Synthesis and evaluation of 9,9-dimethylxanthene tricyclics against trypanothione reductase, Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani

Derivatives of 9,9-dimethylxanthene were synthesised and evaluated against trypanothione reductase (TR) and in vitro against parasitic trypanosomes and leishmania. High in vitro antiparasitic activity was observed for some derivatives with one compound showing high activity against all three parasites (ED50 values of 0.02, 0.48 and 0.32 microM, for Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani, respectively). The lack of correlation between inhibitory activity against TR and ED50 values suggests that TR is not the target.     

Extrachromosomal, homologous expression of trypanothione reductase and its complementary mRNA in Trypanosoma cruzi.

Trypanothione reductase (TR), a flavoprotein oxidoreductase present in trypanosomatids but absent in human cells, is regarded as a potential target for the chemotherapy of several tropical parasitic diseases caused by trypanosomes and leishmanias. We investigated the possibility of modulating intracellular TR levels in Trypanosoma cruzi by generating transgenic lines that extrachromosomally overexpress either sense or antisense TR mRNA. Cells overexpressing the sense construct showed a 4-10-fold increase in levels of TR mRNA, protein and enzyme activity. In contrast, recombinant T.cruzi harbouring the antisense construct showed no significant difference in TR protein or catalytic activity when compared with control cells. Although increased levels of TR mRNA were detected in some of the antisense cells neither upregulation nor amplification of the endogenous trypanothione reductase gene (tryA) was observed. Instead, a proportion of plasmid molecules was found rearranged and, as a result, contained the tryA sequence in the sense orientation. Plasmid rescue experiments and sequence analysis of rearranged plasmids revealed that this specific gene inversion event was associated with the deletion of small regions of flanking DNA.     

Parallel synthesis of a library of 1,4-naphthoquinones and automated screening of potential inhibitors of trypanothione reductase from Trypanosoma cruzi

Solid- and solution-phase parallel syntheses of 1,4-naphthoquinones (1,4-NQ) are described. A library of 1360 amides was constructed from the combination of 12 newly synthesised 1,4-NQ carboxylic acid and 120 amines, and was screened for inhibition of trypanothione reductase (TR) from Trypanosoma cruzi. The most active hits from a primary screening were re-synthesised and confirmed. This approach proves that it is possible to design potent and highly specific TcTR inhibitors deriving from menadione, juglone and plumbagin.     

Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.     

High-throughput screening affords novel and selective trypanothione reductase inhibitors with anti-trypanosomal activity

Trypanothione reductase (TR), an enzyme that buffers oxidative stress in trypanosomatid parasites, was screened against commercial libraries containing approximately 134,500 compounds. After secondary screening, four chemotypes were identified as screening positives with selectivity for TR over human glutathione reductase. Thirteen compounds from these four chemotypes were purchased, and their in vitro activity against TR and Trypanosoma brucei is described.     

In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi

We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exerting its anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.     

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 microg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 microM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 micro M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 microM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.     

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone,a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 M, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 M, indicating a good selectivity against the parasite enzyme.     

Synthesis and antikinetoplastid activity of a series of N,N'-substituted diamines

A series of 25 N,N'-substituted diamines were prepared by controlled reductive amination of free aliphatic diamines with different substituted benzaldehydes. The library was screened in vitro for antiparasitic activity on the causative agents of human African trypanosomiasis, Chagas' disease and visceral leishmaniasis. The most potent compounds were derived from a subset of diamines that contained a 4-OBn substitution, having a 50% parasite growth inhibition in the submicromolar (against Trypanosoma cruzi) or nanomolar (against Trypanosoma brucei and Leishmania donovani) range. We conclude that members of this series of N,N'-substituted diamines provide new lead structures that have potential to treat trypanosomal and leishmanial infections.     

Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design

Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Because this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidized form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 A, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through pi-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with nonplanar molecules, such as clomipramine, where stacking is not possible.     

Inhibition of protein kinase activity from Trypanosoma cruzi and Trypanosoma gambiense by 3'-deoxyadenosine.

3'-Deoxydadenosine was found to be a potent inhibitor of nucleoside-stimulated protein kinase activity from culture forms of Trypanosoma cruzi and bloodstream forms of Trypanosoma gambiense. The type of inhibition by 3'-deoxyadenosine was competitive with respect to ATP. The inhibition constants for 3'-deoxyadenosine were determined to be 0.11mM and 0.085mM for the enzyme from T. cruzi and T. gambiense, respectively. The apparent Km value for ATP was 0.2mM for both enzymes. 2'-Deoxyadenosine was less effective as inhibitor of the protein kinase activity. The inhibition constants were calculated to be 0.8mM and 0.67mM, respectively.     

Cloning and expression of trypanothione reductase from a New World Leishmania species

Trypanothione disulfide (T[S]₂), an unusual form of glutathione found in parasitic protozoa, plays a crucial role in the regulation of the intracellular thiol redox balance and in the defense against oxidative stress. Trypanothione reductase (TR) is central to the thiol metabolism in all trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania. Here we report the cloning, sequencing and expression of the TR encoding gene from L. (L.) amazonensis. Multiple protein sequence alignment of all known trypanosomatid TRs highlights the high degree of conservation and illustrates the phylogenetic relationships. A 3D homology model for L. amazonensis TR was constructed based on the previously reported Crithidia fasciculata structure. The purified recombinant TR shows enzyme activity and in vivo expression of the native enzyme could be detected in infective promastigotes, both by Western blotting and by immunofluorescence. Nucleotide sequence data reported in this paper is available in the GenBank^TM database under accession number DQ530259.     

