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N-benzylethylenediamine derivatives have been prepared in good yields using methodology diferent than that described in the literature. ¹H and 13C NMR spectra were used to characterize those compounds. Nine new platinum(II) complexes, analogs of cisplatin and carboplatin, containing these ligands have been prepared and characterized. Preliminary in vitro tests against buccal human carcinoma cell lines (KB cells) showed that the complexes are cytotoxic.

Iridium (Ir)-(COD)-pentamidine tetraphenylborate (CAS 225-75-4) was selected from a primary screening to be evaluated in vitro on three Leishmania (L.) strains comparatively to pentamidine used as reference compound. The IC50 values obtained from in vitro evaluation on promastigotes of L. major CRE 26, L. donovani DD8 and L. donovani LV9 were 3.9, 23.5, and 3.3 mumol/l for Ir-(COD)-pentamidine tetraphenylborate and 1.6, 7.7, and 3.9 mumol/l for pentamidine isethionate, respectively. Cytotoxicity on mouse peritoneal macrophages led to determine a chemotherapeutic index of 1.7 for Ir-(COD)-pentamidine tetraphenylborate and 4 for pentamidine. Considering L. donovani DD8, the uptake of iridium complex by the promastigotes was shown to be saturable with a Km value of 17.4 mumol/l and Vmax of 1.3 nmol/mg protein/2 h. After 2 and 4 h incubation of treated promastigotes in drug free medium the absence of Ir-complex efflux is in favour of intracellular drug binding. As a matter of fact iridium complex was shown to bind ribosomal subunits in vitro, with no effect on macromolecular biosynthesis.

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.

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.

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.

Amphotericin B (AmB)-resistant Leishmania donovani promastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3beta-ol and not ergosterol as in the AmB-sensitive strain.

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.

Paromomycin is an aminocyclitol aminoglycoside antibiotic used for the treatment of leishmaniasis. In view of the central role of mitochondria in cellular energetics and metabolism, its effect on in vivo mitochondrial activities of Leishmania donovani promastigotes-the parasite flagellate form-was investigated. The approach used flow cytometry, amperometric measure of O2 consumption, and, as a global estimate of mitochondrial dehydrogenases, thiazolyl blue reduction (MTT test); some in vitro controls were also made. When added to promastigote cultures for 24-72 h at 150-200 microM (= LC50), paromomycin doubled the generation time, inhibited respiration, and lowered its associated electric potential difference across mitochondrial membranes, as measured by rhodamine 123 fluorescence. The chemical analogue neomycin was ineffective. Furthermore, the in vivo mitochondrial dehydrogenase activities were lower, seemingly because of the shortage of respiratory substrates. Indeed, succinate addition to paromomycin-treated cultures partly restored mitochondrial membrane potential. However, no immediate effect of paromomycin on respiration was observed, neither inhibition of redox chain nor increase of membrane permeability (uncoupling). It is proposed that paromomycin acts at a metabolic level upstream of the respiratory chain itself. This would have the observed delayed consequence because the cell energy supply would progressively decline since it depends upon the proton gradient-viz., membrane potential-generated by respiration. In conclusion, paromomycin is an antibiotic affecting the cell's energetic metabolism; the respiratory dysfunction it induces may be a crucial aspect of its action against Leishmania and possibly other cells.

Polyamine biosynthesis was studied in wild-type promastigotes of Leishmania donovani and Leishmania amazonensis treated with pentamidine and in the parasites resistant to this drug. Treatment of wild-type clones with low pentamidine concentrations for 24 hr provoked a strong decrease in arginine, ornithine, and putrescine pools, while the level of intracellular spermidine remained unchanged. In these cells, the activity of the enzyme ornithine decarboxylase was found to be decreased. Compared to wild-type cells, resistant clones had a lower level of putrescine, higher pools of arginine and ornithine, and a similar spermidine content. Analysis by Western blot and DFMO-binding showed reduced amount of ornithine decarboxylase. Furthermore, in the resistant cells, the kinetic parameters of the enzyme spermidine synthase were markedly changed, showing increased affinity to putrescine and decreased affinity to pentamidine. Thus, it seems that polyamine biosynthesis pathway is a target of pentamidine in Leishmania and is altered in resistant clones.

Sinefungin is an antibiotic possessing a strong anti-leishmanial activity. Among the most important effects of this molecule on Leishmania donovani promastigotes are morphological modifications and a very rapid and effective inhibition of DNA synthesis. These cells contain a single DNA-rich mitochondrion whose division cycle is coordinated with the nuclear division cycle. We have developed a flow-cytometric procedure based upon mithramycin as fluorochrome that can perform quantitative cell cycle analysis on the nuclear DNA. Cell cycle progression was analyzed to establish that sinefungin irreversibly blocks the promastigotes in early S phase. Sinefungin did not react with stationary cells as they were arrested in G1. Surprisingly, taxol, a microtubule-stabilizing drug, induced the same morphological modifications as sinefungin although it interfered with the G2/M progression. According to immunofluorescence studies, the stable microtubular network is apparently affected neither by taxol nor by sinefungin.

A transport system for pentamidine in Leishmania donovani and Leishmania amazonensis promastigotes and axenic amastigotes has been identified and characterized. Pentamidine is not metabolized by these parasites. Its uptake process is saturable, carrier-mediated and energy-dependent. This drug does not inhibit purine or pyrimidine uptake, whereas it inhibits uptake of several amino acids non-competitively and that of putrescine and spermidine competitively. The results suggest that pentamidine shares polyamine-carrier systems in these parasites.

