anciano , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Colectomía , Colon/patología , Colon/cirugía , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Conjuntos de Datos como Asunto , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Masculino , Metilación/efectos de los fármacos , Metiltransferasas/genética , Persona de Mediana Edad , Estadificación de Neoplasias , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Esferoides Celulares , Tubercidina/farmacologíaRESUMEN
BACKGROUND: The earliest manifestation of alcohol-related liver disease (ALD) is steatosis, characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Findings from our laboratory have indicated that many pathological changes, including steatosis, correlate with the alcohol-induced hepatocellular increases in S-adenosylhomocysteine (SAH). Based on these considerations, we hypothesized that an experimental increase in intracellular SAH alone will result in similar steatotic changes to those seen after alcohol exposure. METHODS: Freshly isolated rat hepatocytes grown on collagen-coated plates were exposed to serum-free medium containing 50 µmol/L oleic acid and varying concentrations of 3-deazaadenosine (DZA) to experimentally elevate intracellular SAH levels. RESULTS: Overnight exposure to DZA treatment dose-dependently increased hepatocellular triglyceride accumulation, which was also evident by morphological visualization of larger-sized LDs. The rise in triglycerides and LDs accompanied increases in mRNA and protein levels of several LD-associated proteins known to regulate LD number and size. Furthermore, DZA treatment caused a decline in the levels of lipases that prevent fat accumulation as well as increased the expression of factors involved in lipogenesis and fatty acid mobilization. Collectively, our results indicate that the elevation of intracellular SAH is sufficient to promote fat accumulation in hepatocytes, which is similar to that seen after alcohol exposure.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Etanol/efectos adversos , Metabolismo de los Lípidos/efectos de los fármacos , Hepatopatías/sangre , Hepatopatías/etiología , S-Adenosilhomocisteína/efectos adversos , Tubercidina/uso terapéutico , Animales , Antibióticos Antineoplásicos/farmacología , Humanos , Hepatopatías/patología , Masculino , Ratas , Ratas Wistar , Tubercidina/farmacologíaRESUMEN
Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-ï¢-D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-ï¢-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-ï¢ -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.
Asunto(s)
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacología , Escherichia coli/efectos de los fármacos , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Tubercidina/análogos & derivados , Tubercidina/farmacología , 2-Aminopurina/síntesis química , Acetaldehído/análogos & derivados , Acetaldehído/química , Antibacterianos/química , Antibacterianos/farmacología , Escherichia coli/enzimología , Pirimidinas/química , Tubercidina/síntesis químicaRESUMEN
The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Tubercidina/análogos & derivados , Animales , División Celular/efectos de los fármacos , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor 4 Similar a Kruppel , Ratones , Fosforilación/efectos de los fármacos , Tubercidina/farmacologíaRESUMEN
The methylation of adenosines at the N6 position (m6A formation) is the most prevalent type of RNA modification in humans. This modification is mediated by methyltransferase like 3 (METTL3)-METTL14 complex, and the methyl group can be removed by RNA demethylases including fat mass and obesity-associated (FTO) and AlkB homolog 5. The formed m6A is recognized by reader proteins such as members of the YT521-B homology (YTH) family, resulting in changes in the splicing, nuclear export, and decay of RNA or translation. In this study, we examined the impact of m6A modification on the expression of drug-metabolizing P450 isoforms. By treatment with 3-deazaadenosine, an inhibitor of RNA methylation, CYP1A2, CYP2B6, and CYP2C8 levels were significantly increased (1.6-fold, 2.2-fold, and 2.7-fold, respectively) in HepaRG cells. In subsequent experiments, we focused on CYP2C8, which showed the largest increase. Consistent with the increase in the mRNA level, CYP2C8 protein level and activity were significantly increased by treatment with 3-deazaadenosine. The CYP2C8 expression levels and activities in HepaRG and Huh-7 cells were increased by knockdown of METTL3/14, whereas they were decreased by knockdown of FTO, suggesting that m6A modification downregulates CYP2C8 expression. With an RNA immunoprecipitation assay using an anti-m6A antibody, it was revealed that the adenosines in the 5'-UTR and the last exon of CYP2C8 are methylated in HepaRG cells and human liver samples. It was demonstrated that YTHDC2, which is known to degrade m6A-containing mRNA, downregulates CYP2C8 expression. In conclusion, we found a novel post-transcriptional regulation mechanism in which the YTHDC2 promotes CYP2C8 mRNA degradation via recognizing the m6A in CYP2C8 mRNA, which is installed by METTL3/14 and removed by FTO.
Asunto(s)
Adenosina/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica/genética , Hígado/metabolismo , Regiones no Traducidas 5'/genética , Línea Celular Tumoral , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C8/genética , Citocromo P-450 CYP2C8/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Metilación/efectos de los fármacos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tubercidina/farmacologíaRESUMEN
Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.
