Biomedical subjects

Fanny Vella

Publications and source records attributed to Fanny Vella.

5 recordsLinked to original sources

PubMed2005

Characterization of the melatoninergic MT3 binding site on the NRH:quinone oxidoreductase 2 enzyme.

Melatonin acts through a series of molecular targets: the G-protein coupled receptors, MT1 and MT2, and a third binding site, MT3, recently identified as the enzyme NRH:quinone oxydoreductase 2 (QR2). The relationship between the multiple physiological functions of melatonin and this enzyme remains unclear. Because of the relationship of QR2 with the redox status of cells, these studies could bring the first tools for a molecular rationale of the antioxidant effects of melatonin. In the present paper, we used a QR2-stably expressing cell line and hamster kidneys to compare the 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine binding data, and to characterize the MT3 binding site. We designed and tested compounds from two distinct chemicals series in a displacement assay of the two MT3 ligands, 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine from their cloned target. We also tested their ability to inhibit QR2 catalytic activity. These compounds were separated into two classes: those that bind within the catalytic site (and being inhibitors) and those that bind outside it (and therefore not being inhibitors). Compounds range from potent ligands (K(i) = 1 nM) to potent inhibitors (14 nM), and include one compound [NMDPEF: N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide] active on both parameters in the low nanomolar range. To dissect the physio-pathological pathways in which QR2, MT3 and melatonin meet, one needs more compounds binding to MT3 and/or inhibitors of QR2 enzymatic activity. The compounds described in the present paper are new tools for such a task.

Animals↗

PubMed2005

NRH:quinone reductase 2: an enzyme of surprises and mysteries.

Quinone reductase 2 has been discovered in 1961 and rediscovered in 1997. Because of its sequence homology with quinone reductase 1, it has been suspected to detoxify quinones. Ten years later, evidences begin to point to a versatile role of this enzyme. Indeed, QR2 is strongly suspected to be the molecular target of anti-malarian drugs such as chloroquin or paraquine, and of red wine-derived resveratrol that might be responsible for the so-called French paradox. It also is identical to the melatonin binding site MT3, and might therefore be a rationale explanation for the antioxidant role of melatonin. Finally QR2 might be implicated in the toxicity, in vivo, of quinones such as menadione. The present commentary attempts to summarize this information and discusses a series of hypotheses.

Amino Acid Sequence↗

PubMed2005

Quinone reductase 2 substrate specificity and inhibition pharmacology.

Quinone reductase 2 is a mammalian cytosolic FAD-dependent enzyme, the activity of which is not supported by conventional nicotinamide nucleotides. An endobiotic substrate has never been reported for this enzyme nor a set of molecular tools, such as inhibitors. In the present work, we used the recombinant human enzyme, expressed in CHO cells for the systematic screening of both co-substrates and substrates. The co-substrates survey showed that the natural occurring compound, N-ribosylnicotinamide, was a poor co-substrate. The synthetic N-benzylnicotinamide is a better one compared to any other compounds tested. We found that tetrahydrofolic acid acted as a co-substrate for the reduction of menadione catalysed by quinone reductase 2, although with poor potency (Km approximately 2 mM). Among a series of commercially available quinones, a single one was found to be substrate of quinone reductase 2, in the presence of N-benzyldihydronicotinamide: coenzyme Q0. Finally, we tested a series of 197 flavonoids as potential inhibitors. We found apigenin, genistein or kaempferol as good inhibitor of quinone reductase 2 activity with IC50 in the 100 nM range. These compounds, co-substrate, substrate and inhibitors will permit to better know this enzyme, the role of which is still poorly understood.

Animals↗

PubMed2004

Organs from mice deleted for NRH:quinone oxidoreductase 2 are deprived of the melatonin binding site MT3.

Two melatonin receptors (MT1 and MT2) have been cloned. A third melatonin binding site, MT3, is known with remarkable and distinct pharmacological properties. We previously reported the purification of MT3 and identified it as the enzyme dihydronicotinamide riboside:quinone reductase 2 (NQO2). To investigate the relationship between NQO2 and MT3, we generated a NQO2-/- mouse strain. These mice no longer present MT3 binding sites as measured with 2-[125I]-iodo, 5-methoxycarbonylamino-N-acetyltryptamine, the specific MT3 radioligand. These data establish NQO2 as part of the MT3 binding sites in vivo and resolve the matter of the nature of the third melatonin binding site.

Animals↗

PubMed2003

A recombinant chimeric epidermal growth factor-like module with high binding affinity for integrins.

Integrins are cell surface receptors involved in numerous pathological processes such as metastasis invasion and abnormal angiogenesis. To target these receptors, the epidermal growth factor (EGF)-like domain of human complement protease C1r was used as a natural scaffold to design chimeric modules containing the RGD motif. Here we report a high yield bacterial expression system and its application to the production of two such modules, EGF-RGD and V2, the latter variant mimicking the RGD-containing domain of disintegrins. These modules were characterized chemically, and their biological activity was investigated by cellular assays using various Chinese hamster ovary cell lines expressing beta1 and beta3 integrins and by surface plasmon resonance spectroscopy. Remarkably, the modifications leading to the V2 variant had differential effects on the interaction with beta3 and beta1 integrins. The disintegrin-like V2 module exhibited enhanced binding affinities compared with EGF-RGD, with KD values of 7.2 nm for alpha5beta1 (a 4-fold decrease) and 3.5 nm for alphavbeta3 (a 1.5-fold decrease), comparable with the values determined for natural integrin ligands. Analysis by NMR spectroscopy also revealed a differential dynamic behavior of the RGD motif in the EGF-RGD and V2 variants, providing insights into the structural basis of their relative binding efficiency. These novel RGD-containing EGF modules open the way to the design of improved variants with selective affinity for particular integrins and their use as carriers for other biologically active modules.

Amino Acid Sequence↗