RESUMEN
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
Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.
Asunto(s)
Barrera Hematorretinal/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/administración & dosificación , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Degeneración Retiniana/tratamiento farmacológico , Animales , Barrera Hematorretinal/efectos de los fármacos , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Liposomas , Ratones , Células Fotorreceptoras/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Degeneración Retiniana/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Melanoma is one of the most aggressive cancers and displays high resistance to conventional chemotherapy underlining the need for new therapeutic strategies. The cGMP/PKG signaling pathway was detected in melanoma cells and shown to reduce migration, proliferation and to increase apoptosis in different cancer types. In this study, we evaluated the effects on cell viability, cell death, proliferation and migration of novel dimeric cGMP analogues in two melanoma cell lines (MNT1 and SkMel28). These new dimeric cGMP analogues, by activating PKG with limited effects on PKA, significantly reduced proliferation, migration and increased cell death. No decrease in cell viability was observed in non-tumor cells suggesting a tumor-specific effect. These effects observed in melanoma are possibly mediated by PKG2 activation based on the decreased toxic effects in tumor cell lines not expressing PKG2. Finally, PKG-associated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP), linked to cell death, proliferation and migration was found increased and with a change of subcellular localization. Increased phosphorylation of RhoA induced by activation of PKG may also contribute to reduced migration ability of the SkMel28 melanoma cell line when treated with cGMP analogues. These findings suggest that the cGMP/PKG pathway can be envisaged as a therapeutic target of novel dimeric cGMP analogues for the treatment of melanoma.
RESUMEN
Activation of the cGMP-dependent protein kinase G (PKG) can inhibit growth and/or induce apoptosis in colon cancer. In this study we evaluated the effects on cell viability, cell death and proliferation of novel dimeric cGMP analogues, compared to a monomeric compound. Three colon cancer cell lines, which only express isoform 2 of PKG, were treated with these novel cGMP analogues and responded with increased PKG activity. cGMP analogues reduced cell viability in the three cell lines and this was due to a cytostatic rather than cytotoxic effect. These findings suggest that activation of PKG2 can be a therapeutic target in the treatment of colon cancer and, most importantly, that dimeric cGMP analogues can further improve the beneficial effects previously observed with monomeric cGMP analogues.
Asunto(s)
Antineoplásicos/farmacología , GMP Cíclico/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Dimerización , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The cyclic dinucleotides (CDNs) cyclic diguanosine monophosphate (c-diGMP) and cyclic diadenosine monophosphate (c-diAMP) with two canonical 3'â5' internucleotide linkages are ubiquitous second messenger molecules in bacteria, regulating a multitude of physiological processes. Recently the noncanonical CDN cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) featuring a mixed linkage, which consists of a 2'â5' and a 3'â5' internucleotide bond, has been identified as a signaling molecule in metazoan species in late 2012. 2'3'-cGAMP formation is biocatalyzed by cGAMP synthase (cGAS) upon sensing of cytosolic double-stranded DNA (dsDNA) and functions as an endogenous inducer of innate immunity by directly binding to and activating the adaptor protein stimulator of interferon genes (STING). Thereby 2'3'-cGAMP can stimulate interferon-ß (INF-ß) secretion, a major signaling pathway of host defense, which is independent of toll-like receptor (TLR) activation. Medicinal chemistry of 2'3'-cGAMP and development of corresponding analogs are still in their infancy, and only a handful of structurally related compounds are available to the scientific community. The aim of this chapter is to summarize synthetic approaches to prepare canonical and noncanonical endogenous CDNs including 2'3'-cGAMP. Furthermore, we will describe syntheses of 2'3'-cGAMP analogs bearing modifications, which will facilitate further studies of the emerging biological functions of 2'3'-cGAMP and to identify additional receptor proteins. Finally, we will review latest developments concerning 2'3'-cGAMP analogs with improved hydrolytic stability in cell cultures and in tissues, putatively qualifying for new therapeutic options on the basis of 2'3'-cGAMP signaling.
