Inhibition by methioninyl adenylate of focus formation by Rous sarcoma virus.

Methioninyl adenylate is a specific and potent inhibitor of the enzyme methionyl-tRNA synthetase and, consequently, of protein biosynthesis. In cultures of chick embryo fibroblasts infected with Rous sarcoma virus, incubation for a 2-day period with 1 to 3 mM concentrations of this inhibitor, as late as 4 days after infection, irreversibly prevented subsequent formation of foci of transformed cells. Later addition could also elicit the irreversible disappearance of already existing foci, by phenotypic reversion and/or cell killing. Virus production in transformed cells and replication in newly infected cells were also inhibited but to a lesser degree. Under the same conditions, the same concentrations of methioninyl adenylate caused only a reversible growth arrest of normal cells. The selective toxicity of the inhibitor for transformed cells is not due to a greater affinity for the target enzyme, but it may be due to the fact that inhibition of protein biosynthesis is only partially reversible in these cells, whereas it is fully reversible in normal cells.

Relationship between inhibition of protein methylase I and inhibition of Rous sarcoma virus-induced cell transformation.

A correlation was found between inhibition of protein methylase I and inhibition of virus-induced cell transformation by structural analogs of S-adenosylhomocysteine; all good inhibitors of this enzyme are also good inhibitors of Rous sarcoma virus-induced chicke embryo fibroblast transformation. The inhibitory effect of these analogs was similar on enzymes from normal and transformed cells; no significant variation of the inhibition constants was observed after purification of protein methylase I. From the kinetic constants obtained, a structure-activity relationship can be established for protein methylase I.

Further studies of the action of methionyl adenylate on chick embryo fibroblasts.

Methionyl adenylate (Met-AMP) inhibits protein synthesis by interacting with methionyl-tRNA synthetase. Addition of 1--3 mM inhibitor to chick embryo fibroblasts rapidly stops protein synthesis and DNA synthesis but not RNA synthesis. These effects can be reversed by renewal of the medium. The extent and reversibility of protein and DNA syntheses depend on the concentration of MetAMP in the cultures, the length of exposure and the cellular density. MetAMP is recognised by several enzymes as substrate and/or as inhibitor. MetAmp is degraded to methionol plus 5'-adenylic acid by 5'-phosphodiesterase. Adenosine deaminase, adenylic acid deaminase and 3':5'-phosphodiesterase cannot use MetAMP as substrate but the last enzyme is inhibited. The presence of MetAMP in cultures provokes a small but reproducible increase in the level of methionyl-tRNA synthetase and 5'-phosphodiesterase.

Association of SIBA treatment and a Met-depleted diet inhibits in vitro growth and in vivo metastatic spread of experimental tumor cell lines.

We have used 5'-deoxy-5'-S isobutyl-thioadenosine (SIBA), an analog of S-adenosylhomocysteine, alone or in association with a methionine-depleted diet in order to obtain an antitumoral effect in two different tumor models: a transplantable rat rhabdomyosarcoma (RMS-J1) induced by i.m. injection of nickel and the well-known Lewis lung carcinoma (3LL) of C57BL/6 mice. Since SIBA has been reported to inhibit the methyl group transfer from methionine to S-adenosylhomocysteine, among other activities, its association with a reduction of methyl donors, achieved by methionine depletion of the diet (in vivo) or the culture medium (in vitro), should logically lead to an additive effect. In vitro, 3LL and RMS-J1 were sensitive to the cytotoxic effect of SIBA and were methionine-dependent for their proliferation. Fibroblast proliferation was not affected by these two treatments alone or in association. In vivo, either SIBA treatment or a low methionine diet led to a significant decrease in the metastatic character of these two tumors; however, local tumor growth was not significantly affected. The median number of 3LL metastases counted in the lungs was reduced from 100 to 18 by SIBA treatment, and to 27 by the low methionine diet. No additive effect could be detected when the treatments were given simultaneously. RMS-J1-bearing rats treated with SIBA and fed a low Met diet underwent primary tumor excision. The median numbers of lung metastatic nodules were 27, 26, 14 and 8 for the control, SIBA-treated rats, methionine-deprived rats and rats receiving the combined therapy. Expressed as percentages 20 per cent were cured, 23 per cent showed a low number of lung metastases (P less than 10), whereas all the rats in the control group developed more than 10 pulmonary nodules. No cytotoxic effect could be observed on the treated rats. The role of SIBA and methionine depletion, as agents interfering with transmethylation processes, in regard to the control of tumor development, namely metastatic invasiveness, is discussed.

