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).     

Methylglyoxal metabolism in trypanosomes and leishmania

Methylglyoxal is a toxic by-product of glycolysis and other metabolic pathways. In mammalian cells, the principal route for detoxification of this reactive metabolite is via the glutathione-dependent glyoxalase pathway forming d-lactate, involving lactoylglutathione lyase (GLO1; EC 4.4.1.5) and hydroxyacylglutathione hydrolase (GLO2; EC 3.2.1.6). In contrast, the equivalent enzymes in the trypanosomatid parasites Trypanosoma cruzi and Leishmania spp. show >200-fold selectivity for glutathionylspermidine and trypanothione over glutathione and are therefore sensu stricto lactoylglutathionylspermidine lyases (EC 4.4.1.-) and hydroxyacylglutathionylspermidine hydrolases (EC 3.2.1.-). The unique substrate specificity of the parasite glyoxalase enzymes can be directly attributed to their unusual active site architecture. The African trypanosome differs from these parasites in that it lacks GLO1 and converts methylglyoxal to l-lactate rather than d-lactate. Since Trypanosoma brucei is the most sensitive of the trypanosomatids to methylglyoxal toxicity, the absence of a complete and functional glyoxalase pathway in these parasites is perplexing. Alternative routes of methylglyoxal detoxification in T. brucei are discussed along with the potential of exploiting trypanosomatid glyoxalase enzymes as targets for anti-parasitic chemotherapy.     

Glutathione and trypanothione in parasitic hydroperoxide metabolism

Thiol-dependent hydroperoxide metabolism in parasites is reviewed in respect to potential therapeutic strategies. The hydroperoxide metabolism of Crithidia fasciculata has been characterized to comprise a cascade of three enzymes, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase, plus two supportive enzymes to synthesize the redox mediator trypanothione from glutathione and spermidine. The essentiality of the system in respect to parasite vitality and virulence has been verified by genetic approaches. The system appears to be common to all genera of the Kinetoplastida. The terminal peroxidase of the system belongs to the protein family of peroxiredoxins which is also represented in Entamoeba and a variety of metazoan parasites. Plasmodial hydroperoxide metabolism displays similarities to the mammalian system in comprising glutathione biosynthesis, glutathione reductase, and at least one glutathione peroxidase homolog having the active site selenocysteine replaced by cysteine. Nothing precise is known about the antioxidant defence systems of Giardia, Toxoplasma, and Trichomonas species. Also, the role of ovothiols and mycothiols reportedly present in several parasites remains to be established. Scrutinizing known enzymes of parasitic antioxidant defence for suitability as drug targets leaves only those of the trypanosomatid system as directly or indirectly validated. By generally accepted criteria of target selection and feasibility considerations tryparedoxin and tryparedoxin peroxidase can at present be rated as the most appealing target structures for the development of antiparasitic drugs.     

A second class of peroxidases linked to the trypanothione metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.     

Host-parasite interaction of tyrosine metabolism in rats infected with trypanosoma brucei

• 1.1. p-Hydroxyphenylpyruvate, the by-product of tyrosine metabolism by T. brusei, was found to be non-toxic to the rat.
• 2.2. A model is proposed for host-parasite interaction in the metabolism of tyrosine.
• 3.3. Oxygen uptake data suggest a conversion of parasite respiration to the Kreb's cycle during glucose deprivation conditions in the host, fueled by products of tyrosine metabolism.

     

Preparative enzymatic synthesis of trypanothione and trypanothione analogues

Trypanosomatids, the causative agents of several tropical diseases, have a unique thiol metabolism based on trypanothione [bis(glutathionyl)spermidine]. Enzymes of the pathway are attractive drug target molecules but the availability of trypanothione remains an obstacle. Here, we present a convenient method for the production of trypanothione and trypanothione disulfide in >200 mg quantities using a mutant of Crithidia fasciculata trypanothione synthetase in which Cys59 has been replaced by an alanine residue. The reagent costs less than 1% of the commercial price of trypanothione disulfide. The protocol also allows the synthesis of related glutathione conjugates. It will greatly facilitate the thorough analysis of this parasite’s metabolism and drug screening approaches against trypanothione-dependent enzymes.     

Rational design of selective ligands for trypanothione reductase from trypanosoma cruz Structural effects on the inhibition by dibenzazepines based on imipramine

Abstract

Trypanothione reductase, the enzyme which in trypanosomal and leishmanial parasites catalyses the reduction of trypanothione disulphide to the redox-protective dithiol and has been identified as a potential target for rational antiparasite drug design, has been found to be strongly inhibited by tricyclic compounds containing the saturated dibenzazepine (imipramine) nucleus, with K_i values in the low micromolar range. This drug lead structure was designed by molecular graphics analysis of a three-dimensional homology model, focussing on the active-site. Inhibition studies were carried out to determine the effect of inhibitor structure on the inhibitory strength towards recombinant trypanothione reductase from Trypanosoma cruzi Hansch analysis showed that inhibitory strength depended on terms in ² and s`_m indicating dependence on both lipophilicity and inductive effect for ring-substituted analogues of imipramine. The side-chain ω-aminoalkyl chain had to be longer than 2-carbon units for inhibition. The effect on inhibition strength of the substituent at the ω-amino position on the side-chain of the central ring nitrogen atom depended markedly on the detailed substitution pattern of the rest of the molecule. This provides kinetic evidence studies of multiple binding modes within a single, blanket binding site for the inhibitor with the tricyclic ring system in the general region of the hydrophobic pocket lined by Trp21, Tyr110, Met113 and Phe114. This aspect of the structural sensitivity of the precise active-site triangulation adopted by the inhibitor is probably a function of the use of hydrophobic interactions of low directional specificity in this pocket combined with an electrostatianchoring by the ω-N+HMe₂ function of the inhibitor, presumably with a glutamate sidethain, such as Glu-18, Glu-466prime; and/or Glu-467prime;.     

