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We previously reported dipeptidomimetic compounds as inhibitors of neuronal and/or inducible NO synthases (n/iNOS) with significant selectivity against endothelial NOS (eNOS). They were composed of an S -ethylisothiocitrullin-like moiety linked to an extension through a peptide bond or a 1,2,4-oxadiazole link. Here, we developed two further series where the extension size was increased to establish more favorable interactions in the NOS substrate access channel. The extension was introduced on the solid phase by the reductive alkylation of an amino-piperidine moiety or an aminoethyl segment in the case of dipeptide-like and 1,2,4-oxadiazole compounds, respectively, with various benzaldehydes. Compared to the previous series, more potent inhibitors were identified with IC 50 in the micromolar to the submicromolar range, with significant selectivity toward nNOS. As expected, most compounds did not inhibit eNOS, and molecular modeling was carried out to characterize the reasons for the selectivity toward nNOS over eNOS. Spectral studies showed that compounds were interacting at the heme active site. Finally, selected inhibitors were found to inhibit intra-cellular iNOS and nNOS expressed in RAW264.7 and INS-1 cells, respectively.

More than 160 arginine analogues modified on the C-terminus via either an amide bond or a heterocyclic moiety (1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,4-triazole) were prepared as potential inhibitors of NO synthases (NOS). A methodology involving formation of a thiocitrulline intermediate linked through its side-chain on a solid support followed by modification of its carboxylate group was developed. Finally, the side-chain thiourea group was either let unchanged, S-alkylated (Me, Et) or guanidinylated (Me, Et) to yield respectively after TFA treatment the corresponding thiocitrulline, S-Me/Et-isothiocitrulline and N-Me/Et-arginine substrate analogues. They all were tested against three recombinant NOS isoforms. Several compounds containing a S-Et- or a S-Me-Itc moiety and mainly belonging to both the dipeptide-like and 1,2,4-oxadiazole series were shown to inhibit nNOS and iNOS with IC 50 in the 1-50 μM range. Spectral studies confirmed that these new compounds interacted at the heme active site. The more active compounds were found to inhibit intra-cellular iNOS expressed in RAW264.7 and INS-1 cells with similar efficiency than the reference compounds L-NIL and SEIT., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new fossil of Ziphiidae from the upper Miocene Gram Formation ( ca 9.9-7.2 Ma) is described herein. Computed tomographic scanning of the specimen was performed to visualize the mandibles and to obtain a three-dimensional digital reconstruction. It possesses several characters of the derived ziphiids, such as the dorsoventral thickening of the anterior process of the periotic, the dorsoventral compression of the pars cochlearis and the short unfused symphysis. The specimen cannot be identified beyond the family level, because of the unusual nature of the preserved parts consisting of the mandibles, earbones and postcranial remains. It differs from other ziphiid species from the Gram Formation, Dagonodum mojnum , in its larger size and the more derived morphology of its mandibles and earbones. Its long and thickened stylohyal, combined with its reduced teeth, suggests that this new specimen relied primarily on suction feeding. By contrast, the other ziphiid species from the Gram Formation, D. mojnum , shows adaptations for a more raptorial feeding strategy. Assuming the two species were coeval, their co-occurrence at the same locality with two different feeding strategies, may represent a case of niche separation. They may have hunted different types of prey, thus avoiding direct competition for the same food resource., Competing Interests: We have no competing interests., (© 2019 The Authors.)

