Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection.

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone-derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.

Two approaches to discovering and developing new drugs for Chagas disease.

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A 'proof of concept' molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Cloning of a virulence factor of Entamoeba histolytica. Pathogenic strains possess a unique cysteine proteinase gene.

Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.

The crystal structure of cruzain: a therapeutic target for Chagas' disease.

Trypanosoma cruzi, a protozoan parasite, is the etiologic agent of American trypanosomiasis or Chagas' disease. Chagas' disease afflicts more than 24 million individuals in South and Central America producing a debilitating life-long disease. It is the leading cause of heart failure in many Latin American countries. Currently, there is no satisfactory treatment for this parasitic infection. Cruzain (also known as cruzipain, gp 57/51), the major cysteine protease present in T. cruzi, is critical for the development and survival of the parasite within the host cells, making this enzyme a target for potential trypanocidal drugs. Here we report the X-ray crystal structure of cruzain complexed with the potent inhibitor Z-Phe-Ala-fluoromethyl ketone. The structure was determined at 2.35 A (Rcryst = 0.15) by molecular replacement using a modified papain as the search model. The refined structure is compared to papain. Features which distinguish cruzain from papain are discussed since they may aid in the design of specificity inhibitors. Fluorescence microscopy shows that a biotinylated form of the bound inhibitor does not effectively reach host proteases in their lysosomal compartment, but is selectively taken up by the parasite. The inhibitor greatly reduces parasitemia in a cell culture system, without adverse effects to mammalian cells. This biological selectivity can be exploited, in conjunction with unique active site features revealed by the crystal structure, to develop chemotherapy for Chagas' disease.

Aryl ureas represent a new class of anti-trypanosomal agents.

BACKGROUND: The trypanosomal diseases including Chagas' disease, African sleeping sickness and Nagana have a substantial impact on human and animal health worldwide. Classes of effective therapeutics are needed owing to the emergence of drug resistance as well as the toxicity of existing agents. The cysteine proteases of two trypanosomes, Trypanosoma cruzi (cruzain) and Trypanosoma brucei (rhodesain), have been targeted for a structure-based drug design program as mechanistic inhibitors that target these enzymes are effective in cell-based and animal models of trypanosomal infection. RESULTS: We have used computational methods to identify new lead scaffolds for non-covalent inhibitors of cruzain and rhodesain, have demonstrated the efficacy of these compounds in cell-based and animal assays, and have synthesized analogs to explore structure activity relationships. Nine compounds with varied scaffolds identified by DOCK4.0.1 were found to be active at concentrations below 10 microM against cruzain and rhodesain in enzymatic studies. All hits were calculated to have substantial hydrophobic interactions with cruzain. Two of the scaffolds, the urea scaffold and the aroyl thiourea scaffold, exhibited activity against T. cruzi in vivo and both enzymes in vitro. They also have predicted pharmacokinetic properties that meet Lipinski's 'rule of 5'. These scaffolds are synthetically tractable and lend themselves to combinatorial chemistry efforts. One of the compounds, 5'(1-methyl-3-trifluoromethylpyrazol-5-yl)-thiophene 3'-trifluoromethylphenyl urea (D16) showed a 3.1 microM IC(50) against cruzain and a 3 microM IC(50) against rhodesain. Infected cells treated with D16 survived 22 days in culture compared with 6 days for their untreated counterparts. The mechanism of the inhibitors of these two scaffolds is confirmed to be competitive and reversible. CONCLUSIONS: The urea scaffold and the thiourea scaffold are promising leads for the development of new effective chemotherapy for trypanosomal diseases. Libraries of compounds of both scaffolds need to be synthesized and screened against a series of homologous parasitic cysteine proteases to optimize the potency of the initial leads.

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi.

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter.

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]-X(4)-[AV]-[AP]-X-[AP]-X(3)-Y-X(9)-[LIVF]-X(2)-[SA]-G-[QS], and the C-terminal: [AT]-R-X(2)-[IVFY]-X-[VC]-X(2)-L-P-X(4)-[LIVM]-E-[IVM] -[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.

Rapid identification of Trypanosoma cruzi isolates by 'dot-spot' hybridization.

The presence of isolate-specific Trypanosoma cruzi minicircles has been shown in the kinetoplast DNA of this parasite. This led to the rapid identification of isolates and clones of trypanosomes by means of 'dot-spot' hybridizations with molecularly cloned minicircle probes. Unexpectedly, whole kDNAs were also suitable as probes for this purpose, provided that filters were washed under stringent conditions. This was attributed to the presence of the above-mentioned isolate-specific minicircle sequences. The fact that parasites could be directly spotted onto nitrocellulose filters simplified the rapid routine screening of a large number of samples.