Potent in vitro anti-Trypanosoma cruzi activity of pyridine-2-thiol N-oxide metal complexes having an inhibitory effect on parasite-specific fumarate reductase

In the search for new therapeutic tools against Chagas disease (American trypanosomiasis) palladium and platinum complexes of the bioactive ligand pyridine-2-thiol N-oxide were exhaustively characterized and evaluated in vitro. Both complexes showed high in vitro growth inhibition activity (IC(50) values in the nanomolar range) against Trypanosoma cruzi, the causative agent of the disease. They were 39-115 times more active than the antitrypanosomal drug Nifurtimox. The palladium complex showed an approximately threefold enhancement of the activity compared with the parent compound. In addition, owing to their low unspecific cytotoxicity on mammalian cells, the complexes showed a highly selective antiparasite activity. To get an insight into the mechanism of action of these compounds, DNA, redox metabolism (intraparasite free-radical production) and two parasite-specific enzymes absent in the host, namely, trypanothione reductase and NADH-fumarate reductase, were evaluated as potential parasite targets. Additionally, the effect of metal coordination on the free radical scavenger capacity previously reported for the free ligand was studied. All the data strongly suggest that trypanocidal action of the complexes could mainly rely on the inhibition of the parasite-specific enzyme NADH-fumarate reductase.     

Characterization of quinonoid-dihydropteridine reductase (QDPR) from the lower eukaryote Leishmania major

Biopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterize Leishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H(4)biopterin pools. LmQDPR shows good homology to metazoan quinonoid-dihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8-9 copies containing LmQDPR plus two other genes. QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H(4)biopterin throughout this family of human parasites.     

CoMFA-SIMCA model for antichagasic nitrofurazone derivatives

The physico-chemical properties of some 5-nitro-2-furaldehyde semicarbazones (nitrofurazones) and thiophene analogues were compared with their in vitro and in vivo trypanocidal activity against Trypanosoma cruzi (Tulahuen strain). 3D-QSAR models were obtained by applying the SIMCA methodology to the electrostatic and steric fields (CoMFA fields) of the molecules. Nitrofurazones bearing N4 substituents. which cover a range of 14-17 A from the nitro group with a thickness of about 6 A when considering the extended conformer. produced complete survival in infected mice. The in vitro model allows larger N4 substituents than the SURVival model, but they must not bear positive centres in the region 15-16 A from the nitro group. Moreover, the in vitro model is in agreement with the active site of trypanothione reductase (TR). Both models can be of use in the design of novel drugs bearing an amide-like group at a distance of 7-9 A from an easily reducible group.     

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).     

Design, synthesis and evaluation of 2,4-diaminoquinazolines as inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper describes the design, synthesis and evaluation of a series of 2,4-diaminoquinazolines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were designed by a generating virtual library of compounds and docking them into the enzyme active site. Following their synthesis, they were found to be potent and selective inhibitors of leishmanial dihydrofolate reductase. The compounds were also found to have potent activity against Trypanosoma brucei rhodesiense, a causative organism of African trypanosomiasis and also against Trypanosoma cruzi, the causative organism of Chagas disease. There was significantly lower activity against Leishmania donovani, one of the causative organisms of leishmaniasis.     

Synthesis and antiprotozoan evaluation of new alkyl-linked bis(2-thioxo-[1,3,5]thiadiazinan-3-yl) carboxylic acids

Two new series of several alkyl-linked bis(2-thioxo-[1,3,5]thiadiazinan-3-yl) carboxylic acids were synthesized in a two step procedure from the corresponding alkyl bis-dithiocarbamic salt intermediary. The novel compounds were evaluated for their activity in vitro against Trypanosoma cruzi strain CL (clone CL B5) and Trichomonas vaginalis strain JH 31A.     

2,4-Diaminopyrimidines as inhibitors of Leishmanial and Trypanosomal dihydrofolate reductase

This paper describes the synthesis of 4'-substituted and 3',4'-disubstituted 5-benzyl-2,4-diaminopyrimidines as selective inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were then assayed against the recombinant parasite and human enzymes. Some of the compounds showed good activity. They were also tested against the intact parasites using in vitro assays. Good activity was found against Trypanosoma cruzi, moderate activity against Trypanosoma brucei and Leishmania donovani. Molecular modeling was undertaken to explain the results. The leishmanial enzyme was found to have a more extensive lipophilic binding region in the active site than the human enzyme. Compounds which bound within the pocket showed the highest selectivity.     

Prolylisoxazoles: potent inhibitors of prolyloligopeptidase with antitrypanosomal activity

Prolylprolylisoxazoles and prolylprolylisoxazolines were synthesized through a 1,3-dipolar cycloaddition reaction. These compounds are potent inhibitors of human and trypanosomal prolyloligopeptidase. They were shown to inhibit Trypanosoma cruzi and Trypanosoma b. brucei in in vitro systems with ED(50)'s in the lower microM range.