Characterization of RNA from Leishmania tropica and Leishmania d.donovani promastigotes.

RNA has been prepared from promastigotes of Leishmania tropica and Leishmania d.donovani using three different methods. Extraction by hot phenol/isothiocyanate gave the best quantitative and qualitative results. The analysis of total RNA on methyl mercuric agarose gels shows that the large rRNA species is nicked: it is composed of a 630 and a 560 kDa molecule. The small rRNA species has a molecular weight of 800,000. Poly(A+) RNA can be translated in a rabbit reticulocyte lysate system. The newly synthesized products comprise high molecular weight proteins and show different patterns using RNA from L. tropica or from L. d. donovani promastigotes.

Proton-ATPase activities involved in the uptake of an S-adenosylmethionine analogue.

Characteristics of the transport of sinefungin (SF) were studied in Leishmania donovani promastigotes grown in vitro in a semi-defined medium. The uptake is time and pH dependent, temperature sensitive, saturable and independent of the growth phase. Metabolic inhibitors decrease the influx, indicating that sinefungin uptake is an energy requiring process. The presence of Na+ is unnecessary for activity. The uptake is sensitive to valinomycin and nigericin and to the H+-ATPases inhibitors such as N'N'-dicyclohexylcarbodiimide, bafilomycin A and oligomycin. Sulfhydryl group(s) are involved in carrier activity. Use of SF analogues shows, stereospecificity of the transporter, recognition of the 6'-amino group and to a lesser degree of the 9'-amino group of the lateral chain, whereas the 9'-carboxyl group of the lateral chain is not implicated in the recognition. Adenosine and ornithine do not interfere with the uptake. No significant amount of SF is tightly bound to macromolecules. In a SF-resistant clone, though the uptake of SF is reduced (the apparent Vmax is 276 pmoles mg protein(-1) 30 min(-1) compared with 2061 pmoles mg protein(-1) 30 min(-1) for the wild-type clone), the apparent affinity for SF is similar to that of wild-type cells (Km 0.7 and 0.6 microM respectively). This lower uptake activity is not the reflection of an increased efflux of the drug. In these resistant cells, the susceptibility of SF uptake to variation of the external pH, as well as to azide, NaF, and valinomycin are decreased, that to nigericin is lost.

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.

Hybrid molecules: growth inhibition of Leishmania donovani promastigotes by thiosemicarbazones of 3-carboxy-beta-carbolines.

The thiosemicarbazones of beta-carboline-3-carboxaldehyde (compound 2) and 3-acetyl-beta-carboline (compound 3) were found to effectively inhibit the in vitro growth of the promastigote form of Leishmania donovani, 50% inhibition being obtained at concentrations of 5.0 and 2.5 microM, respectively, while irreversible growth inhibition was achieved at 40 (compound 2) and 17.5 microM (compound 3). The thiosemicarbazone of pyridine-2-carboxyaldehyde (compound 4) was considerably less active while both methyl beta-carboline-3-carboxylate (compound 1) and the thiosemicarbazone of ethyl 5-formyl-6-azaindole-2-carboxylate (compound 5) were inactive at the highest concentrations tested. At concentrations provoking approximately 50% growth inhibition of promastigotes, compound 2 was observed to preferentially block DNA rather than RNA synthesis, but for compound 3, the reverse was true. Compound 3, the most active analogue studied, may thus act, at least partially, via a novel, though as yet unelucidated, mechanism.

Total synthesis of uracil analogues of sinefungin.

Analogues of sinefungin derivatives 18a and 18b have been prepared from uridine and L-aspartic acid. The key step in the synthesis was the coupling of the radical derived from 14 with the unsaturated amide 13. The latter was produced from the known N-hydroxy-2-thiopyridone ester of L-aspartic acid 12 with the olefin 11. Thus, the essential carbon skeleton was constructed by way of two radical coupling reactions. These analogues as well as 1a and 1b synthesized previously were tested for their antileishmanial effect in vivo and for their inhibitory activity of protein carboxymethylase (protein methylase II). The replacement of the adenine moiety by uracil or dihydrouracil considerably decreases the antiparasitic activity and the affinity for protein methylase II. The synthetic (S)-sinefungin was as active as the natural one. Interestingly, the C-6' epimer 1b was 50% less active in vitro than the natural sinefungin, but both had identical affinities for the target enzyme.

