RESUMEN
Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.
Asunto(s)
Diferenciación Celular/genética , Mesodermo/citología , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Células Madre Pluripotentes/citología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/fisiología , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Inmunofenotipificación , Hibridación in Situ , Técnicas In Vitro , Mesodermo/metabolismo , Mesodermo/fisiología , Ratones , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Matrices Tisulares , Vía de Señalización Wnt/genéticaRESUMEN
A collection of paullones was tested for inhibitory activity against mitochondrial malate dehydrogenase (mMDH) as a biological target for antiproliferative activity. Based on the results of this screening, 5-benzylpaullones and paullone-9-carboxylic acid alkyl esters were developed as selective mMDH inhibitors. The new derivatives did not show noteworthy antiproliferative activity when tested on a panel of cancer cell lines, suggesting that mMDH inhibition is of minor relevance for the growth inhibition caused by paullones.
Asunto(s)
Benzazepinas/síntesis química , Benzazepinas/farmacología , Ácidos Carboxílicos/química , Descubrimiento de Drogas , Malato Deshidrogenasa/antagonistas & inhibidores , Mitocondrias/enzimología , Benzazepinas/química , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Especificidad por SustratoRESUMEN
Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Proteínas Nucleares/fisiología , Fosfoproteínas Fosfatasas/fisiología , Transducción de Señal/fisiología , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Fragmentos de Péptidos/fisiología , Fosforilación , Estructura Terciaria de Proteína/fisiología , Proteínas Supresoras de Tumor/fisiología , Xenopus laevisRESUMEN
Smad transcription factors are key signal transducers for the TGF-beta/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-beta and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Proteína Smad1/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Modelos Biológicos , Osteosarcoma/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Transducción de Señal , Xenopus laevisRESUMEN
(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Purinas/química , Purinas/metabolismo , Piridoxal Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , Supervivencia Celular , Células Cultivadas , Cromatografía de Afinidad , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Estructura Molecular , Estructura Terciaria de Proteína , Piridoxal/metabolismo , Piridoxal Quinasa/antagonistas & inhibidores , Piridoxal Quinasa/genética , Fosfato de Piridoxal/metabolismo , Ratas , Roscovitina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Distribución TisularRESUMEN
Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR-/- and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27(KIP1). Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Crecimiento/farmacología , Indoles/farmacología , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ensayos de Selección de Medicamentos Antitumorales , Fase G1/efectos de los fármacos , Humanos , Indoles/química , Indoles/metabolismo , Ligandos , Neoplasias Hepáticas Experimentales/patología , Ratones , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/farmacología , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/metabolismo , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
Worldwide increasing resistance of Plasmodium falciparum to common anti-malaria agents calls for the urgent identification of new drugs. Glycogen synthase kinase-3 (GSK-3) represents a potential screening target for the identification of such new compounds. We have cloned PfGSK-3, the P. falciparum gene homologue of GSK-3 beta. It encodes a 452-amino-acid, 53-kDa protein with an unusual N-terminal extension but a well-conserved catalytic domain. A PfGSK-3 tridimensional homology model was generated on the basis of the recently crystallised human GSK-3 beta. It illustrates how the regions involved in the active site, in substrate binding (P+4 phosphate binding domain) and in activity regulation are highly conserved. Recombinant PfGSK-3 phosphorylates GS-1, a GSK-3-specific peptide substrate, glycogen synthase, recombinant axin and the microtubule-binding protein tau. Neither native nor recombinant PfGSK-3 binds to axin. Expression and intracellular localisation of PfGSK-3 were investigated in the erythrocytic stages. Although PfGSK-3 mRNA is present in similar amounts at all stages, the PfGSK-3 protein is predominantly expressed at the early trophozoite stage. Once synthesized, PfGSK-3 is rapidly transported to the erythrocyte cytoplasm where it associates with vesicle-like structures. The physiological functions of PfGSK-3 for the parasite remain to be elucidated. A series of GSK-3 beta inhibitors were tested on both PfGSK-3 and mammalian GSK-3beta. Remarkably these enzymes show a partially divergent sensitivity to the compounds, suggesting that PfGSK-3 selective compounds might be identified.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Animales , Proteína Axina , Secuencia de Bases , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Eritrocitos/enzimología , Expresión Génica , Glucógeno Sintasa Quinasa 3/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