Asunto(s)
Nucleótidos Cíclicos/síntesis química , Sistemas de Mensajero Secundario , Animales , Estabilidad de Medicamentos , Humanos , Hidrólisis , Estructura Molecular , Nucleótidos Cíclicos/metabolismo , Nucleótidos Cíclicos/farmacología , Sistemas de Mensajero Secundario/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems, also the noncanonical congeners cyclic cytidine-3',5'-monophosphate (cCMP) and cyclic uridine-3',5'-monophosphate (cUMP) gained more and more interest. Until the late 1980s, only a small number of cCMP and cUMP analogs with sometimes undefined purities had been described. Moreover, most of these compounds had been rather synthesized as precursors of antitumor and antiviral nucleoside-5'-monophosphates and hence had not been tested for any second messenger activity. Along with the recurring interest in cCMP- and cUMP-related signaling in the early 2000s, it became evident that well-characterized small molecule analogs with reliable purities would serve as highly valuable tools for the evaluation of a putative second messenger role of cyclic pyrimidine nucleotides. Meanwhile, for this purpose new cCMP and cUMP derivatives have been developed, and already known analogs have been resynthesized and highly purified. This chapter summarizes early medicinal chemistry work on cCMP and cUMP and analogs thereof, followed by a description of recent synthetic developments and an outlook on potential future directions.
Asunto(s)
CMP Cíclico/síntesis química , Nucleótidos Cíclicos/síntesis química , Profármacos/síntesis química , Uridina Monofosfato/síntesis química , Animales , Cristalización , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , CMP Cíclico/farmacología , Humanos , Estructura Molecular , Nucleótidos Cíclicos/metabolismo , Nucleótidos Cíclicos/farmacología , Permeabilidad , Profármacos/metabolismo , Profármacos/farmacología , Uridina Monofosfato/metabolismo , Uridina Monofosfato/farmacologíaRESUMEN
The Mediterranean Diet (MD) has been proved to exert benefits with respect to the maintenance of the redox balance, and wine is a representative component. Bioactive compounds such as polyphenols, melatonin and hydroxytyrosol act as radical scavengers and regulate the oxidation status of organisms. Oxidative damage to DNA yields a large range of end products. The repair of oxidized DNA entails the removal of the useless bases and/or nucleotides as well as the release of circulating nucleotides and nucleosides. The current research aims to elucidate, for the first time, the DNA protection against oxidative stress provided by three types of red wine - relating it to the intake of bioactive compounds - after the intake of a serving of red wine/must by 18 healthy female volunteers during a short term double-blind, crossover and placebo-controlled study. The novelty of our work is to describe the importance of melatonin and hydroxytyrosol and its metabolites (from gut microflora) in comparison with polyphenols in a red wine matrix (excluding colon derivatives). The results show that the intake of red wine and must secondarily reduces oxidative stress and carcinogenesis due to their content of homovanillic acid, as measured by decreases in the plasmatic concentration of 8-hydroxy-2'deoxyguanosine, 8-hydroxyguanine, and 8-nitroguanosine. Moreover, the intake of wine appears to exert vasodilatory effects, mediated by the action of nitric oxide and increased plasma guanosine-3'-5'-cyclic monophosphate plasmatic levels, owing to the intake of wines higher in melatonin and homovanillic acid. Therefore, the results obtained in the present study revealed that polyphenols, despite being the major compounds in the red wine matrix, are not the most effective compounds protecting DNA from oxidative attack.
