Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival

A trans-dominant mutational strategy was used to down-regulate trypanothione reductase (TR) activity levels in Leishmania donovani, the causative agent of visceral leishmaniasis in humans. TR, regarded as an ideal drug target against trypanosomatid infections, is a homodimeric flavoprotein oxidoreductase unique to these organisms that plays a central role in the enzymatic regeneration of the thiol pool. Extrachromosomal, heterologous expression of a trans-dominant mutant version of the Trypanosoma cruzi enzyme in L. donovani resulted in the formation of inactive cross-species heterodimers and in a dramatic decrease of endogenous TR activity levels. Recombinant cells depleted of up to 85% of TR activity were significantly impaired in their ability to regenerate dihydrotrypanothione from trypanothione disulfide following oxidation with diamide. Nonetheless trans-dominant mutant recombinants were still capable of maintaining a reduced intracellular environment during cell growth in culture and were able to metabolize hydrogen peroxide at wild-type rates in vitro. Importantly, however, cells expressing the trans-dominant mutant enzyme displayed a decreased ability to survive inside activated macrophages in a murine model of Leishmania infection. The apparent inability of Leishmania to modulate the expression of active TR homodimers in response to the expression of trans-dominant mutant protein suggests that specific inhibitors of this enzyme should be useful anti-leishmanial agents.     

Structure-activity relationship of new growth inhibitors of Trypanosoma cruzi

Several drugs bearing the 4-phenoxyphenoxy skeleton and other closely related structures were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the etiologic agent of Chagas' disease. The new class of drugs was envisioned by modifying the nonpolar 4-phenoxyphenoxy moiety replacing selected aromatic protons by different groups via electrophilic aromatic substitution reactions as well as introducing a sulfur atom at the polar extreme. Of the designed compounds, sulfur-containing derivatives were shown to be potent antireplicative agents against T. cruzi. Among these drugs, 4-phenoxyphenoxyethyl thiocyanate (compound 56) proved to be an extremely active growth inhibitor of the epimastigote forms of T. cruzi and displayed an IC50 of 2.2 microM. Under the same assay conditions, this drug was much more active than Nifurtimox, one of the drugs currently in clinical use to control this disease. This thiocyanate derivative was also a very active inhibitor against the intracellular form of the parasite at the nanomolar level. Other sulfur derivatives prepared also exhibited very potent antiproliferative action against T. cruzi. The presence of a sulfur atom at the polar extreme for this family of compounds seems to be very important for biological action because this atom was always associated with high inhibition values. 4-Phenoxyphenoxyethyl thiocyanate presents very good prospective not only as a lead drug but also as a potential chemotherapeutic agent.     

Inhibiting effects of spermidine derivatives on Trypanosoma cruzi trypanothione reductase

Trypanothione reductase is a vital component of the antioxidant defenses of trypanosomes. This enzyme reduces trypanothione, a spermidine-glutathione conjugate. The inhibitory effects of several spermidine derivatives on the reduction of trypanothione by Trypanosoma cruzi trypanothione reductase were assessed. Spermidine derivatives containing hydrophobic aromatic substituents were found to be competitive inhibitors of trypanothione reductase. N4-acylated spermidine derivatives were less effective inhibitors than the corresponding N4-alkylated derivatives. The most effective compounds studied were N1, N8-bis(2-naphthylmethyl)spermidine and N4-(2-naphthylmethyl)spermidine, with Ki values of 9.5 and 108 microM, respectively.     

Enzymatic reduction studies of nitroheterocycles

The nitroimidazole derivative Megazol is a highly active compound used against several strains of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanomiasis). With the aim of gaining an insight into the probable mode of action, the interaction of Megazol with different redox enzymes was studied in comparison to that of Nifurtimox and Metronidazole. The three nitroaromatic compounds are reduced by L-lactate cytochrome c-reductase, adrenodoxin reductase, and NADPH:cytochrome P-450 reductase (EC 1.6.2.4), the efficiencies of the enzymatic reductions being roughly related to the reduction potentials of these pseudo-substrates. As the enzyme responsible for the reduction of Megazol within the parasite has not yet been identified, the nitroimidazole was assayed with T. cruzi lipoamide dehydrogenase and trypanothione reductase. Megazol did not inhibit the physiological reactions but proved to be a weak substrate of both flavoenzymes. The single electron reduction of the compound by NADPH:cytochrome P-450 reductase, by rat liver as well as by trypanosome microsomes was confirmed by ESR experiments. As shown here, Megazol interferes with the oxygen metabolism of the parasite, but its extra activity when compared to Nifurtimox may be related to other features not yet identified.     

