RESUMEN
A capillary zone electrophoretic-electrospray ion trap mass spectrometric method has been developed to assess the stability and pathways of degradation of the cancer therapeutic octapeptide, octreotide. As a somatostatin analogue, octreotide contains a single disulphide bond linking Cys(2)-Cys(7) with the structure of NH2-D-Phe-[Formula: see text]-Thr-OH. Resolution of octreotide from its degradation products was achieved using a capillary zone electrophoretic method with bare fused silica capillaries, a 10mM ammonium formate buffer, pH 3.20, at 25 °C and an applied voltage of 25 kV. An ion trap low energy collision induced dissociation procedure was applied for the characterization of the chemical structures of the degradation products derived from an acidic, alkaline, neutral and thermal solution treatment of octreotide. The results so obtained indicated that linear octreotide degradation products were formed under acidic and alkaline conditions, due to the hydrolysis of a ring amide bond and a hitherto unknown desulfurization of the Cys-Cys disulfide bond, respectively. Degradation under neutral conditions occurred via cleavage of the exocyclic N-((2R,3R)-1,3-dihydroxybutan-2-yl) amide bond which also preceded the ring amide hydrolysis under acidic conditions. The developed method was further successfully applied to assess the kinetics of these octreotide degradations. Overall, this method is suitable for the rapid and precise assessment of the stability and quality control of octreotide as a synthetic peptide-based pharmaceutical product, and has led to the discovery of a new Cys-Cys disulfide degradation pathway.
Asunto(s)
Química Farmacéutica/métodos , Estabilidad de Medicamentos , Electroforesis Capilar , Octreótido/química , Espectrometría de Masa por Ionización de Electrospray , Hidrólisis , Cinética , Octreótido/análisisRESUMEN
The effects of different adenosine receptor antagonists and cyclic nucleotide phosphodiesterase (PDE) inhibitors on monoclonal antibody (mAb) titer and cell viability of murine hybridoma cells in culture were measured as part of our investigations to discover additives that enhance mAb production. Specific adenosine receptor antagonists and PDE inhibitors were found to enhance or decrease the titer of immunoglobulin G1 (IgG1) mAbs relative to negative controls, depending on the specific compound and cell line employed. The observed enhancements or decreases in IgG1 mAb titer appeared to be mainly due to an increase or decrease in specific productivity rates (ngmAb/cell), respectively. The different effects of the selective adenosine antagonists suggest that antagonism at the level of the adenosine A2A and A1 or the adenosine A3 receptors result in either enhancement or suppression of IgG1 mAb production by hybridoma cells. Overall, these studies have identified hitherto unknown activities of specific adenosine antagonists and PDE inhibitors which indicate they may have valuable roles as cell culture additives in industrial biomanufacturing processes designed to enhance the yields of mAbs or other recombinant proteins produced by mammalian cell culture procedures.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/biosíntesis , Inmunoglobulina G/biosíntesis , Inhibidores de Fosfodiesterasa/farmacología , Antagonistas de Receptores Purinérgicos P1/farmacología , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Cafeína/análogos & derivados , Cafeína/farmacología , Supervivencia Celular , Hibridomas , RatonesRESUMEN
Over the past decade, a variety of ionic liquids have emerged as greener solvents for use in the chemical manufacturing industries. Their unique properties have attracted the interest of chemists worldwide to employ them as replacement for conventional solvents in a diverse range of chemical transformations including biotransformations. Biocatalysts are often regarded as green catalysts compared to conventional chemical catalysts in organic synthesis owing to their properties of low toxicity, biodegradability, excellent selectivity and good catalytic performance under mild reaction conditions. Similarly, a selected number of specific ionic liquids can be considered as greener solvents superior to organic solvents owing to their negligible vapor pressure, low flammability, low toxicity and ability to dissolve a wide range of organic and biological substances, including proteins. A combination of biocatalysts and ionic liquids thus appears to be a logical and promising opportunity for industrial use as an alternative to conventional organic chemistry processes employing organic solvents. This article provides an overview of recent developments in this field with special emphasis on the application of more sustainable enzyme-catalyzed reactions and separation processes employing ionic liquids, driven by advances in fundamental knowledge, process optimization and industrial deployment.
