RESUMEN
Among the properties melatonin is claimed to possess, are the immuno-inflammation inductive capacities that would be responsible of some of the paramount of activities melatonin is reported to have in most of the human pathological conditions. In the present paper, we measured the effect of melatonin on established cellular models of immuno-inflammation, and found none. The discrepancies are discussed, especially because those properties are reported at pharmacological concentration (1 µM and beyond) at which the melatonin receptors are desensitized by internalization, leading to putative non-receptor-dependent mechanism of action.
Asunto(s)
Inflamación , Melatonina , Melatonina/farmacología , Melatonina/metabolismo , Humanos , Inflamación/metabolismo , Receptores de Melatonina/metabolismo , AnimalesRESUMEN
Over the years, evidence has accumulated on a possible contributive role of the cytosolic quinone reductase NQO2 in models of dopamine neuron degeneration induced by parkinsonian toxin, but most of the data have been obtained in vitro. For this reason, we asked the question whether NQO2 is involved in the in vivo toxicity of MPTP, a neurotoxin classically used to model Parkinson disease-induced neurodegeneration. First, we show that NQO2 is expressed in mouse substantia nigra dopaminergic cell bodies and in human dopaminergic SH-SY5Y cells as well. A highly specific NQO2 inhibitor, S29434, was able to reduce MPTP-induced cell death in a co-culture system of SH-SY5Y cells with astrocytoma U373 cells but was inactive in SH-SY5Y monocultures. We found that S29434 only marginally prevents substantia nigra tyrosine hydroxylase+ cell loss after MPTP intoxication in vivo. The compound produced a slight increase of dopaminergic cell survival at day 7 and 21 following MPTP treatment, especially with 1.5 and 3 mg/kg dosage regimen. The rescue effect did not reach statistical significance (except for one experiment at day 7) and tended to decrease with the 4.5 mg/kg dose, at the latest time point. Despite the lack of robust protective activity of the inhibitor of NQO2 in the mouse MPTP model, we cannot rule out a possible role of the enzyme in parkinsonian degeneration, particularly because it is substantially expressed in dopaminergic neurons.
Asunto(s)
Intoxicación por MPTP , Neuroblastoma , Ratones , Humanos , Animales , Neuronas Dopaminérgicas/metabolismo , Sustancia Negra/metabolismo , Dopamina/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Melatonin is a small natural compound, so called a neuro-hormone that is synthesized mainly in pineal gland in animals. Its main role is to master the clock of the body, under the surveillance of light. In other words, it transfers the information concerning night and day to the peripheral organs which, without it, could not "know" which part of the circadian rhythm the body is in. Besides its main circadian and circannual rhythms mastering, melatonin is reported to be a radical scavenger and/or an antioxidant. Because radical scavengers are chemical species able to neutralize highly reactive and toxic species such as reactive oxygen species, one would like to transfer this property to living system, despite impossibilities already largely reported in the literature. In the present commentary, we refresh the memory of the readers with this notion of radical scavenger, and review the possible evidence that melatonin could be an in vivo radical scavenger, while we only marginally discuss here the fact that melatonin is a molecular antioxidant, a feature that merits a review on its own. We conclude four things: (i) the evidence that melatonin is a scavenger in acellular systems is overwhelming and could not be doubted; (ii) the transposition of this property in living (animal) systems is (a) theoretically impossible and (b) not proven in any system reported in the literature where most of the time, the delay of the action of melatonin is over several hours, thus signing a probable induction of cellular enzymatic antioxidant defenses; (iii) this last fact needs a confirmation through the discovery of a nuclear factor-a key relay in induction processes-that binds melatonin and is activated by it and (iv) we also gather the very important description of the radical scavenging capacity of melatonin in acellular systems that is now proven and shared by many other double bond-bearing molecules. We finally discussed briefly on the reason-scientific or else-that led this description, and the consequences of this claim, in research, in physiology, in pathology, but most disturbingly in therapeutics where a vast amount of money, hope, and patient bien-être are at stake.