Asunto(s)
ADN/química , Melatonina/farmacología , Alcohol Feniletílico/análogos & derivados , Vino/análisis , Adolescente , Adulto , Cromatografía Líquida de Alta Presión , Estudios Cruzados , ADN/metabolismo , Método Doble Ciego , Femenino , Humanos , Melatonina/química , Mutación , Oxidación-Reducción , Alcohol Feniletílico/química , Alcohol Feniletílico/farmacología , Adulto JovenRESUMEN
In this study we analyzed whether our aronia-citrus juice (ACJ, the composition is based on a mixture of 95% citrus juice with 5% of Aronia melanocarpa juice), rich in polyphenols, and physical exercise had an effect on seven catabolites of DNA identified in plasma and on a urine isoprostane (8-iso-PGF2α). Sixteen elite triathletes on a controlled diet for triathlon training (45 days) were used in this clinical trial. Our results show a decrease in the 8-hydroxy-2'-deoxyguanosine concentration due to chronic physical exercise. The ACJ intake and physical exercise maintained the guanosine-3',5'-cyclic monophosphate plasmatic concentrations and decreased the concentration of 8-hydroxyguanine as well as urinary values of 8-iso-PGF2α. Finally, we observed a significant increase in the 8-nitroguanosine levels in triathletes after ACJ intake, compared to the placebo stage. It is concluded that the combination of the intake of ACJ, rich in polyphenolic compounds, with adequate training was able to influence the plasmatic and urinary values of oxidative stress biomarkers. This suggests a positive effect on the oxidative damage and potential associations with DNA repair mechanisms.
Asunto(s)
Bebidas/análisis , Citrus/metabolismo , ADN/metabolismo , Photinia/metabolismo , Polifenoles/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Atletas , Citrus/química , ADN/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Dieta , Femenino , Humanos , Masculino , Estrés Oxidativo , Photinia/química , Esfuerzo Físico , Adulto JovenRESUMEN
UNLABELLED: High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE: In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.
Asunto(s)
GMP Cíclico/análogos & derivados , Análisis por Matrices de Proteínas/métodos , Pseudomonas aeruginosa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Consenso , GMP Cíclico/química , GMP Cíclico/genética , GMP Cíclico/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Movimiento , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic ß-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.
Asunto(s)
AMP Cíclico/análogos & derivados , AMP Cíclico/química , Factores de Intercambio de Guanina Nucleótido/agonistas , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , AMP Cíclico/farmacología , Diseño de Fármacos , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Modelos Moleculares , Unión ProteicaRESUMEN
As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.
Asunto(s)
Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Espectrometría de Fluorescencia/métodos , Cinética , Especificidad por SustratoRESUMEN
The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)ß(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.
Asunto(s)
Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , CMP Cíclico/metabolismo , Sefarosa/química , Toxina de Adenilato Ciclasa/metabolismo , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , CMP Cíclico/química , Regulación de la Expresión Génica , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Células HEK293 , Células HL-60 , Células HeLa , Humanos , Immunoblotting , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometría de Masas , Unión Proteica , Transducción de SeñalRESUMEN
In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Portadoras/análisis , GMP Cíclico/análogos & derivados , Proteómica/métodos , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Pseudomonas aeruginosa/química , Transducción de Señal , Resonancia por Plasmón de SuperficieRESUMEN
The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , AMP Cíclico/genética , GMP Cíclico/genética , Activación Enzimática/fisiología , Células HEK293 , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Hidrolasas Diéster Fosfóricas/genética , Estructura Terciaria de ProteínaRESUMEN
Immunoassays are routinely used as research tools to measure intracellular cAMP and cGMP concentrations. Ideally, this application requires antibodies with high sensitivity and specificity. The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with two non-radioactive and one radioactive cAMP and cGMP immunoassay. Most of the tested cyclic nucleotide analogs showed low degree competition with the antibodies; however, with Rp-cAMPS, 8-Br-cGMP and 8-pCPT-cGMP, a strong cross-reactivity with the corresponding cAMP and cGMP, respectively, immunoassays was observed. The determined EIA-binding constants enabled the measurement of the intracellular cyclic nucleotide concentrations and revealed a time- and lipophilicity-dependent cell membrane permeability of the compounds in the range of 10-30% of the extracellular applied concentration, thus allowing a more accurate prediction of the intracellular analog levels in a given experiment.
