Gerardo Hurtado

Trypanothione reductase from the human parasite Entamoeba histolytica: a new drug target.

Although there is a general agreement that the protist Entamoeba histolytica lacks glutathione, it has been a matter of dispute as to whether this human parasite contains the glutathione derivative known as trypanothione. In the present study, we describe a gene for the TR (trypanothione reductase) obtained from E. histolytica by PCR amplification of its DNA. After Northern‐blot analysis, the radiolabelled DNA probe from Trypanosoma cruzi hybridizes with the total RNA of Entamoeba, showing that the TR gene is expressed as mRNA. We have demonstrated the presence of the NADPH‐dependent TR activity in vitro with partially purified extracts and showed also that the thiol‐bimane compound isolated and purified from E. histolytica trophozoites, unequivocally corresponds, by matrix‐assisted laser‐desorption ionization–time‐of‐flight MS, to the characteristic monoprotonated ion of trypanothione‐(bimane)2 with m/z 1104.4 and the trypanothione‐(bimane) with m/z 914.3. The PCR product consisted of 1476 bp (491 deduced amino acids), has sequences diagnostic for the reducing catalytic site (CVNVGC) as well as domains for binding NADPH, FAD I and FAD II that are present in all members of this group of disulphide‐reducing enzymes, as well as those unique to TRs. The putative protein sequence is 86% identical with that of TR from T. cruzi and it is also clearly distinguishable from other related reductases by phylogenetic analysis. We can conclude, from these highly reliable experiments, that E. histolytica contains the TR enzyme and the thiol compound trypanothione that was previously supposed to occur only in trypanosomatids.

Identification of trypanothione from the human pathogen Entamoeba histolytica by mass spectrometry and chemical analysis

In this paper, we present definitive data to show, from ESI (electrospray ionization) studies, that the thiol–bimane compound isolated and purified from Entamoeba histolytica trophozoites, corresponds unequivocally to the structure of trypanothione. Trypanothione disulphide was shown to have a molecular ion of m/z 722. It was further demonstrated by MALDI–TOF (matrix‐assisted laser desorption ionization–time‐of‐flight) MS that this thiol compound also corresponds to the characteristic monoprotonated ion of trypanothione‐(bimane)2, which has a molecular ion of m/z 1103.95. The ion pattern of the thiol–bimane compound prepared from the commercial trypanothione standard is identical with the E. histolytica thiol–bimane compound. After HPLC separation, chemical amino acid analysis by dabsylation and dansylation of the thiol bimane compound from Entamoeba showed the presence of the following trypanothione components: glutamic acid, cysteic acid, glycine and spermidine. We can conclude from these highly reliable MS experiments and chemical analyses that E. histolytica contains the thiol compound trypanothione, which was previously thought to occur only in trypanosomatids.

Entamoeba histolytica: a eukaryote with trypanothione metabolism instead of glutathione metabolism

Entamoeba histolytica is a human pathogen that lacks the capacity to synthesize glutathione but can incorporate it, from the growth media or presumably from the human host, to form trypanothione [N1,N8‐bis(glutathionyl)‐spermidine conjugate]. This novel thiol compound has previously been found in trypanosomatids, as has its precursor glutathionyl‐spermidine, which was originally detected in Escherichia coli. Previously we showed the presence of these two thiol compounds in extracts from cultures of Entamoeba histolytica HK9. Here we report that when Entamoeba histolytica HK9 is grown in a culture medium that lacks glutathione (treated with the enzyme γ‐glutamyl transpeptidase), trypanothione is not formed, although the trophozoites can continue dividing for at least 60 h but at 25% lower cell density. The finding of a trypanothione metabolism in Entamoeba histolytica raises many questions: one concerns the possibility of a phylogenetic relationship, in this respect, with trypanosomatids such as Trypanosoma cruzi, T. brucei and Crithidia fasciculata; another concerns its role in cell metabolism; a third concerns it possible use as a target for a rational drug design strategy against this parasite.

Inhibitory and lytic effects of phenothiazine derivatives and related tricyclic neuroleptic compounds, on Entamoeba histolytica HK9 and HM1 trophozoites.

It has been shown previously that tricyclic neuroleptics like clomipramine and chlorpromazine have lethal effects on Leishmania donovani and L. major, and other studies indicate that the phenothiazine inhibitors of trypanothione reductase are potential anti‐trypanosomal and anti‐leishmanial drugs. With this in mind and our original observation on the presence of trypanothione in Entamoeba histolytica HK9, we examined the possible inhibitory effects of various phenothiazine and tricyclic derivatives on this human parasite. We found that drugs like clomipramine (KD002), the most potent in vitro inhibitor of trypanothione reductase among 30 tricyclic compounds tested, at 25 μM after 24 h of culture under aerobic conditions, caused a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15×106 to 5·37×106 cells, and at 100 μM to 0·8×106 cells. A substantial inhibitory effect on cell proliferation could also be demonstrated with metronidazol (used clinically against amoebiasis). Under similar experimental conditions other tricyclic and phenothiazine derivatives (OFKs), designed originally to inhibit the trypanothione reductase of trypanosomatides, had an inhibitory effect of 16 to 95%. For comparison, similar results were obtained using clomipramine and a phenothiazine derivative (OFK006) with Trypanosoma cruzi and Crithidia luciliae, except that with the latter the inhibitory effect of clomipramine was less dramatic. Experiments comparing two E. histolytica strains showed that normal cell proliferation under anaerobiosis was higher in strain HK9 than in HM1, which is highly virulent, but that metronidazol and clomipramine were less effective against HM1. Two other drugs tested, diphenydramine (KD005) and a phenothiazine derivative (OFK008), also had significant but lower inhibitory effects on both strains. The inhibitory activity on cell proliferation and the lytic effects on this human parasite by the tricyclic compounds clomipramine, chlorpromazine and others, as well as by the phenothiazine derivatives, indicate that they can be considered potential anti‐amoebic agents.