Sinefungin, an antileishmanial nucleoside, induces morphological and ultrastructural changes in promastigotes of Leishmania donovani. The most important modifications are the enlargement of the flagellar pocket and the increased activity of the Golgi apparatus. Cytoenzymatic labeling demonstrates an increased activity of the soluble acid phosphatase in the flagellar reservoir of sinefungin-treated cells. The affinity constant remained unchanged. Analyzes by Western blot demonstrate an increased amount of the enzyme in the treated cells. The increased amount was not due to impaired enzyme release, as in the external medium the acid phosphatase was also enhanced but to a lesser extent. Under identical conditions the membrane-bound acid phosphatase was not modified. These results indicate that the enlargement of the flagellar pocket is the consequence of the accumulation of acid phosphatase and other Golgi-mediated enzyme provoking unbalanced cytoplasmic exchange. Sinefungin has the same effects on Leishmania tropica promastigotes. However, these effects are not specific to sinefungin. Another molecule, taxol, also induced cell rounding accompanied by increased acid phosphatase activity. Under conditions where cell rounding is not observed, in stationary phase or with compounds which stopped proliferation without shape change, no increase in the amount of acid phosphatase could be observed. These results clearly demonstrate a correlation between morphological, ultrastructural changes and the stimulated expression of acid phosphatase.

The involvement of a carrier for sinefungin (SF) uptake in Leishmania donovani promastigotes is indicated by saturation kinetics, competition studies and SF accumulation against a 270-fold concentration gradient across the cell membrane. Whether SF uptake occurs via nucleoside- or AdoMet-carrier systems was investigated by competition experiments and comparison of the uptake of various molecules in wild-type and SF-resistant cells. Results show that SF did not inhibit purine or pyrimidine uptake whereas it competitively inhibited AdoMet uptake. Furthermore, the uptake of nucleosides in SF-resistant cells is similar to that in wild-type cells, whereas uptake of SF and AdoMet is lower.

Ribosomes of Leishmania, 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 from L. tropica and L. 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 of L. donovani and 61S and 43S for the 88S cytoplasmic ribosome of L. tropica; 55S and 34S for L. tropica and 60S and 39S for L. 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.

In this study, we have surveyed gp63 expression in sinefungin-(SF)-resistant and wild-type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3-4-fold and 1.5-2-fold increase of gp63 protein in SF-resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild-type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF-resistant compared to wild-type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF-resistant and wild-type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF-resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF-resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post-transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF-resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF-resistant compared to wild-type L. donovani promastigotes.

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.

Promastigotes resistant to sinefungin (SF), a nucleoside antibiotic that is structurally related to S-adenosylmethionine (AdoMet), were obtained starting from two cloned strains of Leishmania 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 resistance 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.

The low-copy-number 8.7-kb plasmid pGIF3 of Streptomyces incarnatus was studied after cloning in the Escherichia coli vector pBR322; a restriction map was constructed. Southern blot analysis showed that pGIF3 in S. incarnatus occurs predominantly as integrated in a larger replicon. The plasmid carries a gene for thiostrepton resistance having no homology with the known thiostrepton resistance gene from Streptomyces azureus.

Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent in vitro and in vivo (Bachrach et al. 1980, FEBS Letters 121, 287-291; Neal et al. 1985, Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 85-122). It was previously shown that this molecule is a competitive inhibitor of AdoMet for transmethylases (Paolantonacci et al. 1986, Molecular and Biochemical Parasitology 21, 47-54; Avila et al. 1987, Molecular and Biochemical Parasitology 26, 69-76) and that it induces shape changes of Leishmania donovani promastigotes as observed by light microscopy (Lawrence and Robert-Gero 1990; Bulletin de la Societé Française de Parasitologie 8, 13-18). In the present work the effect of the antibiotic on the ultrastructure was analyzed by electron microscopy. The main changes induced at sublethal concentrations (0.26 microM sinefungin for 16 hr) were progressive rounding, decreased motility, enlargement of the flagellar pocket, and shortening and loss of the external part of the flagellum. The comparison with control cells showed shorter Golgi saccules and fragmentation of the trans-Golgi network into vesicles, indicating a stimulated Golgi apparatus activity. This result, associated with the enlarged flagellar pocket, suggests an unbalanced cytoplasmic exchange between exocytosis and endocytosis. These effects are quite different from those induced by tunicamycin (Dagger et al. 1984, Biology of the Cell 50; 173-180) or paromomycin. In addition, other nucleoside and nonnucleoside growth inhibitors failed to induce similar changes. AdoMet antagonized the sinefungin-induced shape changes and ultrastructural modifications but had no effect with respect to other growth inhibitors. This suggests that the sinefungin activity at the cellular level is specifically related to competition with AdoMet. A comparative study of N-methylation and carboxylmethylation of proteins in sinefungin-treated promastigotes showed that the antibiotic preferentially inhibits the latter, catalyzed by protein-O-methyltransferases. These enzymes are known to regulate the function of various proteins involved in secretion. Overall the results suggest that one of the main targets of sinefungin in exponentially growing cells is the protein carboxylmethylation involved in membrane transport.

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.

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).

The growth of the malarial parasite P. falciparum was arrested by the adenine containing nucleoside sinefungin at the trophozoite stage. The synthesis of DNA, of polyamines and the specific proteins of the schizont stage were completely blocked by the drug. The inhibition of DNA synthesis was not due to a decrease in the amount of DNA polymerase, but to the depletion of polyamines which are required for DNA synthesis.

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.

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.

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.