Asunto(s)
AMP Cíclico/análogos & derivados , AMP Cíclico/análisis , GMP Cíclico/análogos & derivados , GMP Cíclico/análisis , Técnicas para Inmunoenzimas/métodos , Animales , Unión Competitiva , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Plaquetas/metabolismo , Western Blotting , Células CHO , Permeabilidad de la Membrana Celular , Cricetinae , Cricetulus , Reacciones Cruzadas , AMP Cíclico/inmunología , GMP Cíclico/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , RadioinmunoensayoRESUMEN
Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2'-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2'-O-Me-cAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2'-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2'-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral expression of ß-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/fisiología , Isquemia/complicaciones , Riñón/irrigación sanguínea , Insuficiencia Renal/etiología , Transducción de Señal/fisiología , Estrés Fisiológico , Proteínas de Unión al GTP rap1/fisiología , Uniones Adherentes/fisiología , Animales , Adhesión Celular , Hipoxia de la Célula , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Adhesiones Focales , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sistemas de Mensajero Secundario/fisiología , Proteínas de Anclaje a la Quinasa A/genética , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Enfermedad Crónica , AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Masculino , Contracción Miocárdica/efectos de los fármacos , Ratas , Ratas Endogámicas WKY , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
cAMP (adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C). Antagonist analogues (here: Rp-cAMPS) bind to the holoenzyme without dissociating the complex and were developed to identify interaction partners that bind to the entire complex or to PKA-C. More than 80 different proteins were isolated from tissue extracts including several PKA isoforms and known as well as potentially new interaction partners. Nevertheless, unspecific binding of general nucleotide binding proteins limited the outcome of this chemical proteomics approach. Surface plasmon resonance (SPR) was employed to optimise the entire workflow of pull down proteomics and to quantify the effects of different nucleotides (ATP, ADP, GTP and NADH) on PKA-R binding to affinity material. We could demonstrate that the addition of NADH to lysates improved specificity in pull down experiments. Using a combination of SPR studies and pull down experiments it was shown unambiguously that it is possible to specifically elute protein complexes with cAMP or cGMP from cAMPS analogue matrices. The side-by-side analysis of the PKA-R interactome and the holoenzyme complexed with interacting proteins will contribute to a further dissection of the multifaceted PKA signalling network.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Nucleótidos Cíclicos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Marcadores de Afinidad , Unión ProteicaRESUMEN
BACKGROUND AND PURPOSE: By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so-called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5. EXPERIMENTAL APPROACH: Using fluorophore-tagged, isolated GAF domains of PDE2 and PDE5, promising cGMP analogues were selected. Subsequently, the effects of these analogues on the enzymatic activity of PDE2 and PDE5 were analysed. KEY RESULTS: The PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMP- and cGMP-hydrolysing activities of PDE2. The ligand for the GAF domains of PDE5, 8-Br-cGMP, elicited a 20-fold GAF-dependent activation and moreover revealed a time-dependent increase in PDE5 activity that occurred independently of a GAF ligand. Although GAF-dependent PDE5 activation was fast at high ligand concentrations, it was slow at physiologically relevant cGMP concentrations; PDE5 reached its final catalytic rates at 1µM cGMP after approximately 10min. CONCLUSIONS AND IMPLICATIONS: We conclude that the delayed activation of PDE5 is required to shape biphasic, spike-like cGMP signals. Phosphorylation of PDE5 further enhances activity and conserves PDE5 activation, thereby enabling PDE5 to act as a molecular memory balancing cGMP responses to nitric oxide or natriuretic peptide signals.
Asunto(s)
GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Animales , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Ligandos , Ratones , Péptidos Natriuréticos/metabolismo , Óxido Nítrico/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND: In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins. RESULTS: Two sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR) analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources. CONCLUSION: In summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstrated incomplete elution from the matrix, as well as retaining notable amounts of bound protein contaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.