Synthesis, properties and biological evaluation of substituted furo[3,2-e] and pyrano[3,2-e]pyrido[4,3-b]indoles

Furo[3,2-e]- and pyrano[3,2-e]pyrido[4,3-b] indoles were synthesized from 1,4,5-trisubstituted 8-hydroxy-5H-pyrido[4,3-b]indoles. The intermediates, 10-chloro-6H-furo[3,2-e]pyrido[4,3-b]indole (11), 10-chloro-2,6-dihydro-1H-furo[3,2-e]pyrido-[4,3-b]indole (10) and 11-chloro-2,3-dihydro-3H,7H-pyrano[3,2-e]pyrido[4,3-b]indole (15), were substituted by diamines under thermal conditions (180 degrees C). In contrast, 11-chloro-3H,7H-pyrano[3,2-e]pyrido[4,3-b]indole (14), 9-allyl-1-chloro-4,5-dimethyl-5H-pyrido[4,3-b]indole (9a) and 8-propargyloxy-4,5-dimethyl-5H-pyrido[4,3-b]indole (8) led mainly to 1-aminosubstituted 8-hydroxy-5H-pyrido[4,3-b]indole derivatives resulting from an unexpected C3 unit elimination. When examined in three tumour cell lines (L1210 leukaemia, the B16 melanoma and the MCF7 breast adenocarcinoma) the new amino substituted furo[3,2-e]-, dihydrofuro[3,2-e]- and dihydropyrano[3,2-e]-pyrido[4,3-b]indole derivatives revealed cytotoxic properties, especially important for the 2,6-dihydro-1H-furo[3,2-e]pyrido[4,3-b]indole series. The most active compound (12b) significantly inhibits both DNA topoisomerases I and II, and is as potent as Adriamycin at inhibiting cell proliferation and inducing a massive accumulation of L1210 cells in the G2 + M phase of the cell cycle. However, 12b was less active than Adriamycin when tested in vivo against P388 leukaemia or the B16 melanoma tumour models.     

In vitro antimalarial activity of chalcones and their derivatives

A series of chalcones and their derivatives have been synthesized and identified as novel potential antimalarials using both molecular modeling and in vitro testing against the intact parasite. A large number of chalcones and their derivatives were prepared using one-step Claisen-Schmidt condensations of aldehydes with methyl ketones. These condensates were screened in vitro against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and shown to be active at concentrations in the nanomolar range. The most active chalcone derivative, 1-(2,5-dichlorophenyl)-3-(4-quinolinyl)-2-propen-1-one (7), had an IC50 value of 200 nM against both a chloroquine-resistant strain (W2) and a chloroquine-sensitive strain (D6). The resistance indexes for all compounds were substantially lower than for chloroquine, suggesting that this series will be active against chloroquine-resistant malaria. Structure-activity relationships (SAR) of the chalcones in the context of a homology-based model structure of the malaria trophozoite cysteine protease, the most likely target enzyme, are presented.     

Synthesis and antimalarial activity in vitro and in vivo of a new ferrocene-chloroquine analogue

The antimalarial activities of ferrocenic compounds mimicking chloroquine and active upon chloroquine-resistant strains of Plasmodium falciparum were evaluated. Four 7-chloro-4-[[[2-[(N,N-substituted amino)methyl]ferrocenyl]methyl]amino]quinoline derivatives have been synthesized; one of them, 1a, showed high potent antimalarial activity in vivo on mice infected with Plasmodium berghei N. and Plasmodium yoelii NS. and was 22 times more potent against schizontocides than chloroquine in vitro against a drug-resistant strain of P. falciparum.     

New spermine and spermidine derivatives as potent inhibitors of Trypanosoma cruzi trypanothione reductase

Several spermine and spermidine derivatives containing 2-amino diphenylsulfide substituents were prepared and tested for their inhibiting effects on Trypanosoma cruzi trypanothione reductase. IC50 values were assessed between 0.3 and 3 microM. Compound 32 (Ki = 0.4 microM) is the most potent TR inhibitor described so far.     

2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimid ine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]- 6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 microM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.     

Madelung disease: distribution of cervical fat and preoperative findings at sonography, MR, and CT

A simplified approach to the synthesis of 2-polyamine linked-monoindolylmaleimides has been achieved, leading to a new series of trypanothione reductase inhibitors. The conditions of access to N,2-bis(polyamine)-3-monoindolylmaleimides and N,N'-bis(monoindolylmaleimide) polyamines are described. Measured inhibitory activities towards trypanothione reductase from Tryanosoma cruzi show the importance of both aromatic moieties and polyamine chains for trypanothione reductase recognition.     

In vitro and in vivo activities of trybizine hydrochloride against various pathogenic trypanosome species

Trybizine hydrochloride [O,O'-bis(4,6-diamino-1,2-dihydro-2, 2-tetramethylene-s-triazine-1-yl)-1,6-hexanediol dihydrochloride] was active in vitro against the sleeping sickness-causing agents Trypanosoma brucei subsp. rhodesiense and T. brucei subsp. gambiense; against a multidrug-resistant organism, T. brucei subsp. brucei; and against animal-pathogenic organisms Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma congolense; but not against the intracellular parasites Trypanosoma cruzi and Leishmania donovani. Cytotoxic effects against mammalian cells were observed at approximately 10(6)-fold higher concentrations than those necessary to inhibit T. brucei subsp. rhodesiense. Trybizine hydrochloride was able to eliminate T. brucei subsp. rhodesiense and T. brucei subsp. gambiense in an acute rodent model with four intraperitoneal doses of 0.25 mg kg of body weight-1 or four doses of 1 mg kg-1, respectively, or with four oral doses of 20 mg kg-1. The compound expressed activity against suramin-resistant T. evansi strains in mice. However, these concentrations were not sufficient to cure mice infected with multidrug-resistant T. brucei subsp. brucei. A late-stage rodent model with central nervous system involvement could not be cured, indicating that trybizine may not pass the blood-brain barrier in sufficient quantities.     

