RESUMEN
Autotaxin catalyzes the transformation of lyso-phosphatidylcholine in lyso-phosphatidic acid (LPA). LPA is a phospholipid possessing a large panel of activity, in particular as a motility factor or as a growth signal, through its G-protein coupled seven transmembrane receptors. Indirect evidence strongly suggests that autotaxin is the main, if not the only source of circulating LPA. Because of its central role in pathologic conditions, such as oncology and diabetes/obesity, the biochemical properties of autotaxin has attracted a lot of attention, but confirmation of its role in pathology remains elusive. One way to validate and/or confirm its central role, is to find potent and selective inhibitors. A systematic screening of several thousand compounds using a colorimetric assay and taking advantage of the phosphodiesterase activity of autotaxin that requires the enzymatic site than for LPA generation, led to the discovery of a potent nanomolar inhibitor, [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826). This compound was inhibitory toward the various autotaxin isoforms, using an assay measuring the [(14)C]lyso-phosphatidylcholine conversion into [(14)C]LPA. We also evaluated the activity of S32826 in cellular models of diabesity and oncology. Nevertheless, the poor in vivo stability and/or bioavailability of the compound did not permit to use it in animals. S32826 is the first reported inhibitor of autotaxin with an IC(50) in the nanomolar range that can be used to validate the role of autotaxin in various pathologies in cellular models.
Asunto(s)
Anilidas/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Organofosfonatos/farmacología , Fosfodiesterasa I/antagonistas & inhibidores , Pirofosfatasas/antagonistas & inhibidores , Células 3T3 , Anilidas/síntesis química , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Lisofosfolípidos/biosíntesis , Ratones , Organofosfonatos/síntesis química , Fosfatidilcolinas/metabolismo , Hidrolasas Diéster FosfóricasRESUMEN
Melatonin is a neurohormone implicated in both biorhythm synchronization and neuroprotection from oxidative stress. Its functions are mediated by two G-protein-coupled-receptors (MT1 and MT2) and MT3, which corresponds to quinone oxidoreductase 2 (QR2). To determine the binding site of QR2 for melatonin, point mutations of residues crucial for the enzymatic activity of hQR2 were performed. The substitution of the hydrophobic residues Phe126, Ile128 and Phe178 by tyrosines at the active site significantly increased enzymatic activity and decreased the affinity of a structural analog of melatonin, the 2[(125)I]iodo-MCANAT. The mutation of residues implicated in zinc chelating (His(173); His(177)) had no effect on radioligand binding. Destabilisation of the cofactor FAD by mutation N18E showed that 2[(125)I]iodo-MCANAT binding was closely linked to the conformational integrity of human QR2. Surprisingly, the mutations C222F and N161A, which are distant from the determined binding site of the ligand, increased the affinity of 2[(125)I]iodo-MCANAT for hQR2. What seems to better explain the binding variations among the mutants are the activity recorded with BNAH and coenzyme Q1. Various hypotheses are discussed based on the various parameters used in the study: nature of the substrates and co-substrates and nature of the amino acid changes. This study, which constitutes the first structural analysis of hQR2, should enable to better understand the biological role of melatonin on this enzyme and particularly, the discrepancies between the pharmacologies of the melatonin binding site (MT3) and the QR2 catalytic activity.
Asunto(s)
Melatonina/metabolismo , Quinona Reductasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Western Blotting , Células CHO , Catálisis , Cricetinae , Cricetulus , Humanos , Datos de Secuencia Molecular , Mutagénesis , Quinona Reductasas/química , Quinona Reductasas/genética , Homología de Secuencia de AminoácidoRESUMEN
Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).
Asunto(s)
Kinuramina/análogos & derivados , Melatonina/metabolismo , Quinona Reductasas/metabolismo , Sitios de Unión , Humanos , Kinuramina/química , Kinuramina/metabolismo , Melatonina/química , Resonancia Magnética Nuclear Biomolecular , Estrés Oxidativo , Quinona Reductasas/química , Quinona Reductasas/genética , Especies Reactivas de Oxígeno/metabolismo , Receptores de Melatonina/química , Receptores de Melatonina/metabolismoRESUMEN
Autotaxin is a type II ecto-nucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that autotaxin also catalyses a lyso-phospholipase D activity. This enzyme probably provides most of the extracellular lyso-phosphatidic acid from lyso-phosphatidylcholine. There is almost no pharmacological tools available to study autotaxin. Indeed, all the reported inhibitors, thus far, are uneasy-to-use, lyso-phosphatidic acid derivatives. Initially, autotaxin was recognized as a phosphodiesterase (NPP2) [Bollen et al., Curr. Rev. Biochem. Biol. 35 (2000) 393-432], based on sequence similarity and enzymatic capability of autotaxin to catalyse ecto-nucleotidase activity. Phosphodiesterase forms a large family of enzymes characterized by a large number of chemically diverse inhibitors. None of them have been tested on autotaxin activity. For this reason, we screened those reported inhibitors, as well as a series of compounds, mostly kinase inhibitor-oriented, on autotaxin activity. Only two compounds of the various phosphodiesterase inhibitors (calmidazolium and vinpocetine) were potent enough to inhibit autotaxin catalytic activity. From the kinase inhibitor library, we found damnacanthal and hypericin, inhibiting phosphodiesterase activity in the 100-microM range, comparable to most of other available phospholipid-like inhibitors.