Asunto(s)
Melatonina , Glándula Pineal , Animales , Humanos , Melatonina/metabolismo , Antioxidantes/farmacología , Antioxidantes/metabolismo , Glándula Pineal/metabolismo , Ritmo Circadiano/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
D3/D2 sub-specificity is a complex problem to solve. Indeed, in the absence of easy structural biology of the G-protein coupled receptors, and despite key progresses in this area, the systematic knowledge of the ligand/receptor relationship is difficult to obtain. Due to these structural biology limitations concerning membrane proteins, we favored the use of directed mutagenesis to document a rational towards the discovery of markedly specific D3 ligands over D2 ligands together with basic binding experiments. Using our methodology of stable expression of receptors in HEK cells, we constructed the gene encoding for 24 mutants and 4 chimeras of either D2 or D3 receptors and expressed them stably. Those cell lines, expressing a single copy of one receptor mutant each, were stably constructed, selected, amplified and the membranes from them were prepared. Binding data at those receptors were obtained using standard binding conditions for D2 and D3 dopamine receptors. We generated 26 new molecules derived from D2 or D3 ligands. Using 8 reference compounds and those 26 molecules, we characterized their binding at those mutants and chimeras, exemplifying an approach to better understand the difference at the molecular level of the D2 and D3 receptors. Although all the individual results are presented and could be used for minute analyses, the present report does not discuss the differences between D2 and D3 data. It simply shows the feasibility of the approach and its potential.
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Receptores de Dopamina D2 , Receptores de Dopamina D3 , Receptores de Dopamina D3/genética , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Ligandos , Línea Celular , MutagénesisRESUMEN
A way to study G protein-coupled receptors in a minimal system is to reconstruct artificial membrane mimics, made of detergent and/or of lipids in which the purified receptor is maintained. In particular, it is now possible to generate lipid nanoparticles, such as nanodiscs, in which a single receptor molecule is included. Such objects offer the invaluable potential of studying an isolated receptor stabilized in a finely controlled membrane-like environment to evaluate its pharmacology, its function, and its structure at the molecular level. In this chapter, we detail the different steps from the extraction and isolation of a recombinant MT1 melatonin receptor in detergent, down to its reconstitution into nanodiscs. A G protein activation test is further described in order to exemplify how the functionality of such particles may be investigated.
Asunto(s)
Melatonina , Receptor de Melatonina MT1 , Detergentes/química , Proteínas de Unión al GTP/metabolismo , Lípidos/química , Liposomas , Membranas Artificiales , Nanopartículas , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT1/metabolismoRESUMEN
Melatonin, (N-acetyl-5-methoxytryptamine), is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule. For a long time, melatonin had been described as a ligand of a putative "receptor" present in mammalian brains named MT3. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor." The experimental setup of the binding measurements is unusual. The present chapter aims at describing this technique. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2).
Asunto(s)
Melatonina , Quinona Reductasas , 5-Metoxitriptamina , Animales , Antioxidantes , Sitios de Unión , Ligandos , Mamíferos/metabolismo , Melatonina/metabolismo , Quinona Reductasas/metabolismo , Receptores de Melatonina/metabolismoRESUMEN
Cloning may seem to be a view from the past. The time before software, computers and AI were invented. It seems to us worth discussing these points in view of our favorite target: the melatoninergic system. In a few stances, it might be important to point out that even in the new era of dry science, there is still a need to experiment and to prove at the bench that our in silico assertions are right. Most of the living animals express to some extend the melatonin receptors. Some of these animal genomes were completely or partially sequenced, and it is tempting to extract from this huge information the sequence(s) of our favorite genes (MLT receptors). Then, why bother cloning, as opposed to simply built the gene and express it in a host cell? Because the genetic boundaries of the expressed sequence(s) are not 100% sure. Because the melatonin receptor gene(s) comprise a first exon 25,000 base pair far from the second one and the limits between this Ex1 and In1-as between In1 and Ex2-are subject to changes that might have a huge impact on the biochemical properties of the receptor, once expressed. Because a receptor is a biochemical entity with characteristics that are important for the functioning of this particular pathway, and more generally, for the functioning of life.
