Vilma G. Duschak

An insight on targets and patented drugs for chemotherapy of Chagas disease.

Chagas disease or American Trypanosomiasis, a parasitic infection typically spread by triatomine bugs, affects millions of people throughout Latin America. Current chemotherapy based on the nitroaromatic compounds, benznidazole and nifurtimox provides unsatisfactory results and suffers from considerable side effects and low efficacy. Therefore, there is an urgent need for new drugs to treat this neglected disease. Over the last two decades, new advances and understanding in the biology and the biochemistry of Trypanosoma cruzi has allowed the identification of multiple targets for Chagas disease chemotherapy. This review summarizes antichagasic agents obtained based on i) target metabolic biochemical pathways or parasite specific enzymes, ii) natural products and its derivatives, iii) design and synthesis of lead compounds. Related patents filed and issued from 2000 to early 2006 are also discussed. Most of them claimed inhibitors on specific parasite targets such as cysteine proteinase, sterol biosynthesis, protein farnesyltransferase, etc. Particularly, those related to cysteine proteinase inhibitors were the most represented. Natural products also displayed many anti-T cruzi lead compounds. In addition, a few patents claiming natural or synthetic compounds with antichagasic activity, disclosed no specific target. However, only a small proportion of all these patents displayed specific data of biological trypanocidal activity.

Cruzipain, the Major Cysteine Protease of Trypanosoma cruzi: A Sulfated Glycoprotein Antigen as Relevant Candidate for Vaccine Development and Drug Target. A Review

This review aims to present different aspects related to cruzipain, one of the most important proteins of the etiological agent of Chagas disease that has been extensively studied in the last two decades, including all the particularities of the molecule as well as to highlight its participation in multiple relevant functions of the parasite to favour the cell invasion phenomena, to facilitate host tissues proteolytic degradation and to trigger the evasion mechanism from host immune response. Cruzipain has been related with parasite metabolism and identified as both an important candidate for vaccine development and for trypanocidal drug design. We have reported for the first time that this enzyme is a sulfated glycoprotein. Indeed, the sulfated oligosaccharides are main targets for immune responses and are involved in tissue damage in mice immunized in absence of infection contributing to get deeper into the knowledge of the molecule composition and helping to elucidate its role in the infection and/or pathogenesis of the disease. A whole view including all the aspects related to the major cysteine proteinase of Trypanosoma cruzi studied so far including recent advances as proteinase, antigen and glycoprotein will be discussed.

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi Identification of sulfated high-mannose type oligosaccharides

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C‐terminal extension that contains a number of post‐translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV‐MALDI‐TOF mass spectrometry in conjunction with peptide N‐glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N‐linked oligosaccharides present in this domain. The UV‐MALDI‐TOF MS analysis in the negative‐ion mode, using nor‐harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high‐mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV‐MALDI‐TOF MS analysis (a) a main population of high‐mannose type oligosaccharides was shown in the positive‐ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.

Subcellular localization and post-secretory targeting of TgPI, a serine proteinase inhibitor from Toxoplasma gondii.

Viviana Pszenny , Bibiana E. Ledesma , Mariana Matrajt , Vilma G. Duschak , Esteban J. Bontempi , Jean-Francois Dubremetz , Sergio O. Angel * a Departamento de Parasitologia, ANLIS Dr. Carlos G. Malbran, Av. Velez Sarsfield 563, 1281, Ciudad de Buenos Aires, Argentina b Department of Biology, University of Pennsylvania, Philadelphia, USA c Instituto de Investigaciones Biotecnologicas, UNSAM, Provincia de Buenos Aires, Argentina d UMR5539 CNRS, Universite de Montpellier II, Montpellier, France

Sulfates are main targets of immune responses to cruzipain and are involved in heart damage in BALB/c immunized mice

Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz), with an unusual carboxyl-terminal extension (C-T). We have previously reported the presence of sulfate groups in the N-linked oligosaccharide chains of this domain. In order to evaluate the immune responses to sulfated moieties on Cz, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. Interestingly, the abolishment of IgG2b reactivity when desulfated antigens were used as immunogens demonstrates that esterified sulfate groups are absolutely required for eliciting IgG2b response to Cz. Sera from chronically T. cruzi-infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T in the absence of infection elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not present pathological alterations. This is the first report showing that sulfate-bearing glycoproteins from trypanosomatids are able to elicit specific humoral and cellular immune responses and appeared to be involved in the generation of heart tissue damage. These results represent a further step in the understanding of the role of Cz in the course of T. cruzi infection.

