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Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

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Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

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Trypanosomatids are an important group of parasites that predominate in tropical and subtropical areas of the planet, which cause diseases that are classified as forgotten and neglected by the world health organization. In this group of parasites, we find Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense and Leishmania spp, for which there is no vaccine available, and its control has focused mainly on pharmacological treatment. Due to the poverty situation where these diseases are found and the biological complexity of these parasites, there are multiple variables to control, including the diversity of species, the complexity of their life cycles, drug resistance, cytotoxicity, the limited use in pregnant women, the high costs of treatment and the little-known pharmacological mechanisms of action, among others. It is therefore necessary to find new strategies and approaches for the treatment of these parasitic diseases. Among these new approaches is the rational search for new targets based on the allosteric inhibition of protein kinases, which have been little studied in trypanosomatids. Among these kinases, we find Glycogen Synthase Kinase-3 (GSK-3), a kinase of great pharmacological interest, which is under intense basic and clinical research by pharmaceutical companies for the treatment of cancer. This kinase, highly studied in the PI3K/AKT/mTOR pathway signaling in humans, has an orthologous gene in these parasites (GSK-3 s), which has proven to be essential for them in response to different challenges; Therefore, it is notable to increase research in this kinase in order to achieve a broad structural and functional characterization in the different species of trypanosomatids.

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ABSTRACT Trypanosomatids are an important group of parasites that predominate in tropical and subtropical areas of the planet, which cause diseases that are classified as forgotten and neglected by the world health organization. In this group of parasites, we find Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense and Leishmania spp, for which there is no vaccine available, and its control has focused mainly on pharmacological treatment. Due to the poverty situation where these diseases are found and the biological complexity of these parasites, there are multiple variables to control, including the diversity of species, the complexity of their life cycles, drug resistance, cytotoxicity, the limited use in pregnant women, the high costs of treatment and the little-known pharmacological mechanisms of action, among others. It is therefore necessary to find new strategies and approaches for the treatment of these parasitic diseases. Among these new approaches is the rational search for new targets based on the allosteric inhibition of protein kinases, which have been little studied in trypanosomatids. Among these kinases, we find Glycogen Synthase Kinase-3 (GSK-3), a kinase of great pharmacological interest, which is under intense basic and clinical research by pharmaceutical companies for the treatment of cancer. This kinase, highly studied in the PI3K/AKT/mTOR pathway signaling in humans, has an orthologous gene in these parasites (GSK-3 s), which has proven to be essential for them in response to different challenges; Therefore, it is notable to increase research in this kinase in order to achieve a broad structural and functional characterization in the different species of trypanosomatids.

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As leishmanioses são doenças negligenciadas que afetam mais de um bilhão e meio de pessoas ao redor do mundo, principalmente nos países em desenvolvimento, provocando grandes impactos socioeconômicos. Os fármacos disponíveis para o tratamento dessas doenças são ineficazes e apresentam graves efeitos adversos. O processo de pesquisa de novos fármacos envolve, entre outras coisas, a seleção de alvos bioquímicos essenciais para a sobrevivência e desenvolvimento do agente causador. Neste sentido, a Sirtuína 2, uma enzima epigenética com atividade hidrolase essencial para a sobrevivência dos parasitas do gênero Leishmania se apresenta como um alvo validado na busca de novos fármacos contra essas parasitoses. O planejamento de fármacos baseado na estrutura do receptor requer o conhecimento da estrutura tridimensional da proteína alvo. Desta forma, a elucidação estrutural e um estudo minucioso das Sirtuínas das várias espécies do gênero Leishmania apresenta-se como uma importante abordagem na aplicação desta estratégia na busca por agentes quimioterápicos. Até o momento, na família Trypanosomatidae, a única estrutura tridimensional resolvida experimentalmente de uma enzima Sirtuína 2 é a da espécie L. infantum. Assim, este trabalho aplicou a abordagem de Modelagem Comparativa utilizando o software Modeller na construção de modelos da Sir2rp1 das espécies L. infantum, L. major e L. braziliensis, cujas sequências de aminoácidos foram extraídas do banco de dados UNIProt. Os modelos construídos foram validados por meio da função de escore DOPE do Modeller e dos servidores PROCHECK, MolProbity e QMEAN, avaliando sua qualidade estereoquímica e seu enovelamento. Os ligantes naturais da enzima foram sobrepostos nos modelos construídos por alinhamento estrutural utilizando o software PyMol e os complexos validados foram submetidos a simulações de Dinâmica Molecular através do pacote GROMACS. Os complexos refinados foram então analisados por meio dos softwares PyMol e LigPlotPlus e dos pacotes GROMACS e gmx_MMPBSA, e foram estudados os sítios de ligação dos substratos e os resíduos de aminoácidos relevantes envolvidos em sua ligação e reconhecimento. A Modelagem Comparativa da Sirtuína 2 humana e seus homólogos das espécies L. infantum, L. major e L. braziliensis, as simulações de Dinâmica Molecular realizadas com os modelos enzimáticos construídos e validados complexados com seus ligantes naturais, os cálculos de energia de interação entre os modelos e seus substratos e o estudo estrutural comparativo realizado entre eles nos fornecem uma base teórica para a busca de novos inibidores da Sirtuína 2 que sejam mais seletivos e potentes contra as enzimas parasitárias, abrindo caminho para o desenvolvimento de candidatos a fármacos leishmanicidas mais seguros e eficazes

