Françoise Lawrence

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.

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.

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.

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

Altered transport properties of pentamidine-resistant Leishmania donovani and L. amazonensis promastigotes

Abstract Pentamidine-resistant clones of Leishmania donovani and L. amazonensis promastigotes were developed by increase of the drug pressure in the culture medium and characterized. The resistant clones could grow in 40 and 20 μM pentamidine as determined for L.␣donovani and L. amazonensis, respectively, with 50% inhibitory concentrations (IC50 values) being 140 and 60 μM, which were 18 and 75 times higher than those recorded for the parental clones, respectively. Biochemical analysis of the clones showed that the acquired pentamidine resistance was specific (no cross-resistance to unrelated drugs and no reversibility with verapamil) and stable in vitro and in vivo. Pentamidine resistance is related to decreased drug uptake and highly increased efflux in both clones of Leishmania spp., accompanied by an alteration in polyamine carriers. Furthermore, a modification of the uptake of pyrimidine nucleosides and several amino acids by these resistant clones indicates alterations in the surface membrane.

Protein methylation and protein methylases in Leishmania donovani and Leishmania tropica promastigotes

We studied the content of acid-stable methylated amino acids of soluble proteins in promastigotes of Leishmania donovani and L. tropica. epsilon-N-Trimethyllysine and NG,NG-dimethylarginine were found in both Leishmania species after culture in the presence of [methyl-14C]methionine. In addition, 3-N-methylhistidine was found only in L. tropica and epsilon-N-dimethyllysine only in proteins of L. donovani. As sinefungin, an antileishmanial nucleoside antibiotic, is a known transmethylase inhibitor, its effect on protein methylation was studied, in whole cells and in vitro. In the first case the drug had no effect on the content of methylated amino acid residues of soluble proteins. In vitro, histone methylation by crude extracts was studied at pH 7.2 and 9.0, known in other organisms as optimum pH values for arginine and lysine methylation, respectively. Surprisingly, arginine methylation by extracts of L. donovani was the same at both pH values while lysine residues were more efficiently methylated at pH 7.2 than at pH 9 by the extracts of the two species. These results indicate that the properties of protein methylases I and III of these parasites are different from those of other organisms hitherto studied. The inhibition constants of sinefungin for the leishmanial protein methylases were weak in comparison with those for enzymes from other sources, with the exception of the constant of L. donovani enzyme at pH 9.

Decreased rate of S-adenosyl-L-homocysteine metabolism: an early event related to transformation in cells infected with Rous sarcoma virus.

Abstract An increase of methylase activity is often related to neoplastic transformation. SAH, the natural inhibitor of transmethylases, does not inhibit cell transformation induced by RSV, in contrast to one of its synthetic analogues, SIBA. This inefficiency was thought to be due to the rapid metabolism of SAH by transformed cells. We now show, that, on the contrary, 70 % of the added amount of SAH disappears in one hour in cell-free extracts of normal cell against only 14 % in extracts of transformed cells. This decreased rate of degradation occurred one day post infection. Cells infected with the non transforming RAV1 degrade SAH at the same rate as normal cells. A decrease of SAH-hydrolase and adenosine deaminase activity was also observed in infected cells. The decrease of the first enzyme seems to be related to the transformed state, whereas that of the second enzyme seems to depend only on infection, since it is also observed in cells infected with RAV1.

Distribution of macromolecular methylations in promastigotes of Leishmania donovani and impact of sinefungin

ABSTRACT. Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent both in vitro and in vivo. This molecule, structurally related to S‐adenosylmethionine, is a good competitive inhibitor of methyltransferases in vitro. The aim of this report is to analyze the impact of sinefungin on methylation pattern and the subcellular localisation of methyl groups and various methylases in promastigotes of Leishmania donovani. We have shown the presence of various methylated macromolecules in different subcellular fractions, with somewhat higher concentration in membrane fraction. In vitro, sinefungin inhibits the three main protein methylases, but in cells cultured in its presence the protein carboxylmethylations are specifically inhibited.

Characterization of sinefungin-resistant Leishmania donovani promastigotes

Promastigotes resistant to sinefungin (SF), a nucleoside antibiotic that is structurally related to S-adenosylmethionine (AdoMet), were obtained starting from two cloned strains ofLeishmania donovani. The resistance was induced by increasing the drug pressure gradually until promastigotes capable of growing in the presence of concentrations 10,000 times higher than the 50% growth-inhibitory (IC50) values for the control cells were obtained. The resitance to SF of both clones was specific and stable in the absence of drug pressure. High-performance liquid chromatographic (HPLC) analyses indicated highly reduced levels of SF in the two resistant clones. However, the intracellular SF concentration in these resistant cells was much higher than the IC50 values for wild-type cells. In one clone, the decreased drug uptake was coupled to a decrease in the affinity of two protein methylases for SF, whereas in the other clone the biosynthesis of polyamine precursors was modified. This study demonstrates that resistance to a drug molecule with pleiotropic targets can be developed through various mechanisms by different strains.