Transcriptomics-Guided In Silico Drug Repurposing: Identifying New Candidates with Dual-Stage Antiplasmodial Activity.

In tropical and subtropical areas, malaria stands as a profound public health challenge, causing an estimated 247 million cases worldwide annually. Given the absence of a viable vaccine, the timely and effective treatment of malaria remains a critical priority. However, the growing resistance of parasites to currently utilized drugs underscores the critical need for the identification of new antimalarial therapies. Here, we aimed to identify potential new drug candidates against Plasmodium falciparum, the main causative agent of malaria, by analyzing the transcriptomes of different life stages of the parasite and identifying highly expressed genes. We searched for genes that were expressed in all stages of the parasite's life cycle, including the asexual blood stage, gametocyte stage, liver stage, and sexual stages in the insect vector, using transcriptomics data from publicly available databases. From this analysis, we found 674 overlapping genes, including 409 essential ones. By searching through drug target databases, we discovered 70 potential drug targets and 75 associated bioactive compounds. We sought to expand this analysis to similar compounds to known drugs. So, we found a list of 1557 similar compounds, which we predicted as actives and inactives using previously developed machine learning models against five life stages of Plasmodium spp. From this analysis, two compounds were selected, and the reactions were experimentally evaluated. The compounds HSP-990 and silvestrol aglycone showed potent inhibitory activity at nanomolar concentrations against the P. falciparum 3D7 strain asexual blood stage. Moreover, silvestrol aglycone exhibited low cytotoxicity in mammalian cells, transmission-blocking potential, and inhibitory activity comparable to those of established antimalarials. These findings warrant further investigation of silvestrol aglycone as a potential dual-acting antimalarial and transmission-blocking candidate for malaria control.

Integrative taxonomy and phylogenetic systematics of the Triatoma rubrovaria subcomplex (Hemiptera, Triatominae).

Triatoma rubrovaria subcomplex consists of T. carcavalloi, T. circummaculata, T. klugi, T. limai, T. oliveirai, T. pintodiasi, T. rubrovaria, T. patagonica and T. guasayana, which can be vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease. In this study, morphological, morphometric, and genetic characters of T. circummaculata, T. pintodiasi, T. carcavalloi, T. klugi, and T. rubrovaria were analyzed in view of the integrative taxonomy and phylogeny of the T. rubrovaria subcomplex. Molecular studies were carried out through the sequencing and analysis of the mitochondrial genes COI and CytB, nuclear genes ITS I, ITS 2, 16S, and 28S from rDNA and rescued a monophyletic group. Furthermore, differential morphological characters were found among the five species in the pronotum, scutellum, stridulatory sulcus, male genitalia, and external female genitalia. Finally, morphometric analyses made it possible to differentiate the five species. Phylogenetic analyzes rescued the relationship of T. pintodiasi with members of the T. rubrovaria subcomplex and demonstrated that this subcomplex is a monophyletic group composed of the species T. carcavalloi, T. circummaculata, T. klugi, T. guasayana, T. limai, T. oliveirai, T. patagonica, T. pintodiasi, and T. rubrovaria. Furthermore, through integrative taxonomy, it was possible to confirm the specific status of the species T. carcavalloi, T. circummaculata, T. pintodiasi, T. klugi, and T. rubrovaria, offering new useful morphological characters for the differentiation and characterization of these potential vectors and distributed in Southern Brazil.

Update and elucidation of Plasmodium kinomes: Prioritization of kinases as potential drug targets for malaria.