Leishmaniasis are neglected diseases that affect more than one and a half billion people around the world, mainly in developing countries, causing major socioeconomic impacts. The drugs available for the treatment of these diseases are ineffective and have serious adverse effects. The process of researching new drugs involves, among other things, the selection of biochemical targets essential for the survival and development of the causative agent. In this sense, Sirtuin 2, an epigenetic enzyme with hydrolase activity essential for the survival of parasites of the Leishmania genus, presents itself as a validated target in the search for new drugs against these parasites. Structure-Based Drug Design requires knowledge of the three-dimensional structure of the target protein. In this way, structural elucidation and a detailed study of Sirtuins from various species of the genus Leishmania presents itself as an important approach in the application of this strategy in the search for chemotherapeutic agents. To date, in the Trypanosomatidae family, the only experimentally resolved three-dimensional structure of a Sirtuin 2 enzyme is that of the species L. infantum. Thus, this work applied the Comparative Modeling approach using the Modeller software in the construction of Sir2rp1 models of the species L. infantum, L. major and L. braziliensis, whose amino acid sequences were retrieved from the UNIProt database. The constructed models were validated using Modeller's DOPE score function and the PROCHECK, MolProbity and QMEAN servers, evaluating their stereochemical quality and folding. The enzyme's natural ligands were superimposed on the built models by structural alignment using the PyMol software and the validated complexes were subjected to Molecular Dynamics simulations using the GROMACS package. The refined complexes were then analyzed using the PyMol and LigPlotPlus softwares and the GROMACS and gmx_MMPBSA packages, and the substrate binding sites and relevant amino acid residues involved in their binding and recognition were studied. The Comparative Modeling of human Sirtuin 2 and its homologues from the species L. infantum, L. major and L. braziliensis, the Molecular Dynamics simulations carried out with the constructed and validated enzymatic models complexed with their natural ligands, the interaction energy calculations between the models and their substrates and the comparative structural study carried out between them provide us with a theoretical basis for the search for new Sirtuin 2 inhibitors that are more selective and potent against the parasitic enzymes, paving the way for the development of safer and more effective leishmanicidal drug candidates

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This study focuses on developing accurate immunoassays for diagnosing Chagas disease (CD), a challenging task due to antigenic similarities between Trypanosoma cruzi and other parasites, leading to cross-reactivity. To address this challenge, chimeric recombinant T. cruzi antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) were synthesized to enhance specificity and reduce cross-reactivity in tests. While these antigens showed minimal cross-reactivity with leishmaniasis, their performance with other trypanosomatid infections was unclear. This study aimed to assess the diagnostic potential of these IBMP antigens for detecting CD in patients with Crithidia sp. LVH-60A, a parasite linked to visceral leishmaniasis-like symptoms in Brazil. This study involved seven Crithidia sp. LVH-60A patients and three Leishmania infantum patients. The results indicated that these IBMP antigens displayed 100% sensitivity, with specificity ranging from 87.5% to 100%, and accuracy values between 90% and 100%. No cross-reactivity was observed with Crithidia sp. LVH-60A, and only one L. infantum-positive sample showed limited cross-reactivity with IBMP-8.1. This study suggests that IBMP antigens offer promising diagnostic performance, with minimal cross-reactivity in regions where T. cruzi and other trypanosomatids are prevalent. However, further research with a larger number of Crithidia sp. LVH-60A-positive samples is needed to comprehensively evaluate antigen cross-reactivity.

