RESUMEN
Protein kinase B (PKB) is a serine/threonine kinase that plays a key role in the phosphoinositide 3-kinase (PI3K) pathway-one of the most frequently activated proliferation pathways in cancer. In this pathway, PKB is recruited to the plasma membrane by direct interaction of its pleckstrin homology (PH) domain with the inositol phosphate head-group of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] or phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)]. This recruitment is a critical stage in the activation of PKB, whose downstream effectors play important roles in cell survival, proliferation and growth. It is therefore of great interest to understand PKB's mode of binding, as well as its specificity and affinity for different phosphoinositides. We have used a total of 3 µs of molecular dynamics (MD) simulations to better understand the interactions of the PKB PH domain with the inositol phosphate head-groups of phosphoinositides involved in the PI3K pathway. Our computational models successfully mirror PKB's in vivo selectivity for 3-phosphorylated phosphoinositides. Furthermore, the models also help to rationalize unexpected in vitro data in which inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] binds with a relatively high affinity to the PKB PH domain, despite its parent lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] being known not to bind in vivo. With the support of computational simulations, we propose that when not bonded to a phosphatidate tail Ins(1,4,5)P(3) binds in an orientation in which its inositol ring is flipped with respect to the 3-phosphorylated inositol phosphate ligands and its parent lipid.
Asunto(s)
Fosfatos de Inositol/química , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Sanguíneas/química , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Fosfoproteínas/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , TermodinámicaRESUMEN
Norbadione A (NBA) is a pigment present in edible mushrooms which is presumed to selectively complex Cs(+) cations. Due to a very uncommon complexation mechanism, we used a combination of several experimental techniques, including (1)H-NMR, (133)Cs-NMR, isothermal calorimetric, potentiometric titrations and molecular dynamics MD simulations to determine the nature of the complexed species, as well as their stability constants for the NBA-M(+) systems (M(+) = Cs(+), K(+), Na(+)) in methanol:water 80:20 solutions at 25.0 degrees C. We show that almost no complexation occurs below pH 7.5, as long as a proton, involved in a strong hydrogen bond, bridges both carboxylic and enolic groups of each pulvinic moiety of NBA. Thus, neutralization of that proton is necessary to both set free potential coordination sites and to trigger a conformational change, two conditions needed to bind successively a first, then a second metallic cation. The stability constants determined in this study are in good agreement with each other, leading to the stability order Cs(+) > K(+) > Na(+) for both mono- and bimetallic complexes, which is the reversed order to the one generally observed for low molecular weight carboxylic ligands in water. According to MD simulations in solution, complexation involves a mixture of Z/E isomers and conformers of NBA with a broad diversity of binding modes. Some pH and environment dependent aggregation phenomena are considered to also contribute to the binding process, and to possibly explain the accumulation of radionuclides in mushrooms.
Asunto(s)
4-Butirolactona/análogos & derivados , Cesio/química , Enlace de Hidrógeno , Fenilacetatos/química , Potasio/química , Sodio/química , 4-Butirolactona/química , Calorimetría , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Espectrofotometría UltravioletaRESUMEN
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.
Asunto(s)
Regiones no Traducidas 5'/química , Aminoglicósidos/química , Antivirales/química , VIH-1/genética , ARN Viral/química , Antibacterianos/química , Sitios de Unión , Calorimetría , Cinamatos/química , Dimerización , Higromicina B/análogos & derivados , Higromicina B/química , Modelos Moleculares , Nebramicina/análogos & derivados , Nebramicina/química , Conformación de Ácido Nucleico , Paromomicina/análogos & derivados , Paromomicina/química , TermodinámicaRESUMEN
The binding of the most common metal cations of cytoplasm (Li+, Na+, K+, Mg2+ and Ca2+) to a model molecule having an intramolecular hydrogen-bonding network, myo-inositol-2-monophosphate, was studied using first principles. A strong correlation between the conformation of metal inositol phosphate complexes with the type of metal cation, degree of deprotonation state, and the surrounding environment has been observed. On the basis of the hydrogen-bonding network analysis of the cation-phosphate complexes (Mn+-Ins(2)P1), the alkali cations show little effect on the conformational preference while the conformational preference for the binding of the alkaline earth cations is pH-dependent and solvent-dependent. For example, these calculations predict that Mg2+-Ins(2)P1(0) and Mg2+-Ins(2)P1(2-) favor the 1a/5e form while Mg2+-Ins(2)P1(1-) favors the 5a/1e conformation. The Ca2+-Ins(2)P1(2-) complex prefers the 1a/5e conformation in the gas phase and in a nonpolar protein environment, but inverts to the 5a/1e conformation upon entering the polar aqueous phase. The binding affinities of the cations and the pK(a) values for the cation-phosphate complexes are derived from thermodynamical analysis.
