Malka Robert-Gero

The antifungal antibiotic sinefungin as a ver y active inhibitor of methyltransferases and of the transformation of chick embryo fibroblasts by rous sarcoma virus

Summary The antifungal antibiotic Sinefungin is a structural analogue of S-adenosyl-methionine (SAM) and of S-adenosyl-homocysteine (SAH). It is as active as the synthetic SAH analogue S-isobutyl adenosine (SIBA) in inhibiting Rous Sarcoma Virus (RSV) induced transformation of chick embryo fibroblasts (CEF) in cell culture. Sinefungin, like SAH and SIBA is a competitive inhibitor in vitro of tRNA methylases and of protein methylases I and III of CEF. Its ki value for tRNA methylases of normal and transformed cells is respectively 3 to 10 times lower than the ki of SAH. It is thus in vitro the most active tRNA methylase inhibitor described until now. Protein methylase I and III are inhibited to about the same extent by the two molecules whereas SIBA has much higher ki values for the three enzymes in vitro . When methylation was measured in whole cells SAH, SIBA and Sinefungin inhibited (14C)-methyl incorporation to about the same degree in normal cells, but only SIBA and Sinefungin were active in transformed cells. Download : Download full-size image

Inhibition of virus-induced cell transformation by synthetic analogues of S-adenosyl homocysteine

Summary 1 mM 5′-deoxy-5′-S-isobutyl-adenosine (SIBA) (I), a structural analogue of S-adenosyl-homocysteine (SAH), inhibits the oncogenic transformation of chick embryo fibroblasts (CEF) infected with Rous sarcoma virus (RSV). When SIBA remains in contact with infected cells for 48 hours the inhibition of focus formation is irreversible, at least for 8 days. Virus replication is also strongly inhibited by SIBA. Normal CEF are only reversibly inhibited by this compound. SAH, the natural inhibitor of SAM dependent transmethylases has no effect on cell transformation.

Alterations in membrane fluidity, lipid metabolism, mitochondrial activity, and lipophosphoglycan expression in pentamidine-resistant Leishmania

Abstract Cytoplasmic and mitochondrial membranes were studied in wild-type promastigotes of Leishmania donovani and L. amazonensis treated with pentamidine and in the parasites resistant to this drug. Analyses by flow cytometry showed membrane fluidification in resistant cells and a modification of the lipidic metabolism in pentamidine-treated cells and in resistant clones as compared with wild-type ones. Pentamidine decreased the amounts of membrane polar lipids, i.e., phospholipids, and neutral lipidic droplets in the cytoplasm. Perturbation of lipid constituents did not induce modifications of membrane acid phosphatase and protease activities. Lipophosphoglycan, the major cell-surface glycoconjugate, was no longer detected by Western blotting in resistant cells. The use of rhodamine 123 showed a decrease in transmembrane mitochondrial potential in wild-type cells treated with pentamidine and in resistant parasites. In conclusion, cytoplasmic and mitochondrial membranes and lipidic metabolism are altered in pentamidine-resistant parasites.

Ribosomes ofLeishmania are a target for the aminoglycosides

Ribosomes ofLeishmania, a parasitic protozoan (member of the order of Kinetoplastidae), were purified on a sucrose density gradient. Two different types of ribosomes were isolated from the promastigotes: cytoplasmic (88S and 91S fromL. tropica andL. donovani, respectively) and mitochondrial (75S in both species). Both types of ribosome dissociated into their subunits at low Mg2+ concentration (1–2 mM) as follows: 67S and 49S for the 91S cytoplasmic ribosomes ofL. donovani and 61S and 43S for the 88S cytoplasmic ribosome ofL. tropica; 55S and 34S forL. tropica and 60S and 39S forL. donovani mitochondrial ribosomes, respectively. Paromomycin (aminosidine), an aminoglycoside aminocyclitol antibiotic, interacted with the ribosomes to promote the association of the subunits. Under similar experimental conditions, spermidine and pentamidine were inactive.