Asunto(s)
Adipocitos/metabolismo , Complejos Multienzimáticos/farmacología , Fosfodiesterasa I/farmacología , Pirofosfatasas/farmacología , Humanos , Complejos Multienzimáticos/metabolismo , Fosfodiesterasa I/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas , Pirofosfatasas/metabolismoRESUMEN
Antibodies (Abs) specific to cell-surface receptors are attractive tools for studying the physiological role of such receptors or for controlling their activity. We sought to obtain such antibodies against the type 1 receptor for melatonin (MT1). For this, we injected mice with CHO cells transfected with a plasmid encoding human MT1 (CHO-MT1-h), in the presence or absence of an adjuvant mixture containing Alum and CpG1018. As we previously observed that the immune response to a protein antigen is increased when it is coupled to a fusion protein, called ZZTat101, we also investigated if the association of ZZTat101 with CHO-MT1-h cells provides an immunogenic advantage. We measured similar levels of anti-CHO and anti-MT1-h Ab responses in animals injected with either CHO-MT1-h cells or ZZTat101/CHO-MT1-h cells, with or without adjuvant, indicating that neither the adjuvant mixture nor ZZTat101 increased the anti-cell immune response. Then, we investigated whether the antisera also recognized murine MT1 (MT1-m). Using cloned CHO cells transfected with a plasmid encoding MT1-m, we found that antisera raised against CHO-MT1-h cells also bound the mouse receptor. Altogether our studies indicate that immunizing approaches based on MT1-h-expressing CHO cells allow the production of polyclonal antibodies against MT1 receptors of different origins. This paves the way to preparation of MT1-specific monoclonal antibodies.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Inmunización , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT1/inmunología , Animales , Células CHO , Cricetulus , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Quinones are highly reactive molecules that readily undergo either one- or two-electron reduction. One-electron reduction of quinones or their derivatives by enzymes such as cytochrome P450 reductase or other flavoproteins generates unstable semiquinones, which undergo redox cycling in the presence of molecular oxygen leading to the formation of highly reactive oxygen species. Quinone reductases 1 and 2 (QR1 and QR2) catalyze the two-electron reduction of quinones to form hydroquinones, which can be removed from the cell by conjugation of the hydroxyl with glucuronide or sulfate thus avoiding its autoxidation and the formation of free radicals and highly reactive oxygen species. This characteristic confers a detoxifying enzyme role to QR1 and QR2, even if this character is strongly linked to the excretion capacity of the cell. Using EPR spectroscopy and confocal microscopy we demonstrated that the amount of reactive oxygen species (ROS) produced by Chinese hamster ovary (CHO) cells overexpressing QR1 or QR2 compared to naive CHO cells was determined by the quinone structural type. Indeed, whereas the amount of ROS produced in the cell was strongly decreased with para-quinones such as menadione in the presence of quinone reductase 1 or 2, a strong increase in ROS was recorded with ortho-quinones such as adrenochrome, aminochrome, dopachrome, or 3,5-di-tert-butyl-o-benzoquinone in cells overexpressing QR, especially QR2. These differences could originate from the excretion process, which is different for para- and ortho-quinones. These results are of particular interest in the case of dopamine considering the association of QR2 with various neurological disorders such as Parkinson disease.
Asunto(s)
Benzoquinonas/química , Radicales Libres/química , Quinona Reductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Oxígeno/metabolismo , Quinona Reductasas/químicaRESUMEN
Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC(50) in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatoninAsunto(s)
Inhibidores Enzimáticos/farmacología
, Piridinas/farmacología
, Alcaloides de Pirrolicidina/farmacología
, Quinona Reductasas/antagonistas & inhibidores
, Animales
, Secuencia de Bases
, Cartilla de ADN/genética
, Inhibidores Enzimáticos/química
, Furanos/química
, Furanos/farmacología
, Humanos
, Técnicas In Vitro
, Cinética
, Melatonina/farmacología
, Piridinas/química
, Alcaloides de Pirrolicidina/química
, Quinona Reductasas/genética
, Quinona Reductasas/metabolismo
, Receptores de Melatonina/metabolismo
, Proteínas Recombinantes/antagonistas & inhibidores
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/metabolismo
, Resveratrol
, Spodoptera
, Estilbenos/farmacología
, Especificidad por Sustrato
RESUMEN
The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS.
Asunto(s)
Células/metabolismo , Desoxirribonucleasas/fisiología , Marcación de Gen/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Mutagénesis Sitio-Dirigida/métodos , Animales , Células CHO , Línea Celular , Células/citología , Mapeo Cromosómico/métodos , Cricetinae , Cricetulus , Desoxirribonucleasas/metabolismo , Modelos Biológicos , Factores de Tiempo , TransfecciónRESUMEN
Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.