Strains and clones of Trypanosoma cruzi differ in their expression of a surface antigen identified by a monoclonal antibody.

Four Trypanosoma cruzi strains and 50 clones derived from a total of 10 strains were assayed with a monoclonal antibody, WIC 29.26 Ab, for expression of an epitope previously demonstrated to be on the carbohydrate portion of a 72 000 Da surface glycoprotein of Y strain epimastigotes. WIC 29.26 Ab bound to only 2 of the 4 strains and 23 of the 50 clones tested. A group of 10 cloned isolates from one strain contained both reactive and non-reactive clones. Competitive inhibition studies with soluble extracts of the reactive and non-reactive isolates suggested that in addition to being absent from the surface membrane, the antigenic determinant is not synthesized by the non-reactive parasites. These data indicate that the epitope recognized by WIC 29.26 Ab is not present on all T. cruzi epimastigotes, and provide the first demonstration of clone-specific differences in a parasite antigen detected by a monoclonal antibody. No correlation was found between the reactivity of isolates with WIC 29.26 Ab and the previously investigated parameters of growth rate and modal volume.

Isolate-dependent differences in the oxidative metabolism of Trypanosoma cruzi epimastigotes.

Epimastigote cultures of the two cloned Trypanosoma cruzi stocks, CA-I/72 and HO-3/15, grown under identical conditions, differ both qualitatively and quantitatively in their cytochrome content. The CA-I/72 stock has a four-fold higher cytochrome b content (19.2 nM (mg protein)-1) than the HO-3/15 stock (4.9 nM (mg protein)-1). Cytochrome o is present at 29 nM (mg protein)-1 in the CA-I/72 stock but is below detectable limits in the HO-3/15 stock. There is no inter-stock difference in oxygen utilization (12-15 nM O2 min-1 (mg protein)-1) during exponential growth. However, stationary phase CA-I/72 epimastigotes utilize twice as much oxygen as HO-3/15 epimastigotes. Oxygen utilization by HO-3/15 epimastigotes incubated in Dulbecco's phosphate buffer solution (starvation conditions), was stimulated earlier and to higher levels by the addition of glucose than by CA-I/72 epimastigotes under identical conditions. Under starvation conditions and with the cytochrome chain partially inhibited by antimycin A,(anti-A) the addition addition of glucose also increased oxygen utilization by CA-I/72 epimastigotes. In contrast, anti-A did not influence glucose-stimulated oxygen utilization by HO-3/15 epimastigotes. Following partial inhibition with anti-A, salicylhydroxamic acid produced an additional 50% inhibition in oxygen utilization in both stocks irrespective of the growth phase of the organisms. These data indicate that marked intra-specific differences in oxidative metabolism exist within the T. cruzi population and that an alpha-glycerophosphate oxidase or similar salicylhydroxamic acid-inhibitable compound may be present in the organism.

Polymorphisms within minicircle sequence classes in the kinetoplast DNA of Trypanosoma cruzi clones.

Four minicircle classes were analyzed using cloned minicircles as probes and single-cell cloned Trypanosoma cruzi parasites. The hybridization conditions used allowed identification of minicircle classes within kinetoplast DNA that were non-homologous to each other. Two of these minicircle classes, detected with probes pTckAWP-2 and -3, were present together in several of the CA 1 and Miranda clones, in spite of the fact that either pTckAWP-2 or both minicircle classes were undetectable in other isolates and clones of the parasite. The other two minicircle classes (pTckM-84 and -88) were located in some Miranda cloned parasites which were characterized by the simple restriction endonuclease pattern of their minicircles. Both pTckM-84 and -88 minicircle classes represented 52-71% of the kinetoplast DNA in the latter group of trypanosomes. Restriction endonuclease mapping allowed the identification of polymorphic minicircles in two of the four minicircle classes analyzed (pTckAWP-2 and pTckM-88). The polymorphisms were observed in part of the molecules of one minicircle class within a single trypanosome clone, as well as when different clones or even some of those obtained from the same isolate were compared. In addition, a different proportion of pTckM-88 type of minicircle sequence class was observed in the kinetoplast DNA from two of the Miranda clones analyzed. These observations demonstrated that similar molecules may evolve independently in sequence in each parasite. The polymorphic minicircles detected may arise from sequence variations before expansion of a future homogeneous minicircle sequence class.

End products and enzyme levels of aerobic glucose fermentation in trypanosomatids.