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Melatonina , Animales , Clonación Molecular , Exones , Melatonina/metabolismo , Receptores de Melatonina/genéticaRESUMEN
To ensure the physical interaction between a protein and its ligand, many techniques can be applied. One of them, isothermal titration calorimetry (ITC), measures the heat exchange between a forming molecular complex and its milieu. From this heat exchange, it is possible to acquire the thermodynamic parameters, the binding stoichiometry and the affinity constant (Ka) between the two interacting binding partners, which can then be used to determine the dissociation constant (Kd). We made use of ITC to determine the true Kd of melatonin for its putative receptor MT3, also known as the enzyme quinone reductase 2 (NQO2). In this chapter, we describe the step-by-step procedure for performing this experiment and extend it to 2-iodomelatonin, a melatonin derivative that was used in the initial identification and characterization of MT3. The dissociation constants of melatonin and 2-iodomelatonin toward NQO2 derived from these experiments are in line with data reported previously, albeit using alternative techniques.
Asunto(s)
Melatonina , Quinona Reductasas , Calorimetría/métodos , Humanos , Ligandos , Melatonina/metabolismo , Unión Proteica , Quinona Reductasas/metabolismo , TermodinámicaRESUMEN
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein.
Asunto(s)
Melatonina , Quinona Reductasas , 5-Metoxitriptamina , Animales , Antioxidantes , Clonación Molecular , Cristalización , Humanos , Ligandos , Mamíferos/metabolismo , Melatonina/metabolismo , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , Receptores de Melatonina/metabolismo , Rayos XRESUMEN
The third melatonin binding site MT3 turned out to be an enzyme, NQO2 (E.C. 1.6.99.2). Its catalytic activity is inhibited by melatonin with an IC50 in the 50-100 µM range. Some of the functions of melatonin at pharmacological concentrations (1 µM and above) might be explained by this inhibition capacity of melatonin at NQO2. In order to determine precisely these parameters, it is required to comprehend the basic enzymology of this enzyme. In the following chapter, we present the basic conditions of measuring NQO2 catalytic activities and inhibition.
Asunto(s)
Melatonina , Quinona Reductasas , Sitios de Unión , Melatonina/metabolismo , Melatonina/farmacología , Quinona Reductasas/química , Quinona Reductasas/metabolismoRESUMEN
Melatonin exerts its effects through a series of target proteins/receptors and enzymes. Its antioxidant capacity might be due to its capacity to inhibit a quinone reductase (NQO2) at high concentration (50 µM). Demonstrating the existence of a complex between a compound and a protein is often not easy. It requires either that the compound is an inhibitor-and the complex translates by an inhibition of the catalytic activity-or the compound is radiolabeled-and the complex translates in standard binding approaches, such as in receptology. Outside these two cases, the detection of the protein:small molecule complexes by mass spectrometry has recently been made possible, thanks to the development of so-called native mass spectrometry. Using this approach, one can measure masses corresponding to an intact noncovalent complex between a compound and its target, usually after titration or competition experiments. In the present chapter, we detail the characterization of NQO2:melatonin interaction using native mass spectrometry.
Asunto(s)
Melatonina , Quinona Reductasas , Antioxidantes , Quinona Reductasas/química , Quinona Reductasas/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.
RESUMEN
Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.
Asunto(s)
Calcio , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules. METHODS AND RESULTS: In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N3-lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach. CONCLUSIONS: In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach-the Halo Tag-to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Ligasas/genética , Tiorredoxinas/metabolismo , Química Clic , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Ligasas/metabolismo , Lisina/química , Ingeniería de Proteínas , Procesamiento Proteico-Postraduccional , Ácido Tióctico/química , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.