Asunto(s)
Algoritmos , Fosfatos de Inositol/química , Metales/química , Sitios de Unión , Cationes , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Conformación Molecular , Fosforilación , Proteínas/química , Protones , Solventes/química , TermodinámicaRESUMEN
3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 1,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with an additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the (31)P NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphate-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphatase catalysed dephosphorylation. However, surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatase to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and (1)H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.
Asunto(s)
Imitación Molecular , Organofosfatos/síntesis química , Organofosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Sistemas de Mensajero Secundario , Fenómenos Químicos , Química Física , Concentración de Iones de Hidrógeno , Inositol Polifosfato 5-Fosfatasas , Espectroscopía de Resonancia Magnética , Estructura Molecular , Organofosfatos/química , Fosfatos/química , Fosforilación , Polifosfatos/química , Protones , Colorantes de Rosanilina/química , Colorantes de Rosanilina/metabolismo , Especificidad por Sustrato , VolumetríaRESUMEN
myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.
Asunto(s)
Fosfatos de Inositol/química , Fosfatos de Inositol/síntesis química , Espectroscopía de Resonancia Magnética/métodos , Conformación Molecular , Fosfohidrolasa PTEN/química , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Potenciometría/métodos , Fuerza Protón-Motriz , Electricidad Estática , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The suitability of high-resolution solid-state 31P NMR for a straightforward determination of the protonation state of phosphate groups as well as of their pK2 values extracted from solid state mono : dianionic ratios has been demonstrated.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Fosfatos/química , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Sensibilidad y EspecificidadRESUMEN
The microscopic protonation mechanism, at an inframolecular level, of norbadione A, a pigment extracted from mushrooms and known to complex cesium cations, was determined by using 1H NMR titrations and the cluster expansion method. This study revealed a pH dependent Z to E isomer switch that occurs in both pulvinic moieties. As a consequence, norbadione A can exist in solution in four stereomeric forms (E-E, E-Z, Z-E, and Z-Z), which can be of interest in the development of molecular-level devices. In the presence of 0.15 M NaCl, the calculated microconstants showed an unusual apparent cooperativity between the enol groups, which results from the release of the sodium cations upon protonation of norbadione A.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/química , Fenilacetatos/química , Concentración de Iones de Hidrógeno , Cinética , Resonancia Magnética Nuclear Biomolecular , Potenciometría , Protones , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate [D-6-deoxy-Ins(1,3,4,5)P(4)] 3 is a novel deoxygenated analogue of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)] 2, a central and enigmatic molecule in the polyphosphoinositide pathway of cellular signalling. D-6-Deoxy-Ins(1,3,4,5)P(4) is a moderate inhibitor of Ins(1,4,5)P(3) 5-phosphatase [1.8microM] compared to Ins(1,3,4,5)P(4) [0.15microM] and similar to that of L-Ins(1,3,4,5)P(4) [1.8microM]. In displacement of [(3)H] Ins(1,4,5)P(3) from the rat cerebellar Ins(1,4,5)P(3) receptor, while slightly weaker [IC(50)=800nM] than that of D-Ins(1,3,4,5)P(4) [IC(50)=220nM], 3 is less markedly different and again similar to that of L-Ins(1,3,4,5)P(4) [IC(50)=660nM]. 3 is an activator of I(CRAC) when inward currents are measured in RBL-2H3-M1 cells using patch-clamp electrophysiological techniques with a facilitation curve different to that of Ins(1,3,4,5)P(4). Physicochemical properties were studied by potentiometric (31)P and (1)H NMR titrations and were similar to those of Ins(1,3,4,5)P(4) apart from the observation of a biphasic titration curve for the P1 phosphate group. A novel vicinal phosphate charge-induced conformational change of the inositol ring above pH 10 was observed for D-6-deoxy-Ins(1,3,4,5)P(4) that would normally be hindered because of the central stabilising role played by the 6-OH group in Ins(1,3,4,5)P(4). We conclude that the 6-OH group in Ins(1,3,4,5)P(4) is crucial for its physicochemical behaviour and biological properties of this key inositol phosphate.
