Isolation of an Anti-Tumour Disintegrin: Dabmaurin-1, a Peptide Lebein-1-Like, from Daboia mauritanica Venom.

In the soft treatment of cancer tumours, consequent downregulation of the malignant tissue angiogenesis constitutes an efficient way to stifle tumour development and metastasis spreading. As angiogenesis requires integrin-promoting endothelial cell adhesion, migration, and vessel tube formation, integrins represent potential targets of new therapeutic anti-angiogenic agents. Our work is a contribution to the research of such therapeutic disintegrins in animal venoms. We report isolation of one peptide, named Dabmaurin-1, from the hemotoxic venom of snake Daboia mauritanica, and we evaluate its potential anti-tumour activity through in vitro inhibition of the human vascular endothelial cell HMECs functions involved in tumour angiogenesis. Dabmaurin-1 altered, in a dose-dependent manner, without any significant cytotoxicity, HMEC proliferation, adhesion, and their mesenchymal migration onto various extracellular matrix proteins, as well as formation of capillary-tube mimics on MatrigelTM. Via experiments involving HMEC or specific cancers cells integrins, we demonstrated that the above Dabmaurin-1 effects are possibly due to some anti-integrin properties. Dabmaurin-1 was demonstrated to recognize a broad panel of prooncogenic integrins (αvß6, αvß3 or αvß5) and/or particularly involved in control of angiogenesis α5ß1, α6ß4, αvß3 or αvß5). Furthermore, mass spectrometry and partial N-terminal sequencing of this peptide revealed, it is close to Lebein-1, a known anti-ß1 disintegrin from Macrovipera lebetina venom. Therefore, our results show that if Dabmaurin-1 exhibits in vitro apparent anti-angiogenic effects at concentrations lower than 30 nM, it is likely because it acts as an anti-tumour disintegrin.

DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water.

Peroxidation is an important process both in chemistry and biology, and peroxyl radicals play a crucial role in various pathological situations involving lipid and protein peroxidation. A few secondary and tertiary peroxyl radicals can be detected directly by Electron Spin Resonance (ESR). However, primary and secondary alkylperoxyl radicals have extremely short lifetimes and their direct observation is impossible in biological samples. DMPO has been used to trap alkylperoxyl radicals generated in biological systems and the characterization of DMPO-alkylperoxyl spin adducts has been claimed by different authors. However, it was then clearly shown that all the assignments made previously to DMPO-OOR adducts were actually due to DMPO-OR adducts. We have investigated the potential of DEPMPO to characterize the formation of alkylperoxyl radicals in biological milieu. Various DEPMPO-OOR (R = Me, primary or secondary alkyl group) spin adducts were unambiguously characterized and the formation of DEPMPO-OOCH(3) was clearly established during the reaction of tert-butylhydroperoxide with chloroperoxidase and cytochrome c.

Design of new derivatives of nitrone DEPMPO functionalized at C-4 for further specific applications in superoxide radical detection.

A general synthetic route to prepare derivatives of the DEPMPO nitrone (5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide) functionalized at C-4 was established via the synthesis of 4-HMDEPMPO nitrone (5-diethoxyphosphoryl-4-hydroxymethyl-5-methyl-1-pyrroline-N-oxide) that was obtained from reduction of the nitro compound 1. (4R*,5S*)-4-HMDEPMPO was successfully separated from its minor diastereoisomer and could be used to generate various substituted analogues. Among them, 4-NHSDEPMPO, 5-diethoxyphosphoryl-4-succinimidyloxycarbonyloxymethyl-5-methyl-1-pyrroline-N-oxide, constitutes a NH2-reactive precursor for further conjugation to relevant moieties such as targeting groups, labels, or drugs. From 4-NHSDEPMPO, a biotinylated nitrone was synthesized offering new perspectives for targeted delivery applications. A short study of the trapping behaviors of the (4R*,5S*)-isomer of these 4-HMDEPMPO analogues proved that they are as good as DEPMPO for detecting superoxide. For each isomer, only one diastereoisomer adduct was obtained, resulting from the addition of superoxide on the less hindered face of the nitrone, that is, trans to the phosphoryl group and the C-4 substituent. From spectra simulation and experiments in various solvents, we proved that ESR patterns of each adduct corresponded to the superimposed signals of two sets of conformers in a sufficiently slow chemical exchange to induce a widening and a dissymmetry of some of the signal lines. This phenomenon was drastically reduced when compared with that observed for DEPMPO superoxide and attributed to a similar chemical exchange, and it did not hamper spectrum assignment. Determination of the decay rate of the superoxide adduct of (4R*,5S*)-4-HMDEPMPO proved that it has a 25% longer half-life time than the superoxide adduct of DEPMPO.