Asunto(s)
Biocatálisis , Materiales Biocompatibles/síntesis química , Líquidos Iónicos , Biotransformación , HumanosRESUMEN
We have investigated the binding characteristics of a potent member of the bis-ortho-substituted five-membered nitrogen heterocycle class of ALK-5 kinase inhibitors with lysates of cultured HEK-293 cells to identify protein binding partners of potential biological significance. An affinity chromatographic resin containing an immobilized ALK-5 kinase inhibitor, 2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine, was used to capture specific proteins from the cell lysate. The soluble inhibitor was then used to specifically elute the proteins which selectively bound to the pharmacophore ligand structure. Application of 2-D SDS-PAGE analysis with positive and negative controls demonstrated the inhibitor bound several different proteins via selective molecular recognition processes. The structural features of the specifically eluted proteins were identified by peptide mass fingerprinting (PMF) methods and included proteins with structural, metabolic and chaperone functions. Furthermore, these PMF results identified the therapeutic target in various cancer treatment studies, HSP-70, as a potential high-affinity binding partner. These observations warrant examination of bis-ortho-substituted five-membered nitrogen heterocycles as dual ALK-5/HSP-70 inhibitors for anti-cancer drug development.
Asunto(s)
Antineoplásicos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/síntesis química , Piridinas/química , Piridinas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Superoxide (O(2)(·-)) is the proximal mitochondrial reactive oxygen species underlying pathology and redox signaling. This central role prioritizes development of a mitochondria-targeted reagent selective for controlling O(2)(·-). We have conjugated a mitochondria-targeting triphenylphosphonium (TPP) cation to a O(2)(·-)-selective pentaaza macrocyclic Mn(II) superoxide dismutase (SOD) mimetic to make MitoSOD, a mitochondria-targeted SOD mimetic. MitoSOD showed rapid and extensive membrane potential-dependent uptake into mitochondria without loss of Mn and retained SOD activity. Pulse radiolysis measurements confirmed that MitoSOD was a very effective catalytic SOD mimetic. MitoSOD also catalyzes the ascorbate-dependent reduction of O(2)(·-). The combination of mitochondrial uptake and O(2)(·-) scavenging by MitoSOD decreased inactivation of the matrix enzyme aconitase caused by O(2)(·-). MitoSOD is an effective mitochondria-targeted macrocyclic SOD mimetic that selectively protects mitochondria from O(2)(·-) damage.
Asunto(s)
Materiales Biomiméticos/farmacología , Compuestos Macrocíclicos/química , Mitocondrias/efectos de los fármacos , Aconitato Hidratasa/química , Aconitato Hidratasa/metabolismo , Animales , Ácido Ascórbico/química , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/química , Catálisis , Cristalografía por Rayos X , Cinética , Manganeso/química , Manganeso/farmacología , Microsomas Hepáticos/metabolismo , Mitocondrias/metabolismo , Conformación Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Oxidación-Reducción , Radiólisis de Impulso , Ratas , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
Novel inhibitors of TGF-ß1 and activin A signalling based on a 2-aryl-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridine pharmacophore have been synthesised. Compounds containing phenyl or aromatic nitrogen heterocycle substituents inhibited both types of signalling with HEK-293T cells in culture, with a selectivity preference for TGF-ß1. Synthetic compounds containing pyridin-3-yl, pyrazol-4-yl, pyrazol-1-yl or 1H-imidazoyl-1-yl substituents exhibited structural and functional attributes suitable for further investigation related to the development of more potent TGF-ß inhibitors.
Asunto(s)
Activinas/antagonistas & inhibidores , Compuestos Heterocíclicos/farmacología , Piridinas/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Activinas/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Humanos , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Transducción de Señal/efectos de los fármacos , Estereoisomerismo , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The mechanism for the catalytic dismutation of superoxide by the Mn(II) pentaazamacrocyclic compound M40403 ([manganese(II) dichloro-(4 R,9 R,14 R,19 R)-3,10,13,20,26 pentaazatetracyclo [20.3.1.0 (4,9).0 (14,9)] hexacosa-1(26),-22(23),24-triene], SODm1) and two 2,21-dimethyl analogues has been investigated using pulse radiolysis. The initial rate of reaction between superoxide and the manganese compounds was found to be dependent on structure and pH, with the resulting transient adducts possessing spectral characteristics of the metal center being oxidized to Mn(III). Values for the p K a of the transient adducts (p K a = 5.65 +/- 0.05; 5.3 +/- 0.1 and <5 for SODm1, SODm2 and SODm3, respectively) were obtained from spectrophotometric and conductivity measurements. Reaction of these transient adducts with further superoxide was highly structure dependent with the 2 S,21 S-dimethyl derivative (SODm2) being highly catalytically active at pH 7.4 ( k cat = 2.35 x 10 (8) M (-1) s (-1)) compared to SODm1 ( k cat = 3.55 x 10 (6) M (-1) s (-1)). In contrast the 2 R,21 R-dimethyl derivative (SODm3) showed no dismutation catalysis at all. The reaction rates of the initial complexes with HO 2 (*) were significantly lower than with O 2 (*-), and it is proposed that O 2 (*-) is the main reactant in the catalytic cycle at pH 7.4. Variable temperature studies revealed major differences in the thermodynamics of the catalytic cycles involving SODm2 or SODm1. In the case of SODm2, the observed high entropic contribution to the activation energy is indicative of ligand conformational changes during the catalytic step. These results have provided the basis for a new mechanism for the catalytic dismutation of superoxide by Mn(II)-pentaazamacrocycle SOD mimetics.