Asunto(s)
Canales de Calcio/química , Fosfatos de Inositol/química , Monoéster Fosfórico Hidrolasas/química , Receptores Citoplasmáticos y Nucleares/química , Canales de Calcio/metabolismo , Concentración de Iones de Hidrógeno , Receptores de Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/análisis , Fosfatos de Inositol/metabolismo , Inositol Polifosfato 5-Fosfatasas , Conformación Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Relación Estructura-ActividadRESUMEN
2-O-(2-Aminoethyl)-Ins(1,4,5)P(3), (5), a novel derivative of the Ca(2+)-mobilising second messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], was synthesised from myo-inositol. 5 was found to be a potent mobiliser of intracellular Ca(2+), and an Ins(1,4,5)P(3) affinity matrix synthesised from 5 was effective at selectively binding N-terminal fragments of the Ins(1,4,5)P(3) receptor containing the intact Ins(1,4,5)P(3) binding site. The microprotonation scheme for 5 was resolved and the related constants were determined in comparison with Ins(1,4,5)P(3) and another reactive Ins(1,4,5)P(3) analogue 1-O-(2-aminoethyl-1-phospho)-Ins(4,5)P(2), (2a), by potentiometric and NMR titration methods. The (31)P and (1)H NMR titration curves for compound 5 and Ins(1,4,5)P(3) are remarkably close, indicating analogous acid-base properties and intramolecular interactions for the two compounds. The 1-phosphate-modified Ins(1,4,5)P(3) derivative 2a, on the contrary, behaves as a bisphosphorylated rather than a trisphosphorylated inositol. Thus, 5 is a new reactive Ins(1,4,5)P(3) analogue of considerable potential for investigation of the chemical biology of Ins(1,4,5)P(3)-mediated cellular signalling.
Asunto(s)
Aminas/química , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/síntesis química , Técnicas Biosensibles/métodos , Calcio/química , Calcio/metabolismo , Cromatografía de Afinidad , Hepatocitos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inositol/química , Inositol 1,4,5-Trifosfato/química , Cinética , Ligandos , Modelos Moleculares , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Fósforo , Potenciometría , Unión Proteica , ProtonesRESUMEN
Four adenophostin analogues lacking the adenine moiety were subjected to 31P- and 1H-NMR titrations in order to determine the acid-base behaviour of the individual ionisable groups of the molecules and the complex interplay of intramolecular interactions resulting from the protonation process. For the two trisphosphorylated compounds, the curve pattern of the phosphorus nuclei corresponds to the superimposition of the titration curves of a monophosphorylated polyol and a polyol carrying two vicinal phosphates, suggesting that the two phosphate moieties behave independently. Also, the general shape of 1H-NMR titration curves of the studied compounds is very close to that of adenophostin A, indicating that the adenine moiety does not specifically interact with the phosphorylated sugar moieties. The curves show, however, that both trisphosphorylated compounds adopt slightly different preferential conformations which could contribute to explain the difference in their affinity for Ins(1,4,5)P3 receptor. Their macroscopic as well as the microscopic protonation constants are higher than those of adenophostin A, indicating that the adenine moiety plays a base-weakening effect on the phosphate groups. Further analysis of the microscopic protonation constants confirms that the compound whose conformation is the closest to that of adenophostin A also shows the highest biological activity. The two bisphosphorylated analogues studied behave very similarly, suggesting that the deletion of the hydroxymethyl group on the pentafuranosyl ring only weakly influences the protonation process of the phosphate groups that bear the glucopyranose moiety.
Asunto(s)
Adenina/química , Adenosina/análogos & derivados , Adenosina/química , Agonistas de los Canales de Calcio/química , Conformación Proteica , Protones , Proteínas Bacterianas/química , Inositol 1,4,5-Trifosfato/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Fósforo/química , FosforilaciónRESUMEN
1H NMR hydroxy proton titration experiments for myo-inositol 2-phosphate and myo-inositol 1,2,6-tris(phosphates) in aqueous solution are presented to demonstrate that by following OH signals versus pH, evidence can be brought for HB interaction between the hydroxyl and phosphate groups. The chemical shifts of the OH protons vicinal to phosphate groups appear deshielded by ca. 2.5 ppm with regard to those two centers removed from the phosphates. Remarkably, the deshielded protons are only present when their neighboring phosphate groups are fully deprotonated but persist until high pHs. From these results, C-OH...2-O3P-O type I, C-HO...-HO3P-O type II and C-OH...-HO3P-O type III hydrogen bonds are evidenced and discussed.