Synthesis and biological activity of sinefungin analogues.

A series of nucleosides (2-4) that derive from adenosine by chain extension at the 5'-end have been synthesized starting from the known phosphonate 7. The latter was first combined with 4-pentenal to give 8, which underwent chemical manipulations to provide triacetate 11, which was found suitable for the adenylation step. Further transformations, among them the Hofmann degradation of the amide group of compound 13, and final deprotection gave nucleosides 2-4. They were considered as analogues of sinefungin (1) and tested for their antileishmanial activity together with compounds 5 and 6, which were obtained independently. All the modifications with respect to sinefungin resulted in nearly complete loss of growth inhibitory activity. These results indicate that the 9' terminal amino and carboxyl groups are necessary for the activity and that the presence of the amino group at C-6' is not sufficient to maintain the antileishmanial effect. Some of the analogues however could antagonize or reverse the inhibitory activity of sinefungin (1).

Inhibition of leishmanial DNA synthesis by sinefungin.

RNA, DNA and protein biosynthesis were studied in Leishmania donovani and L. tropica promastigotes cultured with or without sinefungin. Thymidine incorporation was significantly impaired by this compound. Neither the uptake of thymidine nor its phosphorylation were inhibited. Furthermore the ratios of deoxyribonucleotide to the corresponding ribonucleotide were not significantly affected by sinefungin. Analysis of the DNA indicates that the inhibition of thymidine incorporation affects mostly nuclear DNA, kDNA being less affected by this drug. No such effect on thymidine incorporation was observed in macrophages, the host cells of these parasites.

Involvement of plasmids in Streptomyces incarnatus phenotype.

The sinefungin producing, pock forming strain Streptomyces incarnatus was shown to be thiostrepton resistant. However, it does not produce thiostrepton and structurally related antibiotics. In this strain, five low copy plasmids of variable sizes were detected with electron microscopy. The strain S. lividans TK24 became thiostrepton resistant upon transformation by one of the plasmids of S. incarnatus.

Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria.

Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases. Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases.

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.

Enhanced sinefungin production by medium improvement, mutagenesis and protoplast regeneration of Streptomyces incarnatus NRRL 8089.

Increased production of sinefungin, a very potent antifungal and antiparasitic nucleoside antibiotic was achieved by medium and strain improvement. When soybean-meal, dextrin and yeast extract were added as carbon and nitrogen sources to the fermentation medium, instead of corn steep liquor, soya-oil and glucose; the antibiotic yield increased from 40 micrograms/ml to 126 micrograms/ml with low biomass production. Strain improvement was attempted by two methods. The mean antibiotic yield of the variants after multistep mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine and ethyleneimine was 466 micrograms/ml. Protoplasts of the parental strain were prepared by lysozyme digestion from mycelia grown in a medium containing 0.7% glycine. The mean activity of the regenerated protoplasts was 664 micrograms/ml. Thus, the overall sinefungin production could be increased 16-fold.

Biosynthesis of sinefungin by cell-free extract of Streptomyces incarnatus NRRL 8089.

The formation of sinefungin an antifungal and anti-parasitic nucleoside antibiotic was demonstrated in cell-free extracts from Streptomyces incarnatus. Incorporation studies as well as HPLC analysis showed that the immediate biosynthetic precursors of the antibiotic are L-arginine and ATP. The biosynthesis was optimal when the cell-free extract was prepared from 72 hours mycelium and incubated at least 80 minutes with arginine and ATP. The presence of dithiothreitol, pyridoxal phosphate and Mg2+ in the incubation mixture was also necessary. When a membrane fraction was incubated under the same conditions with ATP and L-arginine, sinefungin production was not observed. A working hypothesis is put forward to explain the biosynthesis of this antibiotic.

Development and characterization of paromomycin-resistant Leishmania donovani promastigotes.

Paromomycin is an antileishmanial chemotherapeutic agent. Leishmania donovani promastigotes resistant to 800 microM of paromomycin were selected by increasing drug pressure and cloned. These promastigotes did not acquire multidrug resistance. Paromomycin resistance was stable in the absence of the drug in the culture. It remained stable also in amastigotes isolated after a passage in mice. Furthermore the resistant parasites were still infective to macrophages in vitro and for mice in vivo. A sensitive method to detect and to quantify intracellular paromomycin by HPLC was developed and allowed to show that the main mechanism of resistance seems to be due to decreased drug uptake probably as a consequence of altered membrane composition.