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human α2-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on hosts molecules and hypothetically regulate parasite functions controlled by cruzipain.

A striking common O-linked N-acetylglucosaminyl moiety between cruzipain and myosin

Single units of O‐linked N‐acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C‐terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self‐antigens like myosin are associated with Chagas heart disease. The participation of O‐GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross‐reactivity between Cz and myosin is mainly focused in the C‐T domain. ELISA inhibition assays using rabbit sera specific for Cz and C‐T in conjunction with immune‐gold electron microscopy analysis of heart tissues from mice immunized with C‐T confronted with polyclonal rabbit sera specific for Cz and C‐T prior and after myosin adsorption provided evidence which indicates that O‐GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O‐GlcNAc‐containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease.

UV-MALDI mass spectrometry analysis of NBD-glycosphingolipids without an external matrix

Each day, advances in the instrumentation and operating protocols bring new applications and insights into the molecular processes of ultra violet-matrix assisted laser desorption/ionization-mass spectrometry (UV-MALDI MS), increasing its potential use. We report here an approach in which mass spectrometry analysis of sphingolipids has been performed using a fluorescent tag (nitrobenz-2-oxa-1, 3-diazole, NBD) covalently linked to the sphingoid base as matrix. Thus, different labeled-sphingolipids were analyzed: ceramide, dihydroceramide, acetylceramide, glucosylceramide, galactosylceramide, galactosyldihydroceramide. In addition an extract of glycosphingolipids obtained from epimastigote forms of Trypanosoma cruzi metabolically labeled with NBD-ceramide was analyzed. The goal of this work is to show that no matrix needs to be added for the mass spectrometry analysis as the same tag used to label the lipids may generate efficiently analyte ions to obtain high quality signals.

An anionic synthetic sugar containing 6-SO3 -NAcGlc mimics the sulfated cruzipain epitope that plays a central role in immune recognition.

Cruzipain (Cz), the major cysteine proteinase of Trypanosoma cruzi, is a glycoprotein that contains sulfated high‐mannose‐type oligosaccharides. We have previously determined that these sulfate groups are targets of specific immune responses. In order to evaluate the structural requirements for antibody recognition of Cz, a systematic structure–activity study of the chemical characteristics needed for antibody binding to the Cz sulfated epitope was performed by immunoassays. With this aim, different synthesized molecules were coupled to the proteins BSA and aprotinin and confronted with (a) mouse sera specific for Cz and its carboxy‐terminal (C‐T) domain, (b) antibodies raised in rabbits immunized with Cz and its C‐terminal domain and (c) IgGs purified from human Chagas disease sera. Our results indicate that a glucosamine containing an esterifying sulfate group in position O‐6 and an N‐acetyl group was the preferred epitope for the immune recognition of sera specific for Cz and its C‐T domain. Although to a minor extent, other anionic compounds bearing sulfate groups in different positions and number as well as different anionic charged groups including carboxylated or phosphorylated monosaccharides, disaccharides and oligosaccharides were recognized. In conclusion, we found that synthetic anionic sugar conjugates containing N‐acetyl d‐glucosamine‐6‐sulfate sodium salt (GlcNAc6S) competitively inhibit the binding of affinity purified rabbit anti‐C‐T IgG to the C‐T extension of Cz. Extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N‐glycan‐linked sulfated epitope displayed in the C‐T domain of Cz.