Low-molecular-mass thiol compounds from a free-living highly pathogenic amoeba, Naegleria fowleri.

Acid extracts labelled with the fluorescent reagent monobromobimane and separated by HPLC have enabled the detection of low‐molecular‐mass thiol compounds in Naegleria fowleri for the first time. The amounts detected are expressed in nmol/1×106 trophozoites cultivated at various stages of growth in the appropriate culture medium. N. fowleri is a highly pathogenic free‐living amoeba, in which we found important thiol compounds, some of them in their reduced and oxidized forms. Unlike cysteine and glutathione, a number of these are not represented in normal human lymphocytes. Some of these thiol compounds from Naegleria must have their respective disulphide reductases, although the presence of thiol‐disulphide exchange reactions must be considered. Ovothiol A, with antioxidant properties, is an example of a compound that is kept reduced by trypanothione in trypanosomatids, although no disulphide reductase for ovothiol A has yet been discovered. In our case we were unable to detect this biothiol in Naegleria . The presence of thiol compounds that seem to be particular to this pathogen and which are not present in human lymphocytes opens the possibility of searching for disulphide‐reducing enzymes that can serve as drug targets.

Thiol compounds from a free-living pathogenic opportunistic amoeba, Acanthamoeba polyphaga

New bimane‐reacting compounds from perchloric acid extracts have been detected by HPLC from Acanthamoeba polyphaga. The main compounds detected are cysteine, glutathione and other novel thiol compounds. All of these compounds must be thiols, since they disappear or decrease substantially when treated by N‐ethylmaleimide prior to acetonitrile/bimane derivatization. Cysteine and glutathione increase in quantity when dithiothreitol reduction is applied to the fresh extract. This means that they are likely to be present in their oxidized and reduced form and indicates the possible presence of a corresponding thiol/disulphide enzymic system. There are other compounds that have a different behaviour, since although they can react with bimane, they do not disappear if treated previously by N‐ethylmaleimide. This shows that they are not thiols but can react with bimane. The main thiol compounds found to be present, in both the parasite and the host lymphocyte cells, were cysteine and glutathione. We were unable to detect ovothiol A in Acanthamoeba but instead we found another thiol compound that could be structurally related to trypanothione. The new thiol compounds unique to this parasite and not present in lymphocytes will permit the study of disulphide‐reducing enzymes as potential drug targets.

Detection by HPLC of a trypanothione synthetase activity in vitro from Entamoeba histolytica.

We have previously demonstrated the presence of glutathione‐spermidine (Gsp) and trypanothione [T(SH)2] from Entamoeba histolytica trophozoites, on the basis of results obtained with acid extracts purified by Florisil and DEAE‐cellulose, derivatized with the fluorescent reagent monobromobimane and separated by HPLC. Gsp was originally found in Escherichia coli and later in trypanosomatids such as Trypanosoma cruzi, T. brucei, T. congolense and the insect trypanosomatid Crithidia fasciculata, along with the novel compound T(SH)2, N1,N8‐bis(glutathionyl)‐spermidine. Here we demonstrate the presence of a T(SH)2 synthetase activity in partly purified extracts from Entamoeba histolytica HK9, incubated at two pH values (6.5 and 7.5) with reduced glutathione (GSH), spermidine and ATP, in the presence of Mg2+ at different time intervals. The thiol products were detected by HPLC in picomole amounts and compared with commercial Gsp and T(SH)2 standards. We have used also an extract of Crithidia luciliae as a reference, to compare our results with C. fasciculata, in which the presence of this enzyme has previously been demonstrated and was later purified and separated into two synthetase activities from the same source: one for Gsp and the other for T(SH)2. The presence of a T(SH)2 synthetase activity in Entamoeba histolytica means that this protozoan has a similar metabolism to that of the trypanosomatids and opens the possibility of establishing a rational drug design against this human parasite.

In vivo Inhibition of Reduced Thiol Compounds from Entamoeba histolytica HK9 by Phenothiazines and Related Tricyclic Drugs

The need to find new drugs is more demanding each day. The various strategies include the search for metabolic routes unique to the pathogen and that are not present in the host, and the analysis of the molecular structure of enzymes that can be inhibited by rationally designed compounds. This strategy has already been applied to the specific in vitro inhibition of trypanothione reductase, an enzyme present in trypanosomatides such as T. cruzi, T. brucei , and T. congolense , as well as in various species of Leishmania , by phenothiazine derivatives such as clomipramine and others prepared recently (1). Because Entamoeba histolytica does not have the capacity to form glutathione because it lacks glutathione reductase (2), the parasite incorporates glutathione from the culture medium and initiates a trypanothione metabolism similar to that of trypanosomatides (3). We have studied the inhibitory effect of 10 phenothiazineand tricyclic-derived compounds on the reduced thiols of this human parasite. Similar to the effect observed in trypanosomatides, Entamoeba histolytica trophozoites were susceptible to the phenothiazine and tricyclic derivatives at a concentration of 100 m M during 24 h of culture incubation.

Mass spectrometry characterization of trypanothione and novel peptides of medical importance isolated from Acanthamoeba polyphaga.

This paper presents unequivocal results about the presence of trypanothione and its precursor glutathionespermidine from the opportunistic human pathogen Acanthamoeba polyphaga. They were isolated by RP-HPLC as thiolbimane derivatives and characterized using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF). Additionally RP-HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were also present in A. polyphaga. Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH-dependent disulfidereducing enzymes. Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites. In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets.