Chagas disease treatment: From new therapeutic targets to drug discovery and repositioning

Chagas disease, caused by the protozoan Trypanosoma cruzi, currently affects millions of people worldwide although it is only endemic in America. Chagas is considered a neglected tropical disease because it afflicts the low-income and poorest populations in developing regions of the Americas, particularly in remote, rural areas where infrastructure such as adequate housing, sanitation and clinical resources are limited. In addition, governments pay scarce or no attention to this health problem.Benznidazole and nifurtimox, both discovered more than 50 years ago, are the only two drugs available to treat Chagas disease and not only present severe side effects but also are ineffective in the chronic phase of the disease when most of the patients are diagnosed. Recent efforts to develop new treatments for Chagas disease, including posaconazole repositioning and the prodrug of ravuconazole (E1224) trial, have been unsuccessful and remark the urgent need to develop new therapeutic alternatives. The scientific effort must be focused in finding simple, safe and effective drugs that directly target the parasite without harming the patients.It is known that the identification of reliable molecular targets for drug development is challenging, with high rates of failure at the stage of validating potential candidates. Thus, we propose in this Special Issue to revise different potential drug targets of the parasite T. cruzi and to explore the latest advances in drug design and drug repositioning.The first review introduces the challenges of Chagas disease and evidenced the requirement of new therapies to treat it. Egui et al. [1] focus on the immunologic profile associated with the clinical status and evolution of Chagas disease patients as wells as the effectiveness of the current treatment including different biomarkers to monitor the T. cruzi infection. In the next revisions, different metabolic pathways and proteins are explored as therapeutic targets against the parasite T. cruzi, and the final two reviews include the latest strategies to identify new potential targets.Alonso et al. [2] explore the bromodomain-containing proteins as potential targets in protozoa since some of these proteins are essential for viability and diverge from the mammalian ones, which are also approach in their review. Cordeiro [3] describes the importance of the NADPH producing enzymes in biosynthetic processes as well as in the neutralization of reactive oxygen and nitrogen species. He propose them as potential targets, highlighting the role of glucose-6-phosphate dehydrogenase and the cytosolic malic enzyme.Schoijet et al. [4] propose the signal transduction pathway in trypanosomatids as a novel therapeutic target, particularly the cAMP signaling pathway. The authors shed light in phosphodiesterases (PDEs) as druggable target, because of the prominent roles they play in the life cycle and the essentiality for parasite survival. Despite they are highly conserved enzymes, authors highlight the potentiality of differential inhibition from their human orthologs and suggest the drug repositioning approach as a promising strategy to find inhibitors among the numerous drugs against human PDEs that are available in the market.Sangenito et al. [5] introduce the aspartyl peptidase inhibitors used to treat the infection with the human immunodeficiency virus (HIV) as a drug repositioning strategy. Co-infection of patients with HIV with other microorganisms, such as protozoan parasites, is common and the use of HIV peptidase inhibitors evidenced a decrease both in prevalence and incidence of these co-infections. Indeed, several of these inhibitors have been tested in T. cruzi showing multiple pathophysiological effects on the parasite.Talevi et al. [6] present the thiol-polyamine metabolism, a well-known and validated T. cruzi target because many of its components are absent or significantly differ from the host homologs offering interesting candidates for a rational design of selective drugs. In this review, the authors critically revise the state of the art of the thiol-polyamine metabolism deepening in the pharmacological potential of its components and, properly introducing to the different computer-aided approximations to assist systematic drug repositioning strategies. Sayé et al. [7], propose the most represented family of amino acid and derivative transporters in T. cruzi as drug targets, focalizing in proline and polyamine permeases. This family is absent in the human host and their members are responsible of the acquisition of relevant nutrients for the survival of the parasite. The review also discusses the latest advances in drug repositioning strategy applied to these transporters. Saavedra et al. [8] explore new fields in drug target development and open new interrogations about target prioritization. The authors analyse the fundamentals of Metabolic Control Analysis and kinetic modelling of metabolic pathways and apply them to the trypanothion metabolism of T. cruzi. They conclude that the enzymes with the highest pathway control are the most convenient targets for therapeutic intervention, leaving under discussion if the agreed criterion of gene essentiality is enough to guarantee a valid target. Last, Salas-Sarduy et al. [9] introduce the initiative made by public-private programs for drug discovery against Chagas disease. This strategy consists in taking advantage of the resources invested by the pharmaceutical industry in other commercial areas allowing the evaluation of libraries of millions of low-molecular weight synthetic compounds. These strategies require high-throughput screenings and setting up of robust enzymatic assays to identify and validate small molecule inhibitors, a matter as well addressed by the authors.Recognizing that Chagas disease requires urgent attention is the first step to develop new alternative treatments with less toxic effects, and this Special Issue is responding directly to this need.Fil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Repurposing of terconazole as an anti Trypanosoma cruzi agent