The leishmania ARL-1 and Golgi traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.     

Antitumor quinol PMX464 is a cytocidal anti-trypanosomal inhibitor targeting trypanothione metabolism

Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class ("quinols") and identified several with potent trypanocidal activity (EC(50) < 100 nM). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)(2)), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)(2), TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and L-cysteine bound in a 2:1 ratio with PMX464 with K(d) values of 6 and 27 μM, respectively, whereas T(SH)(2) bound more tightly in a 1:1 ratio with a K(d) value of 430 nM. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)(2) is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.     

The natural heterogeneity of trypanosoma cruzi: Biological and medical implications

Trypanosoma cruzi is a heterogeneous group of parasites. The imposition of natural or artificial pressures can result in the selection of subsets of the population with concomitant changes in characteristics used to evaluate the group. In order to ascertain the extent of heterogeneity, stocks of single-cell clones were prepared from various sources. Selected cell biological, biochemical, immunochemical, parasitological, and histopathological parameters of these clones have been studied. A ten-fold difference in the rate of growth of the epimastigote stage of T cruzi clones has been observed. The extracellular growth rates of the clones correlate with the rate of growth of the obligate intracellular amastigote stage and consequently, the length of intracellular cycle of the parasite. A 40% difference in the amount of total DNA/parasite has been found between clones. Although the amount of DNA/kinetoplast and nucleus varies between clones, the major contribution to the differences in total DNA/parasite appears to be the nucleus. From 16 to 35 antigens have been demonstrated in the T cruzi clones assayed to date. Five to seven of these antigens are common to all of the stocks assayed. However, both isolate- and clone-specific antigens have also been demonstrated. The susceptibility of inbred strains of mice to T cruzi clones varies with the clone of the parasite. These data imply that the genetics of the parasite as well as the host modulate both the course and outcome of a T cruzi infection. The influence of monosaccharides on the receptor-mediated infection of vertebrate cells by trypomastigotes of T cruzi also varies between clones. The implications of these findings upon our concept and understanding of present and future problems in Chagas disease are discussed.     

Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466′, Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777–TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.     

Interaction of leishmania metacyclics with macrophages

Metacyclics of L. major and putative metacyclics of L. m. mexicana survived better in explanted murine macrophages than promastigotes from mid-log phase cultures. The latter forms, however, attached in greater numbers to macrophages and, in the case of L. major, also more became intracellular. Only a small percentage of the macrophages infected with amastigotes exhibited a respiratory burst (as detected by nitroblue tetrazolium (NBT) reduction), whereas this occurred with most of the macrophages infected with either metacyclic or non-infective promastigotes of L. major. Approximately half of the macrophages infected with L. m. mexicana promastigotes reduced NBT. The results suggest that avoidance of the oxygen metabolite arm of the host cell's microbicidal activity is not the main survival strategy for metacyclics entering macrophages. Metacyclics of L. major, however, were found to be less sensitive than mid-log phase promastigotes to hydrogen peroxide and also human serum; properties which may aid their survival.     

Class and subclass selection in parasite-specific antibody responses

Antibodies are characteristically induced in many parasitic infection processes. The class and subclass of the antibody response is instrumental because each isotype has a distinct biological function. It is thus crucially important for an infected individual to mount the most appropriate secondary antibody response--that is the response that has the best chance of clearing the infection and/or controlling disease. This represents a fundamental of vaccine strategies. Immuno-epidemiological surveys and in vitro models of antibody production have helped to understand some of the goals which should be achieved when designing antiparasitic vaccines.     

Reduction of pentavalent antimony by trypanothione and formation of a binary and ternary complex of antimony(III) and trypanothione

Several pentavalent antimony compounds have been used for the treatment of leishmaniasis for decades. However, the mechanism of these antimony drugs still remains unclear. One of their targets is thought to be trypanothione, a major low molecular mass thiol inside the parasite. We show that pentavalent antimony (Sb^V) can be rapidly reduced to its trivalent state by trypanothione at mildly acidic conditions and 310 K (k=4.42 M^–1 min^–1 at pH 6.4), and that Sb^III can be bound to trypanothione to form an Sb^III-trypanothione complex. NMR data demonstrate that Sb^III binds to trypanothione at the two thiolates of the cysteine residues, and that the binding is pH dependent and is strongest at biological pH with a stability constant logK=23.6 at 298 K (0.1 M NaNO₃). The addition of low molecular monothiol ligands such as glutathione and cysteine to the Sb^III-trypanothione complex results in the formation of a ternary complex. Thiolates from both trypanothione and monothiol bind to the Sb^III center. The formation of the ternary complex is important, as the antileishmanial properties of the drugs are probably due to a complex between of Sb^III-trypanothione and enzymes. Although thermodynamically stable, the complex is kinetically labile and the free and bound forms of thiolates exchange on the ¹H NMR timescale. Such a facile exchange may be crucial for the transport of Sb^III within parasites.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations amastigote the parasites culture at pH 5.0 and 310 K to resume the intracellular form - BPR bromopyrogallol - ESI-MS electrospary ionization mass spectrometry - GSH glutathione - pH^* pH meter reading in D₂O without correction for isotope effects - promastigote the parasites culture at pH 7.4 and 298 K to resume the extracellular stage - T(SH)₂ reduced form of trypanothione - T(S-S) oxidized form of trypanothione (disulfide form) - TR trypanothione reductase - tart tartrate