Background: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine., Methods: We have expressed CYP2U1 in E. coli and performed UV-vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS., Results: The UV-vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate Fe III state. Imidazole (Im) derivatives, such as miconazole, acted as Fe III ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to "reverse type I" difference UV-vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model., Major Conclusion and General Significance: Our study describes for the first time the mode of interaction of several Fe III -heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although modern beaked whales (Ziphiidae) are known to be highly specialized toothed whales that predominantly feed at great depths upon benthic and benthopelagic prey, only limited palaeontological data document this major ecological shift. We report on a ziphiid-fish assemblage from the Late Miocene of Peru that we interpret as the first direct evidence of a predator-prey relationship between a ziphiid and epipelagic fish. Preserved in a dolomite concretion, a skeleton of the stem ziphiid Messapicetus gregarius was discovered together with numerous skeletons of a clupeiform fish closely related to the epipelagic extant Pacific sardine (Sardinops sagax). Based on the position of fish individuals along the head and chest regions of the ziphiid, the lack of digestion marks on fish remains and the homogeneous size of individuals, we propose that this assemblage results from the death of the whale (possibly via toxin poisoning) shortly after the capture of prey from a single school. Together with morphological data and the frequent discovery of fossil crown ziphiids in deep-sea deposits, this exceptional record supports the hypothesis that only more derived ziphiids were regular deep divers and that the extinction of epipelagic forms may coincide with the radiation of true dolphins., (© 2015 The Author(s).)

Background: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin., Methods: We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions., Results: We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site., Major Conclusion: Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model., General Significance: These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.

We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.

A series of new 7-monosubstituted and 3,7-disubstituted indazoles have been prepared and evaluated as inhibitors of nitric oxide synthases (NOS). 1H-indazole-7-carbonitrile (6) was found equipotent to 7-nitro-1H-indazole (1) and demonstrated preference for constitutive NOS over inducible NOS. By contrast, 1H-indazole-7-carboxamide (8) was slightly less potent but demonstrated a surprising selectivity for the neuronal NOS. Further substitution of 6 by a Br-atom at carbon-3 of the heterocycle enhanced 10-fold the inhibitory effects. Inhibition of NO formation by 6 appeared to be competitive versus both substrate and the cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). In close analogies with 1, compound 6 strongly inhibited the NADPH oxidase activity of nNOS and induced a spin state transition of the heme-Fe(III). Our results are explained with the help of the X-ray structures that identified key-features for binding of 1 at the active site of NOS.

Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of L-arginine to L-citrulline with formation of the signaling molecule nitric oxide (NO). In addition to their fundamental role in NO biosynthesis, NOSs are also involved in the formation of reactive oxygen and nitrogen species (RONS) and in the interactions with some drugs. 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) is a dinitroaromatic compound tested as an antitumor prodrug that requires reduction to the 2- and 4-hydroxylamines to be cytotoxic. Here, we studied the interaction of neuronal, inducible, and endothelial NOSs with CB1954. Our results showed that the three purified recombinant NOSs selectively reduced the 4-nitro group of CB1954 to the corresponding 4-hydroxylamine with minimal 2-nitroreduction. Little further two-electron reduction of the hydroxylamines to the corresponding 2- and 4-amines was observed. The reduction of CB1954 catalyzed by the neuronal NOS (nNOS) was inhibited by O 2 and a flavin/NADPH binding inhibitor, diphenyliodonium (DPI), but insensitive to the addition of the heme ligands imidazole and carbon monoxide and of l-arginine analogues. This reduction proceeded with intermediate formation of a nitro-anion free radical observed by EPR. Involvement of the reductase domain of nNOS in the reduction of CB1954 was confirmed by the ability of the isolated reductase domain of nNOS to catalyze the reaction and by the stimulating effect of Ca (2+)/calmodulin on the accumulation of 4- and 2-hydroxylamines. The recombinant inducible and endothelial NOS isoforms reduced CB1954 with lower activity but higher selectivity for the cytotoxic 4-hydroxylamine compared with nNOS. Finally, CB1954 did not modify the formation of l-citrulline and RONS catalyzed by nNOS. Our results show that all three NOS isoforms are involved in the nitroreduction of CB1954, with predominant formation of the cytotoxic 4-hydroxylamine derivative. This nitroreduction could be of interest for the selective activation of prodrugs by NOSs overexpressed in tumor cells.