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Complejo Antígeno-Anticuerpo/química , Antígenos/química , Fragmentos Fc de Inmunoglobulinas/química , Receptor ErbB-2/química , Proteínas Recombinantes de Fusión/química , Anticuerpos de Dominio Único/química , Secuencia de Aminoácidos , Animales , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos/genética , Antígenos/metabolismo , Camelus , Línea Celular Tumoral , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Peso Molecular , Unión Proteica , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/metabolismoRESUMEN
The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols-if not conjugated-re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells' detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings.
RESUMEN
Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.
Asunto(s)
Fármacos Antiobesidad/química , Obesidad/tratamiento farmacológico , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Tejido Adiposo/efectos de los fármacos , Alquinos/química , Aminobutiratos/química , Animales , Fármacos Antiobesidad/farmacología , Apetito/efectos de los fármacos , Ácido Aspártico/química , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Ácido Glutámico/química , Glicina/análogos & derivados , Glicina/química , Células HEK293 , Hepatocitos/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Insulina/metabolismo , Lactamas/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Relación Estructura-Actividad , Distribución Tisular , Triazoles/químicaRESUMEN
N-ribosyldihydronicotinamide:quinone oxidoreductase 2 (NQO2/QR2, Enzyme Commission number 1.10.99.2) is a cytosolic enzyme, abundant in the liver and variably expressed in mammalian tissues. Cloned 30 years ago, it was characterized as a flavoenzyme catalyzing the reduction of quinones and pseudoquinones. To do so, it uses exclusively N-alkyl nicotinamide derivatives, without being able to recognize NADH, the reference hydrure donor compound, in contrast to its next of a kind, NAD(P)H:quinone oxidoreductase 1 (NQO1). For a long time both enzymes have been considered as key detoxifying enzymes in quinone metabolism, but more recent findings point to a more toxifying function of NQO2, particularly with respect to ortho-quinones. In fact, during the reduction of substrates, NQO2 generates fairly unstable intermediates that reoxidize immediately back to the original quinone, creating a futile cycle, the byproducts of which are deleterious reactive oxygen species. Beside this peculiarity, it is a target for numerous drugs and natural compounds such as melatonin, chloroquine, imiquimod, resveratrol, piceatannol, quercetin, and other flavonoids. Most of these enzyme-ligand interactions have been documented by numerous crystallographic studies, and now NQO2 is one of the best represented proteins in the structural biology database. Despite evidence for a causative role in several important diseases, the functional role of NQO2 remains poorly explored. In the present review, we aimed at detailing the main characteristics of NQO2 from a molecular pharmacology perspective. By drawing a clear border between facts and speculations, we hope to stimulate the future research toward a better understanding of this intriguing drug target. SIGNIFICANCE STATEMENT: Evidence is reviewed on the prevalent toxifying function of N-ribosyldihydronicotinamide:quinone oxidoreductase 2 while catalyzing the reduction of ortho-quinones such as dopamine quinone. The product of this reaction is unstable and generates a futile but harmful cycle (substrate/product/substrate) associated with reactive oxygen species generation.
Asunto(s)
Quinona Reductasas/metabolismo , Quinonas/metabolismo , Animales , Humanos , Hígado/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Cadenas Pesadas de Inmunoglobulina , Receptor ErbB-2/inmunología , Albúmina Sérica Humana/inmunología , Anticuerpos de Dominio Único , Animales , Antígenos/inmunología , Clonación Molecular , Receptores ErbB/inmunología , Escherichia coli/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Proteínas Recombinantes/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificaciónRESUMEN
Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts as a substrate for quinone-reductases that may be associated with its antimalarial properties. Following our exploration of reactive oxygen species-producing compounds such as indolones, as possible new approaches for the research of new ways to treat this parasitosis, we explored derivatives of this natural product and their possible antiplasmodial and antimalarial properties, in vitro and in vivo, respectively. Apart from one compound, all the products tested had weak to moderate antiplasmodial activities, the best IC50 value being equal to 0.58 µM. In vivo activities in the murine model were moderate (at a dose of 50 mg/kg/mice, five times higher than the dose of chloroquine). These results encourage further pharmacomodulation steps to improve the targeting of the parasitized red blood cells and antimalarial activities.