Lucio H. Freitas-Junior

The mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles.

Trypanosoma cruzi expresses at its surface large amounts of mucin-like glycoproteins. The T. cruzi mucins (TcMUC), a group of highly glycosylated GPI-anchored proteins rich in Thr, Ser, and Pro residues, are expressed in high copy numbers in both insect and mammalian stages of the parasite. These molecules are encoded by a multigene family and contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. The TcMUC are important because of their role in host cell invasion and the ability to induce secretion of proinflammatory cytokines and nitric oxide in activated macrophages. The TcMUC are also significant in being the major substrate for the cell surface trans-sialidase. In this review, we summarize the recent knowledge on the molecular structure and function of this family of T. cruzi glycoproteins.

Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development

Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. In this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors – posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compound- and strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease.

Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcriptional level by labile protein factors.

To adapt to different environments,Trypanosoma cruzi, the protozoan parasite that causes Chagas’ disease, expresses a different set of proteins during development. To begin to understand the mechanism that controls this differential gene expression, we have analyzed the levels ofamastin and trans-sialidase mRNAs and the mRNAs encoding members of the 85-kDa glycoprotein gene family, which are differentially expressed in the T. cruzi stages found in the mammalian host. Amastin mRNA is expressed predominantly in intracellular and proliferative amastigotes.trans-Sialidase mRNAs are found mostly in forms undergoing transformation from amastigotes to trypomastigotes inside infected cells, whereas mRNAs encoding the 85-kDa glycoproteins appear only in the infective trypomastigotes released from the cells. The genes coding for these mRNA species are constitutively transcribed in all stages of T. cruzi cells, suggesting that expression is controlled post-transcriptionally during differentiation. Inhibition of transcription by actinomycin D revealed that each mRNA species has a relatively long half-life in stages where it accumulates. In the case of the trans-sialidase and 85-kDa glycoprotein genes, mRNA accumulation was induced by treatment with the protein synthesis inhibitor cycloheximide at the stages that preceded the normal accumulation. Therefore, mRNA stabilization may account for mRNA accumulation. mRNA degradation could be promoted by proteins with high turnover, or stabilization could be promoted by forming a complex with the translational machinery at defined times in development. Identification of the factors that induce mRNA degradation or stabilization is essential to the understanding of control of gene expression in these organisms.

Two distinct groups of mucin-like genes are differentially expressed in the developmental stages of Trypanosoma cruzi

Sialic acid acceptors of Trypanosoma cruzi are abundant mucin-like glycoproteins linked to the parasite membrane by a glycosylphosphatidyl inositol (GPI) anchor. They are heterogeneous and variable in different parasite stages. The protein portion of these mucins contains many threonine residues, and is thought to be encoded by a heterogeneous gene family. To investigate whether the high degree of heterogeneity in the mucin gene family is responsible for the diversity of mucins expressed on the parasite surface, we have studied the expression of mucin genes in several developmental stages of T. cruzi. We have found that mucins are expressed in all parasite stages. By using conserved sequences at 3 end of translated sequences of the gene family and the splice leader sequence, we have isolated 120 mucin-like cDNAs by RT-PCR from epimastigote and trypomastigote mRNAs. All transcribed genes contain conserved 5 and 3 regions, which code for the signal peptide, the sequence for GPI anchor addition, and a conserved domain rich in threonine residues. The internal portions of these genes are highly variable in size and sequence, and can be grouped in two major categories. One group contains KP(1-2)T(6-8) repeats, a motif found in mammalian mucins in the central region. This group is expressed preferentially in the trypomastigote forms ready to be released from the infected mammalian cell. The other has highly variable sequences in the central portion, and is expressed in all parasite stages. Because the number of synonymous substitutions is equivalent to the non-synonymous substitutions in the second group, they are probably evolving neutrally. On the other hand, the KP(1-2)T(6-8) containing genes have more synonymous substitutions and are most likely under a strong selective pressure. We propose that the group of KP(1-2)T(6-8) motif corresponds to the highly glycosylated mucins of the trypomastigote stages. In the other group proteolysis may remove the central domain yielding small mucins, such as the mucins found in insect derived stages of T. cruzi.

Design, synthesis and antitrypanosomal activity of some nitrofurazone 1,2,4-triazolic bioisosteric analogues

Chagas disease, caused by Trypanosoma cruzi, is a parasitosis that predominates in Latin America. It is estimated that 25 million people are under the risk of infection and, in 2008, more than 10 thousand deaths were registered. The only two drugs available in the therapeutics, nifurtimox and benznidazole, showed to be more effective in the acute phase of the disease. However, there is no standard treatment protocol effective for the chronic phase. Nitrofurazone (NF), an antimicrobial drug, has activity against T.xa0cruzi, although being toxic. Considering the need for new antichagasic drugs, the existence of promising new therapeutic targets, as 14α-sterol demethylase and cruzain, and employing the bioisosterism and molecular hybridization approaches, four novel compounds were synthesized, characterized by melting point range, elemental analysis, IR and NMR spectroscopy. The compounds were tested against T.xa0cruzi amastigotes in infected U2OS cells. All compounds showed selectivity towards T.xa0cruzi and showed trypanomicidal activity in low micromolar range. The compound 3 showed potency similar to benznidazole, but lower efficacy. These results highlight the importance of the 1,2,4-triazole, thiosemicarbazonic and nitro group moieties for designing new efficient compounds, potentially for the chronic phase of Chagas disease.

Highly improved antiparasitic activity after introduction of an N-benzylimidazole moiety on protein farnesyltransferase inhibitors.

In our search for new protein farnesyltransferase inhibitors with improved antiparasitic activities, we modified our previously developed 3-arylthiophene series of inhibitors by replacing the thioisopropyl group by different substituted imidazolylmethanamino moieties. Twenty four new derivatives were synthesized and evaluated against human and parasite farnesyltransferases, and their anti-parasitic activity was determined against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani. Introduction of a N-p-substituted-benzylimidazole led to significantly increase the inhibition of parasite proliferation in the submicromolar range. The structure of the best inhibitors was parasite dependent. Three compounds possess IC50 values at the same range as the reference miltefosine against L. donovani proliferation and other new derivatives display high level of anti-trypanosomal activity against T.xa0cruzi, higher or in the same order of magnitude as the reference compounds benznidazole and nifurtimox.

Synthesis and trypanocidal activity of a library of 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols.

A library of 16 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols 17-32 has been synthesized for use in biological testing against Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. The 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols 17-32 were subjected to biological testing to evaluate their efficacy against intracellular Trypanosoma cruzi (Y strain) amastigotes infecting U2OS human cells, with benznidazole as a reference compound. The assay was performed in duplicate (two independent experiments) and submitted to High Content Analysis (HCA) for determination of trypanocidal activity. Three of the tested compounds presented relatively high trypanocidal activity (19, 22 and 29), however severe host cell toxicity was observed concomitantly. Chemical optimization of the highly active compounds and the synthesis of more compounds for biological testing against Trypanosoma cruzi will be required to improve selectivity and so that a structure-activity relationship can be generated to provide a more insightful analysis of both chemical and biological aspects.

A comparative study of warheads for design of cysteine protease inhibitors

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.