Caracterización energética de la unión de 1,3,6- naftalén trisulfonato al factor de crecimiento para fibroblastos ácido humano: implicaciones para su uso como agente anti-angiogénico

The equilibrium interaction of anti-cancer agent 1,3,6-naphatalene trisulfonate with human acidic fi broblast growthfactor has been studied by calorimetry. The affi nity decreases with increasing ionic strength. At pH 7.0 and 0.15 MNaCl concentration, a binding constant of the protein with the ligand was estimated in the 102 – 103 M-1 range, anaffi nity two orders of magnitude lower than that of aFGF with heparin. The interaction is enthalpically driven, andthe entropy change is unfavorable. A small heat capacity change with an unusual positive value of 90 cal K-1mol-1 wasdetermined from the temperature dependence of the enthalpies. Changes in accessible apolar and polar surface areasin the interaction were calculated from the thermodynamic data obtained and parametric equations in the literature.The results were compared with those measured from NMR data. The study includes structural bioenergetic considerationsabout the possible use of 1,3,6-naphatalene trisulfonate as an anti-angiogenic agent itself, or as a lead for thedevelopment of anti-angiogenic drugs.Se ha estudiado, mediante calorimetría isotérmica de reacción, la interacción del agente anticancerígeno 1,3,6-naftaléntrisulfonato con el factor de crecimiento para fi broblastos ácido humano. La afi nidad decrece con el aumento de lafuerza iónica. A pH 7,0 y NaCl 0,15 M, la constante de unión de la proteína con el ligando se encuentra en el rango102 – 103 M-1, una afi nidad dos órdenes de magnitud menor que la del FGFa por heparina. El cambio de entalpíafavorece la interacción, siendo el cambio de entropía desfavorable. De la dependencia del cambio de entalpía con latemperatura se calculó un pequeño cambio en la capacidad calorífi ca del proceso, con un valor excepcionalmentepositivo de 90 cal K-1mol-1. A partir de los datos termodinámicos medidos y de ecuaciones paramétricas establecidasen la literatura, se calcularon cambios en la superfi cie accesible al disolvente, tanto polar como apolar, que acompañana la interacción. Los resultados se compararon con los medidos mediante resonancia magnética nuclear. Elestudio incluye consideraciones de bioenergética estructural sobre el posible uso de 1,3,6-naftalén trisulfonato comoagente antiangiogénico o como molécula líder para el desarrollo de fármacos anti-angiogénicos

Energetic characterization of 1,3,6-naphtalene trisulfonate binding to human acidic fi broblast growth factor: implications for its anti-angiogenic use

Se ha estudiado, mediante calorimetría isotérmica de reacción, la interacción del agente anticancerígeno 1,3,6-naftalén trisulfonato con el factor de crecimiento para fi broblastos ácido humano. La afi nidad decrece con el aumento de la fuerza iónica. A pH 7,0 y NaCl 0,15 M, la constante de unión de la proteína con el ligando se encuentra en el rango 102 – 103 M-1, una afi nidad dos órdenes de magnitud menor que la del FGFa por heparina. El cambio de entalpía favorece la interacción, siendo el cambio de entropía desfavorable. De la dependencia del cambio de entalpía con la temperatura se calculó un pequeño cambio en la capacidad calorífi ca del proceso, con un valor excepcionalmente positivo de 90 cal K-1mol-1. A partir de los datos termodinámicos medidos y de ecuaciones paramétricas establecidas en la literatura, se calcularon cambios en la superfi cie accesible al disolvente, tanto polar como apolar, que acompañan a la interacción. Los resultados se compararon con los medidos mediante resonancia magnética nuclear. El estudio incluye consideraciones de bioenergética estructural sobre el posible uso de 1,3,6-naftalén trisulfonato como agente antiangiogénico o como molécula líder para el desarrollo de fármacos anti-angiogénicos.The equilibrium interaction of anti-cancer agent 1,3,6-naphatalene trisulfonate with human acidic fi broblast growth factor has been studied by calorimetry. The affi nity decreases with increasing ionic strength. At pH 7.0 and 0.15 M NaCl concentration, a binding constant of the protein with the ligand was estimated in the 102 – 103 M-1 range, an affi nity two orders of magnitude lower than that of aFGF with heparin. The interaction is enthalpically driven, and the entropy change is unfavorable. A small heat capacity change with an unusual positive value of 90 cal K-1mol-1 was determined from the temperature dependence of the enthalpies. Changes in accessible apolar and polar surface areas in the interaction were calculated from the thermodynamic data obtained and parametric equations in the literature. The results were compared with those measured from NMR data. The study includes structural bioenergetic considerations about the possible use of 1,3,6-naphatalene trisulfonate as an anti-angiogenic agent itself, or as a lead for the development of anti-angiogenic drugs

Binding curves by continuous gradient flow-mix calorimetry

1 table, 4 figures.teady-state flow-mix calorimetric methods require large amounts of sample and time to obtain data at variable reactant ratios. The use of continuous concentration gradients allows rapid generation of data with chemicals consumption comparable to other calorimetric techniques. An experimental set-up able to generate a linear concentration gradient of one reactant, while keeping the other concentration constant during the experiment, permits a complete titration in a single experiment. Since the calorimeter is not operating under steady-state conditions, the output is affected by the dynamics of the instrument. Therefore, it is necessary to correct the experimental curve by deconvolution to obtain the true signal. Titration of the carboxyl group of glycine with hydrochloric acid, as well as the binding of ribonuclease A to two different nucleotides prove the efficacy of this new methodology.We thank to M. Balmacz for revising the English text. This work was financed by grant BIO2003-04274 from the Spanish Ministry of Science and Education, and grant HPRN-CT-2002-00241 from the European Union; it was also financed, in part, by FEDER funds from the European Union. J.A.G. was supported by the Andalusian Regional government, Spain. We also acknowledge Dr. Lee Hansen for his careful and exhaustive revision of the manuscript.Peer reviewe

Antifreeze glycopeptide analogues: microwave-enhanced synthesis and functional studies

Heggemann C, Budke C, Schomburg B, et al. Antifreeze glycopeptide analogues: microwave-enhanced synthesis and functional studies. Amino Acids. 2010;38(1):213-222.Antifreeze glycoproteins enable life at temperatures below the freezing point of physiological solutions. They usually consist of the repetitive tripeptide unit (-Ala-Ala-Thr-) with the disaccharide alpha-D-galactosyl(1-3)-beta-N-acetyl-D-galactosamine attached to each hydroxyl group of threonine. Monoglycosylated analogues have been synthesized from the corresponding monoglycosylated threonine building block by microwave-assisted solid phase peptide synthesis. This method allows the preparation of analogues containing sequence variations which are not accessible by other synthetic methods. As antifreeze glycoproteins consist of numerous isoforms they are difficult to obtain in pure form from natural sources. The synthetic peptides have been structurally analyzed by CD and NMR spectroscopy in proton exchange experiments revealing a structure as flexible as reported for the native peptides. Microphysical recrystallization tests show an ice structuring influence and ice growth inhibition depending on the concentration, chain length and sequence of the peptides