In the embryonic day 19 organs of Corti, we showed that roscovitine, a chemical inhibitor of cyclin-dependent kinases (CDKs), significantly increased the number of hair cells (HCs) and corresponding supporting cells (SCs) by triggering differentiation of precursor cells without interacting with cell proliferation. The effect of roscovitine was mimicked by other CDK1, 2, 5, and 7 inhibitors but not by CDK4/6 and mitogen-activated protein kinase pathway antagonists. Immunohistochemical analysis indicated that roscovitine-specific intracellular targets, CDK1, 2, 5, and 7, were expressed in the organ of Corti and especially in Hensen's cells. Affinity chromatography studies showed a tight correlation between the protein levels of CDK1/2 and 5 and the rate of roscovitine-induced supernumerary cells in the organ of Corti. In addition, we demonstrated that basal CDK activity was higher and more roscovitine-sensitive at developmental stages that are selectively permissive for the emergence of supernumerary cells. These results suggest that CDKs are involved in the normal development of the organ of Corti and that, at least in E19 embryos, inhibition of CDKs is sufficient to trigger the differentiation of HCs and corresponding SCs, presumably from the Hensen's cell progenitors and/or from progenitors located in the greater epithelial ridge area.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Células Ciliadas Auditivas/embriología , Órgano Espiral/citología , Órgano Espiral/embriología , Animales , Apoptosis , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas CDC2-CDC28/antagonistas & inhibidores , Diferenciación Celular , División Celular , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores Enzimáticos/farmacología , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/enzimología , Immunoblotting , Inmunohistoquímica , Modelos Biológicos , Órgano Espiral/enzimología , Órgano Espiral/crecimiento & desarrollo , Purinas/farmacología , Ratas , RoscovitinaRESUMEN
Gastropod mollusks have been used for over 2500 years to produce the "Tyrian purple" dye made famous by the Phoenicians. This dye is constituted of mixed bromine-substituted indigo and indirubin isomers. Among these, the new natural product 6-bromoindirubin and its synthetic, cell-permeable derivative, 6-bromoindirubin-3'-oxime (BIO), display remarkable selective inhibition of glycogen synthase kinase-3 (GSK-3). Cocrystal structure of GSK-3beta/BIO and CDK5/p25/indirubin-3'-oxime were resolved, providing a detailed view of indirubins' interactions within the ATP binding pocket of these kinases. BIO but not 1-methyl-BIO, its kinase inactive analog, also inhibited the phosphorylation on Tyr276/216, a GSK-3alpha/beta activation site. BIO but not 1-methyl-BIO reduced beta-catenin phosphorylation on a GSK-3-specific site in cellular models. BIO but not 1-methyl-BIO closely mimicked Wnt signaling in Xenopus embryos. 6-bromoindirubins thus provide a new scaffold for the development of selective and potent pharmacological inhibitors of GSK-3.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Indoles/aislamiento & purificación , Indoles/farmacología , Animales , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Inhibidores Enzimáticos/química , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/metabolismo , Indoles/química , Modelos Moleculares , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Mariscos , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Proteínas Wnt , Xenopus/embriología , Xenopus/metabolismoRESUMEN
Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription and differentiation in addition to functions in the nervous system. Deregulation of CDKs in various diseases has stimulated an intensive search for selective pharmacological inhibitors of these kinases. More than 50 inhibitors have been identified, among which >20 have been co-crystallized with CDK2. These inhibitors all target the ATP-binding pocket of the catalytic site of the kinase. The actual selectivity of most known CDK inhibitors, and thus the underlying mechanism of their cellular effects, is poorly known. Pharmacological inhibitors of CDKs are currently being evaluated for therapeutic use against cancer, alopecia, neurodegenerative disorders (e.g. Alzheimer's disease, amyotrophic lateral sclerosis and stroke), cardiovascular disorders (e.g. atherosclerosis and restenosis), glomerulonephritis, viral infections (e.g. HCMV, HIV and HSV) and parasitic protozoa (Plasmodium sp. and Leishmania sp.).
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Animales , Quinasas Ciclina-Dependientes/fisiología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription, differentiation and many functions in the nervous system. The frequent deregulation of CDKs in cancers and in numerous other pathologies justifies the active search for chemical inhibitors capable of reversibly and selectively inhibiting this class of enzymes. Intensive screening of collections of natural and synthetic compounds has led to the identification of several families of ATP competitive CDK inhibitors. As the therapeutic potential of the most promising compounds is currently being evaluated in preclinical and clinical trials, their mechanism of action is still unclear. In particular, the real spectrum of their intracellular targets remains largely unknown. Determination of the selectivity of the compounds and identification of their intracellular targets constitute a prerequisite to understand their cellular effects and to improve their efficiency on a rational basis. The classical method for the determination of a compound's selectivity consists in testing the compound in a panel of purified kinases. However, the selectivity study is then restricted to the panel's enzymes. As a consequence, many, if not most other potential targets are not evaluated. As an alternative way to investigate the range of true targets of CDK inhibitors, we propose an affinity chromatography approach based on immobilized inhibitors. Briefly, the inhibitor is covalently bound to a resin and cellular extracts are batch loaded on this inhibitor matrix. After extensive washing, the bound proteins are resolved by SDS-PAGE and identified by microsequencing. In addition to confirming the interaction of CDK inhibitors with CDKs, this method has led to the identification of additional, sometimes unexpected, targets. We here illustrate the potential of this technique through a few examples.