Malaria is a tropical disease caused by Plasmodium spp. and transmitted by the bite of infected Anopheles mosquitoes. Protein kinases (PKs) play key roles in the life cycle of the etiological agent of malaria, turning these proteins attractive targets for antimalarial drug discovery campaigns. As part of an effort to understand parasite signaling functions, we report the results of a bioinformatics pipeline analysis of PKs of eight Plasmodium species. To date, no P. malariae and P. ovale kinome assemble has been conducted. We classified, curated and annotated predicted kinases to update P. falciparum, P. vivax, P. yoelii, P. berghei, P. chabaudi, and P. knowlesi kinomes published to date, as well as report for the first time the kinomes of P. malariae and P. ovale. Overall, from 76 to 97 PKs were identified among all Plasmodium spp. kinomes. Most of the kinases were assigned to seven of nine major kinase groups: AGC, CAMK, CMGC, CK1, STE, TKL, OTHER; and the Plasmodium-specific group FIKK. About 30% of kinases have been deeply classified into group, family and subfamily levels and only about 10% remained unclassified. Furthermore, updating and comparing the kinomes of P. vivax and P. falciparum allowed for the prioritization and selection of kinases as potential drug targets that could be explored for discovering new drugs against malaria. This integrated approach resulted in the selection of 37 protein kinases as potential targets and the identification of investigational compounds with moderate in vitro activity against asexual P. falciparum (3D7 and Dd2 strains) stages that could serve as starting points for the search of potent antimalarial leads in the future.

QSAR-Based Virtual Screening of Natural Products Database for Identification of Potent Antimalarial Hits.

With about 400,000 annual deaths worldwide, malaria remains a public health burden in tropical and subtropical areas, especially in low-income countries. Selection of drug-resistant Plasmodium strains has driven the need to explore novel antimalarial compounds with diverse modes of action. In this context, biodiversity has been widely exploited as a resourceful channel of biologically active compounds, as exemplified by antimalarial drugs such as quinine and artemisinin, derived from natural products. Thus, combining a natural product library and quantitative structure-activity relationship (QSAR)-based virtual screening, we have prioritized genuine and derivative natural compounds with potential antimalarial activity prior to in vitro testing. Experimental validation against cultured chloroquine-sensitive and multi-drug-resistant P. falciparum strains confirmed the potent and selective activity of two sesquiterpene lactones (LDT-597 and LDT-598) identified in silico. Quantitative structure-property relationship (QSPR) models predicted absorption, distribution, metabolism, and excretion (ADME) and physiologically based pharmacokinetic (PBPK) parameters for the most promising compound, showing that it presents good physiologically based pharmacokinetic properties both in rats and humans. Altogether, the in vitro parasite growth inhibition results obtained from in silico screened compounds encourage the use of virtual screening campaigns for identification of promising natural compound-based antimalarial molecules.

The reservoir system for Trypanosoma (Kinetoplastida, Trypanosomatidae) species in large neotropical wetland.

Distinct species of Trypanosoma have been documented sharing the same hosts in different environments in intricate transmission networks. Knowing this, this study investigated the role of different hosts in the transmission cycles of Trypanosoma species in the Pantanal biome. The mammals were sampled from November 2015 to October 2016. We sampled a total of 272 wild mammals from 27 species belonging to six orders and 15 families, and three species of triatomines (n = 7). We found high parasitemias by Hemoculture test for Trypanosoma cruzi (TcI), Trypanosoma rangeli, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, and high parasitemias by Microhematocrit Centrifuge Technique for Trypanosoma evansi. The carnivore Nasua nasua is a key host in the transmission cycles since it displayed high parasitemias for T. cruzi, T. evansi and T. rangeli. This is the first report of high parasitemias in Tamandua tetradactyla and cryptic infection in Dasypus novemcinctus by T. cruzi; cryptic infection by T. evansi in Eira barbara, Euphractus sexcinctus and Dasyprocta azarae. The collection of Panstrongylus geniculatus increased the geographic distribution of this vector species in the South America. Our results indicate that Trypanosoma species circulate in a complex reservoir system including different host species with different infective competences.

Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx) in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments

Abstract Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5 kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58 kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5 kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.

Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx) in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments.

Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.