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Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Piridazinas/farmacología , Tubercidina/análogos & derivados , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis , Proteínas Serina-Treonina Quinasas/genética , Tubercidina/farmacologíaRESUMEN
African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3'-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3'-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3'-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.
Asunto(s)
Desoxiadenosinas/farmacología , Proteínas de Transporte de Nucleósidos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Tubercidina/farmacología , Animales , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/farmacología , Línea Celular , Supervivencia Celular , Desoxiadenosinas/química , Quimioterapia Combinada , Femenino , Humanos , Ratones , Estructura Molecular , Proteínas de Transporte de Nucleósidos/genética , Proteínas Protozoarias/genética , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiología , Tripanosomiasis Africana/parasitología , Tubercidina/químicaRESUMEN
Enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, has been recognized to play a pivotal role in regulating the immune response in various diseases. However, its role in the inflammatory response induced by ischaemic stroke remains to be further investigated. The aim of this study was to determine the role of EZH2 in microglia-associated inflammation in ischaemic stroke and to further detect the effects of the EZH2 inhibitor, 3-deazaadenosine A (DZNep), in ischaemic brain injury. Here, we found that both in vivo ischemic/reperfusion (I/R) injury and in vitro oxygen-glucose deprivation (OGD) treatment induced a marked upregulation of EZH2 in microglia. The administration of the EZH2 inhibitor DZNep improved behavioural performance and reduced the infarct volume in mice after experimental stroke. Furthermore, we showed that DZNep blocked pro-inflammatory (CD86+) microglial activation and triggered anti-inflammatory (CD206+) microglial polarization in experimental stroke. Pro-inflammatory cytokines such as IL-1ß, IL-6, TNF-α and CXCL10 were also significantly downregulated by DZNep. In addition, it was found that DZNep blocked the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in microglia, which was increased by I/R injury and OGD. Collectively, we demonstrated that EZH2 is implicated in regulating microglial activation and exacerbates neurological deficits after ischaemic stroke, probably via activating STAT3, and that the EZH2 inhibitor DZNep can exert neuroprotective effects after ischaemic stroke.
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Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Infarto de la Arteria Cerebral Media/patología , Microglía/efectos de los fármacos , Microglía/patología , Fármacos Neuroprotectores/farmacología , Tubercidina/farmacología , Animales , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión/complicaciones , Factor de Transcripción STAT3/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC50 ≥ 6.5 nM) and high affinity ligands (KD ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.
Asunto(s)
Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Nucleósidos/análogos & derivados , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Dipiridamol/farmacología , Evaluación Preclínica de Medicamentos , Activadores de Enzimas/química , Holoenzimas/metabolismo , Leishmania/efectos de los fármacos , Simulación del Acoplamiento Molecular , Fosforilación/efectos de los fármacos , Transducción de Señal , Trypanosoma brucei brucei/efectos de los fármacos , Tubercidina/farmacologíaRESUMEN
OBJECTIVE: Over one-third of all patients with epilepsy are refractory to treatment and there is an urgent need to develop new drugs that can prevent the development and progression of epilepsy. Epileptogenesis is characterized by distinct histopathologic and biochemical changes, which include astrogliosis and increased expression of the adenosine-metabolizing enzyme adenosine kinase (ADK; EC 2.7.1.20). Increased expression of ADK contributes to epileptogenesis and is therefore a target for therapeutic intervention. We tested the prediction that the transient use of an ADK inhibitor administered during the latent phase of epileptogenesis can mitigate the development of epilepsy. METHODS: We used the intrahippocampal kainic acid (KA) mouse model of temporal lobe epilepsy, which is characterized by ipsilateral hippocampal sclerosis with granule cell dispersion and the development of recurrent hippocampal paroxysmal discharges (HPDs). KA-injected mice were treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 1.6 mg/kg, b.i.d., i.p.) during the latent phase of epileptogenesis from day 3-8 after injury; the period when gradual increases in hippocampal ADK expression begin to manifest. HPDs were assessed at 6 and 9 weeks after KA administration followed by epilepsy histopathology including assessment of granule cell dispersion, astrogliosis, and ADK expression. RESULTS: 5-ITU significantly reduced the percent time in seizures by at least 80% in 56% of mice at 6 weeks post-KA. This reduction in seizure activity was maintained in 40% of 5-ITU-treated mice at 9 weeks. 5-ITU also suppressed granule cell dispersion and prevented maladaptive ADK increases in these protected mice. SIGNIFICANCE: Our results show that the transient use of a small-molecule ADK inhibitor, given during the early stages of epileptogenesis, has antiepileptogenic disease-modifying properties, which provides the rationale for further investigation into the development of a novel class of antiepileptogenic ADK inhibitors with increased efficacy for epilepsy prevention.