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.     

Direct and indirect exposure to air pollution

Glutathione (GSH), trypanothione (T(SH)2) and glutathionyl spermidine (GSH-SP) concentrations were determined in the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The concentrations of GSH, T(SH)2 and GSH-SP, expressed as nmol of GSH per g of parasite fresh weight, were 60.1, 397.8 and 103.9, respectively, for the Tulahuén strain. For the DM 28c clone, the values were 113.9, 677.9 and 164.1, respectively, and for the LQ strain they were 199.1, 1100.5 and 55.3, respectively. When the parasites were treated with 10 microM nifurtimox or 50 microM benznidazole for 2 h, the concentrations of all three reduced thiols decreased strongly. The total amount of T(SH)2 decreased by more than 50%. Treatment of the parasites with 5 mM buthionine sulfoximine, an inhibitor of GSH synthesis, for 6 h diminished the concentrations of the reduced thiols by between 27% and 53% with respect to the controls. Cyclohexylamine, an inhibitor of spermidine synthesis, decreased the concentrations of T(SH)2 and GSH-SP but not that of GSH. It is possible to conclude from this study that trypanothione is the most important thiol involved in the detoxication of nifurtimox and benznidazole in T. cruzi and that electrophilic reduced metabolites of both drugs are most probably conjugated with GSH, T(SH)2 and GSH-SP, thus decreasing their concentrations. GSH biosynthesis is an important drug target.     

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.     

Antiparasitic effects of the intra-Golgi transport inhibitor megalomicin

The macrolide antibiotic megalomicin (MGM) has been shown to inhibit vesicular transport between the medial- and trans-Golgi, resulting in the undersialylation of cellular proteins (P. Bonay, S. Munro, M. Fresno, and B. Alarcón, J. Biol. Chem. 271:3719-3726, 1996). Due to the effects of MGM on the Golgi and on the replication of enveloped viruses, we decided to test whether it has any antiparasitic activity. The results showed that MGM has potent activity against the epimastigote stage of Trypanosoma cruzi, producing a 50% inhibitory concentration (IC50) of 0.2 microg/ml. Furthermore, MGM was also active against the intracellular replicative, amastigote form of T. cruzi, completely preventing its replication in infected murine LLC/MK2 macrophages at a dose of 5 microg/ml. Although less potent, MGM was also active against Trypanosoma brucei epimastigotes (IC50, 2 microg/ml) and Leishmania donovani and Leishmania major promastigotes (IC50, 3 and 8 microg/ml, respectively). MGM also blocked intracellular replication of the asexual stage of Plasmodium falciparum-infected erythrocytes at 1 microg/ml. Finally, MGM was active in an in vivo model, resulting in the complete protection of BALB/c mice from death caused by acute T. brucei infection and significantly reducing the parasitemia. These results suggest that MGM is a potential drug for the treatment of veterinary and human parasitic diseases.     

Trypanosoma cruzi-induced immunosuppression: blockade of costimulatory T-cell responses in infected hosts due to defective T-cell receptor-CD3 functioning

A model of experimental Trypanosoma cruzi murine infection with chemically induced metacyclic forms (opossum clone Dm28c) showed a marked state of T-cell unresponsiveness during acute phase, but lacked evidence of suppressor cell activity. Spleen cells from infected mice were suppressed in vitro in responses to T-cell activators concanavalin A, anti-Thy1 monoclonal antibody (MAb), and anti-CD3 MAb compared with spleen cells from control littermates. Activation with accessory cell-independent stimulus provided by immobilized anti-CD3 was defective in splenic CD4-positive T cells from infected mice, but not in such cells from control mice. No evidence of splenic suppressor cell activity was found in cell-mixing experiments using nylon-passed T cells from control and infected donors. Kinetic experiments showed that there was a discrete stage in infection when T cells were already suppressed in response to anti-CD3 but still responded to anti-CD69 MAb. In these T cells, immobilized anti-CD3 failed to enhance simultaneous CD69 responses, although anti-CD3 enhanced CD69 responses in control T cells from uninfected donors. These results demonstrate an intrinsic defect in T-cell receptor-mediated T-cell activation, which could be a mechanism generating T-cell suppression during infection by T. cruzi.     

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.     

2,3-Dihydro-6,7-dichloro-pyrido[2,3-b]pyrazine-8-oxide as selective glycine antagonist with in vivo activity

2,3-Dihydro-6,7-dichloro-pyrido[2,3-b]pyrazine-8-oxide was synthesized and evaluated for in vitro/in vivo antagonistic activity at the strychnine insensitive glycine binding site on the NMDA receptor revealing it to be a useful tool to evaluate the effectiveness of glycine antagonists in vivo.