Triatominae survey (Hemiptera: Reduviidae: Triatominae) in the south-central region of the state of Bahia, Brazil between 2008 and 2013.

Triatomine surveillance in rural areas, artificial ecotypes, and natural ecotopes of the cities of Caturama, Ibipitanga, Macaúbas, and Seabra in the south-central region of the Brazilian state of Bahia was carried out between 2008 and 2013. Natural infection by Trypanosoma cruzi was evaluated in the specimens collected to monitor vectors of Chagas disease. A total of 1,357 specimens were collected, and four species were identified: Triatoma sordida (83%), Triatoma lenti (16.4%), Triatoma pseudomaculata (0.5%), and Panstrongylus geniculatus (0.1%). Triatoma sordida was found in four cities, only 0.7% in intradomiciliary environments. Triatoma lenti was found only in Macaúbas; 8.5% were found in intradomiciliary environments, 88.3% in peridomiciliary environments, and 3.1% in sylvatic environments. Natural infection by T. cruzi was 0.5% for T. sordida and 3.1% T. lenti. All of these cases were found in peridomiciliary environments of Macaúbas. As the results show, triatomines were found in intradomiciliary environments in three cities that were surveyed in the south-central region of the state of Bahia. Thus, an epidemiologic survey should be performed to avoid the risk of transmission to the population.

Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Chagas disease is one of the main public health problems in Latin America. Since the available treatments for this disease are not effective in providing cure, the screening of potential antiprotozoal agents is essential, mainly of those obtained from natural sources. This study aimed to provide an evaluation of the trypanocidal activity of 92 ethanol extracts from species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae against the Y and Bolivia strains of Trypanosoma cruzi. Additionally, cytotoxic activity on LLCMK2 fibroblasts was evaluated. Both the trypanocidal activity and cytotoxicity were evaluated using the MTT method, in the following concentrations: 500, 350, 250, and 100 µg/mL. Benznidazole was used for positive control. The best results among the 92 samples evaluated were obtained with ethanol extracts of Ocotea paranapiacabensis (Am93) and Aegiphila lhotzkiana (Am160). Am93 showed trypanocidal activity against epimastigote forms of the Bolivia strain and was moderately toxic to LLCMK2 cells, its Selectivity Index (SI) being 14.56, while Am160 showed moderate trypanocidal activity against the Bolivia strain and moderate toxicicity, its SI being equal to 1.15. The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from Ocotea paranapiacabensis and Aegiphila lhotzkiana against Chagas disease.

Morphological, biological and molecular characterization of three strains of Trypanosoma cruzi Chagas, 1909 (Kinetoplastida, Trypanosomatidae) isolated from Triatoma sordida (Stal) 1859 (Hemiptera, Reduviidae) and a domestic cat.

A study was conducted of the biological, morphological and molecular characters of 3 strains of Trypanosoma cruzi (SI(5), SI(8) and SIGR(3)) isolated from specimens of Triatoma sordida collected in Santo Inácio and a domestic cat. In order to carry out the study, the following parameters were evaluated: pre-patent period, parasitaemia curves, morphology of the parasites, mortality rates, histopathological lesions and molecular typing. The strains presented variable pre-patent periods, low parasitaemia and no animal mortality. The morphological study of trypomastigotes showed a predominance of intermediate-width and short-length forms, as well as low nuclear index. Epimastigotes presented a low nuclear index, intermediate-width forms in strains SI(5) and SI(8), and large-width forms in SIGR(3). A shorter length could be noted in strains SI(8) and SIGR3, whereas SI(5) displayed an intermediate length. The histopathological study did not detect amastigote nests in tissues. The amplification of the divergent domain of 24Sα rRNA, HSP60 and GPI genes of strains SI(5), SI(8) and SIGR(3) classified the 3 strains into Group II. Biological parameters made it possible to classify the strains isolated in Santo Inácio (BA) into Biodeme III, Zymodeme 1 and Group II of T. cruzi.