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. Currently there are no effective treatments for the chronic phase of the disease, when most patients are diagnosed, therefore the development of new drugs is a priority area. Several triazoles, used as fungicides, exhibit trypanocidal activity both in vitro and in vivo. The mechanism of action of such drugs, both in fungi and in T. cruzi, relies in the inhibition of ergosterol biosynthesis affecting the cell viability and growth. Among them, terconazole was the first triazole antifungal drug for human use. In this work, the trypanocidal activity of terconazole was evaluated using in vitro assays. In epimastigotes of two parasites strains from different discrete typing units (Y and Dm28c) the calculated IC50 were 25.7 μM and 21.9 μM, respectively. In trypomastigotes and amastigotes (the clinically relevant life-stages of T. cruzi) a higher drug susceptibility was observed with IC50 values of 4.6 μM and 5.9 μM, respectively. Finally, the molecular docking simulations suggest that terconazole inhibits the T. cruzi cytochrome P450 14-α-demethylase, interacting in a similar way that other triazole drugs. Drug repurposing to Chagas disease treatment is one of the recommended approach according to the criterion of international health organizations for their application in neglected diseases.Fil: Reigada, Chantal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Identification of Trypanosoma cruzi Polyamine Transport Inhibitors by Computational Drug Repurposing

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. In T. cruzi the transport of polyamines is essential because this organism is unable to synthesize these compounds de novo. Therefore, the uptake of polyamines from the extracellular medium is critical for survival of the parasite. The anthracene-putrescine conjugate Ant4 was first designed as a polyamine transport probe in cancer cells. Ant4 was also found to inhibit the polyamine transport system and produced a strong trypanocidal effect in T. cruzi. Considering that Ant4 is not currently approved by the FDA, in this work we performed computer simulations to find trypanocidal drugs approved for use in humans that have structures and activities similar to Ant4. Through a similarity ligand-based virtual screening using Ant4 as reference molecule, four possible inhibitors of polyamine transport were found. Three of them, promazine, chlorpromazine and clomipramine, showed to be effective inhibitors of putrescine uptake, and also revealed a trypanocidal activity in epimastigotes (IC50 values of 69.0, 50.8 and 49.4 μM, respectively) and trypomastigotes (IC50 values of 3.5, 2.8 and 1.4 μM, respectively). Finally, molecular docking simulations suggest that the interactions between the T. cruzi polyamine transporter TcPAT12 and all the identified inhibitors occur in the same region of the protein. However, this location is different from the site occupied by the natural substrates. The value of this effort is that repurposing known drugs in the treatment of other pathologies, especially neglected diseases such as Chagas disease, significantly decreases the time and economic cost of implementation.Fil: Reigada, Chantal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Phanstiel, Otto. University Of Central Florida; Estados UnidosFil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico

The amino acid proline has special relevance for the parasite Trypanosoma cruzi,\nthe etiological agent of Chagas disease. This amino acid can be used as an energy and\ncarbon source alternative to glucose, it participates in different stress resistance\nmechanisms, and it is also essential during the cell invasion of metacyclic\ntrypomastigotes and for the life cycle progression inside the mammalian cells.\nIn this work the TcAAAP069 gene of T. cruzi was identified as a proline permease,\nfirst through yeast complementation and then by overexpression in epimastigotes\n(Tc069 parasites). Although the transporter TcAAAP069 proved to be mono-specific, the\nstereoisomer D-proline was able to significantly inhibit L-proline uptake, suggesting the\nexistence of D-amino acid transport processes in the parasite.\nUsing an in silico approach, the TcAAAP733 gene was identified as another\nputative proline permease. Its heterologous expression in defective yeasts for the\nproline transporter showed that despite it is not a permease of this amino acid, it\nencodes a functional permease since significant differences when compared to controls\nwere observed when the uptake of an amino acid mixture was assessed.\nThe TcAAAP069 permease overexpression caused an augment not only on\nproline intracellular concentration but also on ATP levels. When the parasites were\nchallenged against oxidative and nitrosative stress situations, or when were treated with\nthe trypanocidal drugs currently used, nifurtimox and benznidazole, it was evidenced\nthat proline itself participates on the response mechanisms to these conditions since\nTc069 parasites were significantly more resistant than control parasites in all the assays.\nThe study of proline transport throughout the culture progress, as an emulating\nsituation of the different nutritional conditions along the parasites life cycle, proved that\nthe permease TcAAAP069 is regulated both in its activity and expression as in its\nsubcellular localization and this produces variations on proline intracellular\nconcentration too. The maximal activity and expression of TcAAAP069 transporter was observed at the beginning of the exponential growth phase, together with the\nlocalization along the plasmatic membrane besides its presence in the flagellar pocket,\nbeing the latter location a common feature to all the members of the TcAAAP family so\nfar characterized. As the culture proceeded, expression and activity of the transporter\ngradually diminished until undetectable levels were reached on the stationary phase.