Asunto(s)
Cromatografía de Afinidad/métodos , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores Enzimáticos/aislamiento & purificación , Animales , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Conformación Molecular , XenopusRESUMEN
Chemical inhibitors of cyclin-dependent kinases (CDKs) have a great therapeutic potential against various proliferative and neurodegenerative disorders. Intensive screening of a combinatorial chemistry library of 2,6,9-trisubstituted purines has led to the identification of purvalanol, one of the most potent and selective CDK inhibitors to date. In preliminary studies, this compound demonstrates definite anti-mitotic properties, consistent with its nanomolar range efficiency towards purified CDK1 and CDK2. However, the actual intracellular targets of purvalanol remain to be identified, and a method for the determination of its in vivo selectivity was developed. In this technique, cell extracts were screened for purvalanol-interacting proteins by affinity chromatography on immobilized inhibitor. In addition to CDK1, p42/p44 MAPK were found to be two major purvalanol-interacting proteins in five different mammalian cell lines (CCL39, PC12, HBL100, MCF-7 and Jurkat cells), suggesting the generality of the purvalanol/p42/p44 MAPK interaction. The Chinese hamster lung fibroblast cell line CCL39 was used as a model to investigate the anti-proliferative properties of purvalanol. The compound inhibited cell growth with a GI(50) value of 2.5 microM and induced a G2/M block when added to exponentially growing cells. It did not appear to trigger massive activation of caspase. We next tested whether CDKs and p42/p44 MAPK were actually targeted by the compound in vivo. p42/p44 MAPK activity was visualized using an Elk-Gal4 luciferase reporter system and CDK1 activity was detected by the phosphonucleolin level. When cells were treated with purvalanol, p42/p44 MAPK and CDK1 activities were inhibited in a dose-dependent manner. Furthermore, purvalanol inhibited the nuclear accumulation of p42/p44 MAPK, an event dependent on the catalytic activity of these kinases. We conclude that the anti-proliferative properties of purvalanol are mediated by inhibition of both p42/p44 MAPK and CDKs. These observations highlight the potency of moderate selectivity compounds and encourage the search for new therapeutics which simultaneously target distinct but relevant pathways of cell proliferation.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Adenina/química , Animales , Western Blotting , Células Cultivadas/efectos de los fármacos , Cricetinae , Técnica del Anticuerpo Fluorescente Indirecta , Luciferasas/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Estructura Molecular , Fosfoproteínas/metabolismo , Fosforilación , Proteínas de Unión al ARN/metabolismo , Transfección , NucleolinaRESUMEN
Paullones 3 and 4 with aminoalkyl side chains in 2- or 3-position were synthesized as derivatives of kenpaullone 1. Both 3 and 4 showed the characteristic CDK1-inhibitory activity of the paullones and a modest antiproliferative activity on cultured human tumor cell lines. Hence, 3 and 4 appear to be suitable tools for affinity studies directed to find additional intracellular paullone targets.
Asunto(s)
Benzazepinas/síntesis química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Benzazepinas/química , Benzazepinas/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Estructura Molecular , Células Tumorales CultivadasRESUMEN
Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock- and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.
Asunto(s)
Adenina/análogos & derivados , Antivirales/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Simplexvirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adenina/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Quinasas Ciclina-Dependientes/metabolismo , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , VIH-1/genética , VIH-1/fisiología , Humanos , Purinas/farmacología , Roscovitina , Simplexvirus/genética , Simplexvirus/fisiología , Células Vero , Proteínas Virales/metabolismoRESUMEN
Numerous inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3 (GSK-3) are being developed in view of their potential applications against cancers and neurodegenerative disorders. Among these, paullones constitute a family of potent and apparently selective cyclin-dependent kinase and GSK-3 inhibitors. However, their actual intracellular targets remain to be identified. To address this issue we have immobilized a paullone, gwennpaullone, on an agarose matrix. Extracts from various cell types and tissues were screened for proteins interacting with this matrix. This approach validated GSK-3alpha and GSK-3beta as major intracellular paullone targets and also mitochondrial, but not cytoplasmic, malate dehydrogenase (MDH). Mitochondrial MDH was indeed inhibited by micromolar concentrations of paullones. Mitochondrial MDH was the major paullone-binding protein in the parasitic protozoon Leishmania mexicana, and paullones inhibited growth of the parasite. This simple batchwise affinity chromatography approach constitutes a straightforward method for the identification of intracellular targets of this particular class of novel anti-mitotic compounds. It has revealed an unexpected target, mitochondrial MDH, the inhibition of which may participate in the pharmacological effects of paullones.