Asunto(s)
Manganeso/química , Radiólisis de Impulso , Superóxido Dismutasa/química , Catálisis , Química Física/métodos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Metales/química , Modelos Químicos , Estrés Oxidativo , Oxígeno/química , Superóxidos/química , TermodinámicaRESUMEN
Mitochondrial oxidative damage contributes to a range of degenerative diseases. Ubiquinones have been shown to protect mitochondria from oxidative damage, but only a small proportion of externally administered ubiquinone is taken up by mitochondria. Conjugation of the lipophilic triphenylphosphonium cation to a ubiquinone moiety has produced a compound, MitoQ, which accumulates selectively into mitochondria. MitoQ passes easily through all biological membranes and, because of its positive charge, is accumulated several hundred-fold within mitochondria driven by the mitochondrial membrane potential. MitoQ protects mitochondria against oxidative damage in vitro and following oral delivery, and may therefore form the basis for mitochondria-protective therapies.
Asunto(s)
Mitocondrias/metabolismo , Compuestos Organofosforados/metabolismo , Quinonas/química , Ubiquinona/análogos & derivados , Administración Oral , Animales , Cationes , Membrana Celular/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Potenciales de la Membrana , Enfermedades Mitocondriales/terapia , Modelos Biológicos , Modelos Químicos , Oxígeno/metabolismo , Ubiquinona/metabolismoRESUMEN
Mitochondrial production of peroxides is a critical event in both pathology and redox signaling. Consequently their selective degradation within mitochondria is of considerable interest. Here we have explored the interaction of the peroxidase mimetic ebselen with mitochondria. We were particularly interested in whether ebselen was activated by mitochondrial glutathione (GSH) and thioredoxin, in determining whether an ebselen moiety could be targeted to mitochondria by conjugating it to a lipophilic cation, and in exploring the nature of ebselen binding to mitochondrial proteins. To achieve these goals we synthesized 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (MitoPeroxidase), which contains an ebselen moiety covalently linked to a triphenylphosphonium (TPP) cation. The fixed positive charge of TPP facilitated mass spectrometric analysis, which showed that the ebselen moiety was reduced by GSH to the selenol form and that subsequent reaction with a peroxide reformed the ebselen moiety. MitoPeroxidase and ebselen were effective antioxidants that degraded phospholipid hydroperoxides, prevented lipid peroxidation, and protected mitochondria from oxidative damage. Both peroxidase mimetics required activation by mitochondrial GSH or thioredoxin to be effective antioxidants. Surprisingly, conjugation to the TPP cation led to only a slight increase in the uptake of ebselen by mitochondria due to covalent binding of the ebselen moiety to proteins. Using antiserum against the TPP moiety we visualized those proteins covalently attached to the ebselen moiety. This analysis indicated that much of the ebselen present within mitochondria is bound to protein thiols through reversible selenenylsulfide bonds. Both MitoPeroxidase and ebselen decreased apoptosis induced by oxidative stress, suggesting that they can decrease mitochondrial oxidative stress. This exploration has led to new insights into the behavior of peroxidase mimetics within mitochondria and to their use in investigating mitochondrial oxidative damage.
Asunto(s)
Azoles/metabolismo , Mitocondrias Hepáticas/metabolismo , Imitación Molecular , Compuestos de Organoselenio/síntesis química , Compuestos de Organoselenio/metabolismo , Peroxidasa/metabolismo , Animales , Glutatión/metabolismo , Isoindoles , Espectroscopía de Resonancia Magnética , Compuestos de Organoselenio/química , Oxidación-Reducción , Ratas , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , Tiorredoxinas/metabolismoRESUMEN
Mitochondrial-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These antioxidants are ubiquinone and tocopherol derivatives and are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, these cations accumulated within mitochondria inside cells, where the antioxidant moiety prevents lipid peroxidation and protects mitochondria from oxidative damage. The mitochondrially localized ubiquinone also protected mammalian cells from hydrogen peroxide-induced apoptosis while an untargeted ubiquinone analogue was ineffective against apoptosis. When fed to mice these compounds accumulated within the brain, heart, and liver; therefore, using these mitochondrial-targeted antioxidants may help investigations of the role of mitochondrial oxidative damage in animal models of aging.