Trypanosoma cruzi (epimastigotes), Crithidia fasciculata and Leishmania mexicana (promastigotes) were grown in a brain-heart-tryptose medium supplemented with heat-inactivated fetal calf serum. T. cruzi and C. fasciculata utilized glucose completely during the log phase of growth, whereas L. mexicana used significant amounts of the carbohydrate only at the end of the log phase and at the beginning of the stationary phase. In all cases glucose consumption resulted in excretion of succinate, and much smaller amounts of acetate. C. fasciculata and L. mexicana produced very small amounts of pyruvate. C. fasciculata produced ethanol, which was taken up again and metabolysed after glucose was exhausted. Lactate and malate were not produced. The cells were disrupted by sonic disintegration, and the activities of some key enzymes of carbohydrate and amino acid catabolism were assayed in the whole homogenates. Phosphoenolpyruvate carboxykinase was present in the three organisms; L. mexicana presented the highest specific activity. The activity of this enzyme was maximal during glucose consumption, and slightly decreased after glucose was exhausted. This suggests that the role played by the enzyme is glycolytic and not gluconeogenic; the latter is the case in most higher organisms. Hexokinase and pyruvate kinase presented their highest levels in C. fasciculata and T. cruzi during glucose consumption. L. mexicana, which was in active glycolysis during the whole experimental period, presented the highest specific activities of both enzymes. Citrate synthase, on the other hand, increased in C. fasciculata and, to a lesser extent, in T. cruzi, after glucose was exhausted; the enzyme could not be detected in L. mexicana. The NAD-linked glutamate dehydrogenase increased considerably in C. fasciculata and T. cruzi after glucose was exhausted, suggesting a catabolic role for the enzyme. This increase coincided with an increase in NH3 production by both organisms after glucose consumption. The NADP-linked glutamate dehydrogenase, on the other hand, presented a maximum about the time when glucose was exhausted, and then decreased again, which suggests a catabolic role for the enzyme. Both glutamate dehydrogenases had low activities in L. mexicana; this fits in well with the low NH3 production throughout the culture of this organism. The results are in good agreement with current ideas on the mechanism of aerobic glucose fermentation by trypanosomatids, and suggest that, under the experimental conditions used, both T. cruzi and C. fasciculata used glucose perferentially over amino acids for growth.

Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi.

The major proteolytic activity of Trypanosoma cruzi is a cathepsin L-like cysteine protease expressed in all stages of the parasite. As an initial step in identifying possible functions of this enzyme in the life cycle of T. cruzi, and examining its potential as a target for rational drug design, two fluoromethyl ketone-derivatized cysteine protease inhibitors were studied for their effects on T. cruzi infection of mammalian cells. Both inhibitors are irreversible substrate analogues with high specificity for cysteine proteases and minimal toxicity to mammalian cells. While micromolar concentrations of inhibitors had some effect on replication of all parasite stages, the most dramatic arrest of parasite replication occurred at the transformation of trypomastigote to amastigote, and also from amastigote to trypomastigote. It is therefore proposed that the enzyme functions in intracellular protein degradation in some stages of T. cruzi, but also in remodeling of the parasite during transformation between stages. Concentrations of inhibitors necessary to interrupt the parasite life cycle had no observable toxicity to macrophages, fibroblasts or epithelial cells in culture. Differential susceptibility of T. cruzi versus host cysteine proteases to fluoromethyl ketone protease inhibitors suggests that inhibition of the T. cruzi cysteine protease is a potential lead for new chemotherapy of Chagas' disease.

Characterization of the mechanism of protein glycosylation and the structure of glycoconjugates in tissue culture trypomastigotes and intracellular amastigotes of Trypanosoma cruzi.

Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-linked Man7GlcNAc2 and Man9GlcNAc2. Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man9GlcNAc2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man9GlcNAc2 and Man8GlcNAc2 occurred in epimastigotes and trypomastigotes but the structure of the main Man7GlcNAc2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man7GlcNAc2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N-glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.

Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks.

Concentrates of the epimastigote stage of Trypanosoma cruzi stocks derived from single cell clones and cultured under identical conditions display a spectrum of 'colors' varying from dark brown to milk white. The color of the concentrate is reproducible for a parasite stock. An essential component of the culture medium for epimastigote growth is hemin, an iron-containing compound. Consequently, it seemed possible that the color spectrum of the epimastigote stocks reflected differences in the uptake, concentration or utilization of iron. This report describes the quantitative studies utilizing electron probe X-ray elemental mapping, energy dispersive X-ray microanalysis, and energy dispersive X-ray fluorescence spectrometry of the epimastigote stage of two T. cruzi stocks (CA-I/72 and HO-3/15) which display extreme color differences. Striking and statistically significant quantitative differences were found in the levels of Fe, Zn, and K between the two stocks. On the basis of atomic ratios, the CA-I/72 stock contains approximately two-fold more Fe per cell than the HO-3/15 stock. However, in the case of Zn the ratio is reversed; the HO-3/15 stock contains approximately two-fold more Zn per cell than the CA-I/72 stock. The marked inter-stock differences which exist in the levels of several elements could modulate the pathogenicity, survival, or adaptability of T. cruzi and, consequently, be important factors in our understanding of the complex problem of Chagas' disease.

Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins.

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.

Aerobic glucose fermentation by Trypanosoma cruzi axenic culture amastigote-like forms during growth and differentiation to epimastigotes.

Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28 degrees C, reach a stationary phase after two generations, and differentiate to epimastigotes, which then resume growth. Axenic culture amastigotes readily ferment glucose to succinate and acetate, and do not excrete NH3; they have high activities of hexokinase and phosphoenolpyruvate carboxykinase, and very low citrate synthase activity; cytochrome o is absent, and cytochrome b-like is present at a very low level. Epimastigotes catabolize glucose and produce succinate and acetate at a considerably lower rate; they exhibit lower levels of hexokinase and carboxykinase, and much higher levels of citrate synthase and cytochromes o and b-like. They catabolize amino acids, as shown by excretion of NH3 to the medium. The results suggest that axenic culture amastigotes have an essentially glycolytic metabolism, and they acquire the ability to oxidize substrates such as amino acids only after differentiation to epimastigotes.

Cellular and molecular biological analyses of nifurtimox resistance in Trypanosoma cruzi.

We induced nifurtimox resistance in both epimastigotes and tissue culture-derived trypomastigotes of several Trypanosoma cruzi strains. The magnitude of nifurtimox resistance was strain-dependent. A variety of karyotype changes occurred in the nifurtimox-resistant (NR) strains. Chromosome-specific DNA probes identified karyotype changes common to the NR strains that were moderately resistant to nifurtimox but not the NR strains that were highly resistant to nifurtimox. A marked increase in nifurtimox resistance in one NR strain was accompanied by a 100% increase in nuclear DNA mass and a 50% increase in kinetoplast DNA mass. These data suggest that nifurtimox resistance can be accompanied by a wide spectrum of DNA changes. Both trypanothione reductase and heat-shock proteins may modulate the effects of exposure of T. cruzi to nifurtimox. However, we did not detect qualitative or quantitative differences in these genes or their transcripts between the NR strains and the sensitive strains from which they were derived. An understanding of the spectrum of diversity in nifurtimox resistance at the cellular and molecular levels demonstrated in this report is critical in the development of drug therapies against Chagas' disease.

A unique cysteine proteinase gene of pathogenic Entamoeba histolytica correlates with virulence.

Extracellular neutral cysteine proteinases are an important virulence factor of E. histolytica. Experimental evidence supporting its role in invasion includes the ability to degrade components of the extracellular matrix and activate complement by specifically cleaving C3. We had previously reported the isolation of fragments encoding cysteine proteinase genes from HM-1 (ACP1) and a nonpathogenic strain (REF291, ACP2) by PCR using consensus sequences based on conserved structural motifs of eukaryotic cysteine proteinases. Using similar techniques, we have now identified a third gene encoding a cysteine proteinase which is present in both pathogenic and nonpathogenic strains and have correlated cysteine proteinase specific-mRNA levels with enhanced proteolytic activity and cytopathic effect on a fibroblast cell monolayer, a quantitative assay of virulence.

[Trypanocidal effect of cysteine protease inhibitors in vitro and in vivo in experimental Chagas disease]. / Efecto antiparasitario de inhibidores de cisteín proteasas in vitro e in vivo en la enfermedad de Chagas experimental.

Endemic in most American countries, Chagas' disease causes high morbidity and mortality. Recent experimental and clinical evidence shows the importance of chemotherapy in both the acute and chronic phases of this disease. However, treatment is yet limited by the toxicity associated to available drugs. This review describes the design, evolution, and selection of dipeptides that interrupt the intracellular cycle of T. cruzi and cure acute experimental infections in laboratory animals. Peptido-mimetic inhibitors specifically bind cruzain, a T. cruzi cystein protease. The inhibitors cause alterations in the Golgi complex and ER, accumulation of unprocessed enzyme within Golgi cisternae, and decrease of mature cruzain within lysosomes. The most effective compound, N-Pip-F-hF-VS phi, cured an acute lethal infection in experimental animals. Myocardial lesions, lymphocyte infiltration and intracellular amastigote clusers were absent in treated animals. Preliminary toxicology and pharmacokinetic analyses suggest the lack of toxicity associated to high doses and prolonged treatment regimes. Protease inhibitors may soon become good chemotherapeutic alternatives for acute and chronic Chagas' disease.