Mito-DEPMPO synthesized from a novel NH2-reactive DEPMPO spin trap: a new and improved trap for the detection of superoxide.

Mito-DEPMPO, a new DEPMPO analogue bearing a triphenylphosphonium group, was synthesized via a novel NH2-reactive DEPMPO. The half-life of the Mito-DEPMPO superoxide adduct was estimated to be ca. 40 min. Using Mito-DEPMPO, reactive oxygen species generated in intact mitochondria were detected and characterized by EPR.

Diastereoselective synthesis and ESR study of 4-phenylDEPMPO spin traps.

[reaction: see text] The cis and trans diastereoisomers of 5-(diethoxyphosphoryl)-5-methyl-4-phenylpyrroline N-oxide (4-PhDEPMPOt 8 and 4-PhDEPMPOc 9) were prepared stereoselectively and used as spin traps for hydroxyl and superoxide radicals. The spin adduct formed by reaction of the cis stereoisomer 9with superoxide radical anion exhibited an 8-line ESR spectrum, showing only a reduced alternating line width phenomenon. This spectrum is simpler than the 12-line spectrum of DEPMPO-OOH, which exhibits a strong alternating line width phenomenon. The half-life times of the 4-PhDEPMPOc-OOH and DEPMPO-OOH adducts were of the same order: 14.5 and 15.5 min, respectively.

ESR study of spin-trapping with two glycosylated analogues of PBN able to target cell membrane lectins.

The spin trapping behaviour of the two galactosylated nitrones LAMPBN and TA1PBN, both of which are able to target cells through recognition by cell membrane lectins, were widely investigated on a variety of free radicals in aqueous media. The ESR spectra of the more amphiphilic nitrone, TA1PBN, were interpreted in the light of the LAMPBN trapping results. The spin adducts of nitrone TA1PBN, which may be better distributed inside the cell, were surrounded by two distinct environments due to the tensioactive organisation of the trap and gave two different ESR signals for each radical trapped.

ESR study of a biological assay on whole blood: antioxidant efficiency of various vitamins.

This study deals with the activity of various vitamins against the radical-mediated oxidative damage in human whole blood. We have used a biological method that allows both the evaluation of plasma and that of red blood cell resistance against the free radicals induced by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Spin trapping measures using mainly 5-(diethoxyphosphoryl)-5-methyl-1-pyrolline N-oxide nitrone (DEPMPO) were carried out under several conditions to identify the free radicals implicated in this test. Only the oxygenated-centred radical generated from AAPH was found highly reactive to initiate red blood cell lysis. With DEPMPO only alkoxyl radicals were observed and no evidence was found for alkylperoxyl radicals. The antioxidant activity of several lipid- and water-soluble vitamins has been assessed by the biological assay and through two chemical methods. We have noticed high antioxidant activities for tocopherols (in the order delta>gamma>alpha) in the biological test but not through chemical methods. At 1 microM, the delta-tocopherol efficiency in inhibiting radical-induced red blood cell hemolysis was three times as high as the alpha-tocopherol efficiency. For beta-carotene no significant activity even in whole blood was shown. Highly surprising antioxidant activities were observed for acid folic and pyridoxine, compared to ascorbic acid. At 10 microM, the effectiveness of folic acid was almost three times as high as vitamin C. The biological test seems clinically more relevant than most other common assays because it can detect several classes of antioxidants.

Synthesis of a Glycolipidic Amphiphilic Nitrone as a New Spin Trap.

The synthesis of a new amphiphilic nitrone, A, derived from a digalactosyl tris(hydroxymethyl)aminomethane bearing a perfluorocarbon chain is described. A exhibited a surfactant behavior (cmc = 1.6 x 10(-)(5) mol/L), and the specific recognition of the galactosyl moiety grafted on A by the KbCWL1 membrane lectin was established. Preliminary experiments showed that A was able to trap free radicals in aqueous media, the shape of the observed ESR spectra being strongly dependent upon the nature of the trapped free radical.