Inhibitory activity of sinefungin and siba (5′‐deoxy‐5′‐S‐isobutylthio‐adenosine) on the growth of promastigotes and amastigotes of different species of leishmania

The naturally occurring polyamlnes putrescine, spermidine and spermine are organic cations widely distributed in both procaryotic and eucaryotic organisms, including parasitic species, e.g., trypanosomes [ 1,2] and leishmanias [3,4]. In eucaryotic organisms, ornithine decarboxylase catalyses the conversion of ornithine into putrescine, while the synthesis of spermidine and spermine involves S-adenosylmethionine decarboxylase (fig.1). In Leishmunia, as in other organisms, polyamine

Biochemical alterations in paromomycin-treated Leishmania donovani promastigotes

Abstract Paromomycin is used for the treatment of leishmaniasis in humans, but little is known about its mechanism of action. Investigating the effect of this antibiotic on promastigotes of Leishmania donovani, we showed that inhibition of the multiplication of these parasites could be related to its effect on RNA synthesis and to modifications of membranous polar lipids and membrane fluidity, leading to altered membrane permeability.

Structural requirements of quassinoids for the inhibition of cell transformation

Abstract The effects of eleven quassinoids on Rous sarcoma virus induced cell transformation and on growth of normal cells were examined. At concentrations of 0.15-1 μg/ml they inhibited foci formation (76–99 %) without toxic effects on normal cells. The most active compounds also affected virus production by transformed cells. In intact normal and transformed cells, protein and DNA synthesis was equally affected after 3 hours of exposure to quassinoids of both cell types. RNA synthesis was not inhibited. This study has shown that the structural requirement of a C-15 ester in the quassinoids for antileukemic activity in vitro and in vivo is not essential for their antitransforming activity.

Effect of 5′‐deoxy‐5′‐S‐isobutyl adenosine on polyoma virus replication

5’-deoxy-5’-S-isobutyl adenosine (SIBA) was shown to inhibit cell transformation induced by oncogenic RNA viruses, such as Rous sarcoma virus [ 1] or murine sarcoma virus (Yoshikura et al. unpublished results). Further studies showed that SIBA inhibits also mitogen induced blastogenesis of human and rabbit lymphocytes [Z] . SIBA is a synthetic analogue [3] of S-adenosylhomocysteine (SAH), the natural inhibitor of S-adenosylmethionine dependent transmethylases [4]. It inhibits the incorporation of [3H]methyl from methionine into chick embryo fibroblasts (Berneman et al. unpublished results) but it is not yet known whether the primary action of SIBA is on methyl transfer reaction, as the compound inhibits also protein RNA and DNA syntheses in chick embryo cells. To explore further the activity of SIBA as an oncostatic agent we investigated its effect on some biochemical events resulting from the lytic infection of mouse cells by polyoma (PyV), an oncogenic DNA virus. Winocour et al. [5] have found a several fold increase of [‘HI thymidine and [3H]methyl incorporation into DNA of mouse kidney cultures upon

Modification of kinetoplast DNA minicircle composition in pentamidine-resistant Leishmania

Pentamidine, an antiprotozoal drug, was shown to have various cellular and molecular targets depending on the organism. In Leishmania, ultrastructural modifications of kinetoplast and mitochondria have been observed but no data is available on cellular and molecular events involved in development of pentamidine-resistance. The absence of modification of minicircle DNA in pentamidine treated L. donovani and L. amazonensis promastigotes suggested that topoisomerase II activity is not a target. This result was confirmed by quantitation of the enzyme by immunodetection. Southern blot experiments indicated that the kDNA network was altered in resistant clones. Molecular cloning and sequence analysis of kDNA minicircles showed transkinetoplastidy hitherto reported only for arsenite- and tunicamycin-resistant Leishmania. Comparison of wild-type and resistant sequences showed only 32-51% homology. The AT-rich regions, known as binding sites, of the drug occurred less frequently in the resistant clones and their locations were different. These minicircle sequence modifications leading to decreased binding sites for the drug might contribute to pentamidine-resistance in Leishmania.

Expeditious enantioselective synthesis of carbocyclic nucleosides with antileishmanial activity

A short synthesis of the neplanocin A analogue 1, lacking the hydroxymethylene at C-4 of the parent naturally occurring carbocyclic nucleoside, is described. Moreover, the non toxic noraristeromycin 3, an intermediate in the synthetic scheme, was shown to exhibit a promising anti-leishmanial activity.