Asunto(s)
Fosfatos de Inositol/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Fosforilación , Protones , VolumetríaRESUMEN
In our effort to identify potent purinergic P2Y(1) receptor antagonists as potent platelet aggregation inhibitors with enhanced metabolic stability, we developed an efficient route for the large-scale preparation of 2'-deoxy-C-nucleosides of pyrazolo[1,5-a]-1,3,5-triazine. The key strategic elements of this novel synthetic approach involved the following: (i) the use of a novel activating group, the N-methyl-N-phenylamino group, which was easily generated in high yield by treatment of the pyrazolo[1,5-a]-1,3,5-triazin-4-one (5) with phosphorus oxychloride and dimethylaniline under high pressure, (ii) a regio- and stereospecific palladium-mediated coupling reaction of the readily available unprotected glycal 1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol (4b) and the 8-iodo derivative (16), and (iii) the stereoselective reduction of the ketone group of the furanosyl ring followed by the subsequent displacement of the N-methyl-N-phenylamino group upon treatment with methylamine. The beta configuration at the anomeric C-1' position of the glycal moieties was perfectly retained throughout this conversion. This procedure afforded 8-(2'-deoxy-beta-D-ribofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (21) and 8-(2'-deoxy-beta-D-xylofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (24) with an overall yield of 50% and 39%, respectively. Finally, the conversion of nucleosides 21 and 24 to the pyrazolotriazine C-nucleotides 3',5'-bisphosphate 2 and 3',5'-cyclophosphate 26 is also described herein and represents the first reported nucleotide derivatives within the pyrazolo[1,5-a]-1,3,5-triazine series. Preliminary biological testing has shown that compound 2 strongly inhibits ADP-induced human platelet aggregation and shape change and possesses significant efficacies 30 min after injection in rat, highlighting a strong P2Y(1)-receptor antagonist activity in vitro combined with a prolonged duration of action in vivo.
Asunto(s)
Nucleósidos/síntesis química , Nucleótidos/síntesis química , Antagonistas del Receptor Purinérgico P2 , Adenosina Difosfato/farmacología , Animales , Plaquetas/efectos de los fármacos , Humanos , Nucleósidos/administración & dosificación , Nucleósidos/farmacología , Nucleótidos/administración & dosificación , Nucleótidos/farmacología , Ácidos Fosfatidicos , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/farmacología , Ratas , Receptores Purinérgicos P2Y1 , Triazinas/administración & dosificación , Triazinas/síntesis química , Triazinas/farmacologíaRESUMEN
The human P2Y(1) receptor is widely distributed in many tissues and has a classical structure of a G protein-coupled receptor. Activated by adenosine-5'-diphosphate (ADP), this receptor is essential for platelet aggregation. In the present paper, we describe the synthesis of novel P2Y(1) antagonists that could be of interest at least as tools to define the physiological roles of the P2Y(1) receptor, at best as new antithrombotic agents. Thus, we prepared the 2,N(6)-dimethyl-2'-deoxyadenosine-3',5'-bisphosphate derivative, 1e. The biological activity was demonstrated by the ability of compound 1e to inhibit ADP-induced platelet aggregation, shape change, and intracellular calcium rise. This compound was a full antagonist at the P2Y(1) receptor with a pA(2) value of 7.11 +/- 0.11 and was found to be 4-fold more potent than the reference N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (1a, pA(2) = 6.55 +/- 0.05), revealing the potency-enhancing effects of the 2-methyl group. The better activity of 1e as compared to 1a was analyzed using both potentiometric and nuclear magnetic resonance titration techniques, which highlighted specific conformational features of this compound. These results clearly indicate the preference for both compounds for an anti conformation at the N-glycosyl linkage. Furthermore, the percentage of S conformer of 1e is close to that of 1a, which is nearly 70% at pH = 2.8 and increases dramatically when pH increases. From the macroprotonation constants, it can be noted that compound 1e is significantly more basic than 1a. This is indeed expected for the N1 adenine nitrogen due to the electron-donating character of the methyl moiety. By considering the microconstants of the phosphate groups, the higher basicity of P3 and P5 for 1e may be due to the decrease in the local dielectric constant induced by the substitution of the hydrogen atom by a more lipophilic methyl group. Thus, it may be suggested that the gain in activity of 1e when compared to the reference compound 1a would result from its gain in basicity rather than steric and conformational modifications. The synthesis of the first selective radioligand acting at the P2Y(1) receptor ([(33)P]-N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate, 17) is also reported and will be used in the future for efficient screening needed for drug optimization.