\nThese decreases were accompanied by the loss of the plasma membrane subcellular\nlocalization and finally by the disappearance of the protein, even in the flagellar pocket.\nIn addition, the proline transport was not regulated by substrate availability. Finally, the\nassays involving medium modification or artificially altered cellular density suggest that\nthe observed changes for the proline permease would be directly influenced by an\nunknown density-dependent factor.\nTaking together the biological relevance of proline for the parasite T. cruzi and\nour advances on the knowledge of its transport, synthetic analogues of this amino acid\nwere evaluated in order to study their effect on the permease TcAAAP069 activity. Of\nthe four analogues tested, only the compounds named ITP-1B and ITP-1G significantly\ninhibited the proline transport and also of other amino acid and/or polyamines, but not\nthe uptake of other metabolites. However, exclusively with the analogue ITP-1G the\nability to inhibit the transport was linked with a trypanocidal action. The study with the\nproline analogues confirmed the amino acid and derivatives transporter family TcAAAP\nas a multiple and promising therapeutic target for the development of new treatments\nagainst Chagas disease.Fil: Martínez Sayé, Melisa Soledad. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, ArgentinaLa prolina tiene especial relevancia para el parásito Trypanosoma cruzi, agente\ncausal de la enfermedad de Chagas. Este aminoácido puede ser utilizado como fuente\nde carbono y energía alternativa a la glucosa, participa en mecanismos de resistencia a\ndistintos estreses, y es necesario durante la invasión celular de tripomastigotes\nmetacíclicos y para progresar en el ciclo de vida dentro de las células de mamífero.\nEn este trabajo se identificó el gen TcAAAP069 de T. cruzi como una permeasa\nde prolina, primero por complementación en levaduras y luego por sobre-expresión en\nepimastigotes (parásitos Tc069). Si bien la permeasa demostró ser mono-específica el\nestereoisómero D-prolina fue capaz de inhibir significativamente la incorporación de Lprolina,\nsugiriendo la presencia de procesos de transporte de D-aminoácidos en el\nparásito.\nMediante un análisis bioinformático se identificó el gen TcAAAP733 como otra\nposible permeasa de prolina. Su expresión heteróloga en levaduras deficientes para el\ntransportador de prolina demostró que si bien no es una permeasa de este aminoácido,\ncodifica una permeasa funcional ya que se observaron diferencias significativas respecto\nal control cuando se evaluó el transporte de una mezcla de aminoácidos.\nLa sobre-expresión del transportador causó un aumento, no sólo en la\nconcentración intracelular de prolina, sino también en los niveles de ATP. Al enfrentar\nestos parásitos ante situaciones de estrés oxidativo, nitrosativo o con las drogas\ntripanocidas actualmente disponibles, nifurtimox y benznidazol, se evidenció que la\nprolina participa en los mecanismos de respuesta a estas condiciones ya que los\nparásitos Tc069 fueron significativamente más resistentes en todos los casos.\nEl estudio del transporte de prolina durante el progreso del cultivo, como\nsituación que emula las distintas condiciones nutricionales de los parásitos en su ciclo\nde vida, demostró que la permeasa TcAAAP069 está regulada tanto en su actividad y\nexpresión como en su localización subcelular, y esto produce además variaciones en la\nconcentración intracelular de prolina. La máxima actividad y expresión del\ntransportador TcAAAP069 se observó al inicio de la fase exponencial, coincidiendo con la localización de la permeasa a lo largo de la membrana plasmática además de su\npresencia en el bolsillo flagelar, siendo esta última localización una característica común\na todos los miembros de la familia TcAAAP caracterizados hasta el momento. A medida\nque el cultivo progresó, la expresión y la actividad del transportador disminuyeron\ngradualmente hasta alcanzar niveles indetectables en la fase estacionaria. Estas\ndisminuciones fueron acompañadas por la pérdida de la localización subcelular en la\nmembrana y finalmente por la desaparición de la proteína, incluso en el bolsillo flagelar.\nSe observó además que el transporte de prolina no es regulado por la disponibilidad de\nsustrato. Por último, los estudios realizados modificando el medio o alterando\nartificialmente la densidad celular del cultivo sugieren que los cambios observados para\nla permeasa de prolina TcAAAP069 estarían relacionados directamente con un factor\ndensidad-dependiente de naturaleza aún desconocida.\nTeniendo en cuenta la importancia biológica de la prolina en el parásito T. cruzi\ny nuestros avances en el conocimiento de su transporte, se utilizaron análogos sintéticos\ndel aminoácido a fin de estudiar su efecto sobre la actividad de la permeasa TcAAAP069.\nDe los cuatro análogos evaluados, sólo los compuestos denominados ITP-1B e ITP-1G\ninhibieron el transporte de prolina y también de otros aminoácidos y/o poliaminas pero\nno de otros metabolitos. Sin embargo, únicamente con el análogo ITP-1G la capacidad\nde inhibir el transporte estuvo vinculada con una acción tripanocida. El estudio con los\nanálogos de prolina permitió confirmar que la familia de transportadores de\naminoácidos y derivados TcAAAP es un blanco terapéutico múltiple y prometedor para\nel desarrollo de nuevos tratamientos contra la enfermedad de Chagas