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Background: Chagas disease and human African trypanosomiasis cause substantial death and morbidity, particularly in low- and middle-income countries, making the need for novel drugs urgent. Methodology & results: Therefore, an explainable multitask pipeline to profile the activity of compounds against three trypanosomes (Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense and Trypanosoma cruzi) were created. These models successfully discovered four new experimental hits (LC-3, LC-4, LC-6 and LC-15). Among them, LC-6 showed promising results, with IC50 values ranging 0.01-0.072 µM and selectivity indices >10,000. Conclusion: These results demonstrate that the multitask protocol offers predictivity and interpretability in the virtual screening of new antitrypanosomal compounds and has the potential to improve hit rates in Chagas and human African trypanosomiasis projects.

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(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O2 consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H2O2 following oxidation of the Amplex® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD+ reduction, and glucokinase activity recording NADP+ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease.

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Acetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2 (AF2)-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MORF4 related gene (MRG) domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes. The results identified a structurally conserved complex, TcTINTIN, which is orthologous to human and yeast trimer independent of NuA4 for transcription interaction (TINTIN) complexes; and another trimeric complex involving an MRG domain, only seen in trypanosomatids. The identification of a key component of TcTINTIN, TcMRGBP, would not have been possible through traditional homology-based methods. We also conducted molecular dynamics simulations, revealing a conformational change that potentially affects its affinity for TcBDF6. The study also revealed a novel way in which an MRG domain participates in simultaneous interactions with two MRG binding proteins binding two different surfaces, a phenomenon not previously reported. Overall, this study demonstrates the potential of using AF2-processed interactomic datasets to identify protein complexes in deeply branched eukaryotes, which can be challenging to study based on sequence similarity. The findings provide new insights into the acetylation signaling pathways in trypanosomatids, specifically highlighting the importance of MRG domain-containing proteins in forming complexes, which may have important implications for understanding the biology of these organisms and developing new therapeutics. On the other hand, our validation of AF2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology.

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Introduction: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., commonly referred to as TriTryps, are a group of protozoan parasites that cause important human diseases affecting millions of people belonging to the most vulnerable populations worldwide. Current treatments have limited efficiencies and can cause serious side effects, so there is an urgent need to develop new control strategies. Presently, the identification and prioritization of appropriate targets can be aided by integrative genomic and computational approaches. Methods: In this work, we conducted a genome-wide multidimensional data integration strategy to prioritize drug targets. We included genomic, transcriptomic, metabolic, and protein structural data sources, to delineate candidate proteins with relevant features for target selection in drug development. Results and Discussion: Our final ranked list includes proteins shared by TriTryps and covers a range of biological functions including essential proteins for parasite survival or growth, oxidative stress-related enzymes, virulence factors, and proteins that are exclusive to these parasites. Our strategy found previously described candidates, which validates our approach as well as new proteins that can be attractive targets to consider during the initial steps of drug discovery.

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ß-D-glucopyranosyloxymethiluracil (Base J) is a modified thymidine base found in kinetoplastids and some related organisms. Interestingly, Base J distribution into the genome can vary depending on the organism and its life stage. Base J is reported to be found mostly at telomeric repeats, on inactive variant surface glycoproteins (VSG's) expression sites (e.g., T. brucei), in RNA polymerase II termination sites and sub-telomeric regions (e.g., Leishmania). This hypermodified nucleotide is synthesized in two steps with the participation of two distinct thymidine hydroxylases, J-binding protein 1 and 2 (JBP1 and JBP2, respectively) and a ß-glucosyl transferase. A third J-binding protein, named JBP3, was recently identified as part of a multimeric complex. Although its structural similarities with JBP1, it seems not to be involved in J biosynthesis but to play roles in gene expression regulation in trypanosomatids. Over the years, with the characterization of JBP1 and JBP2 mutant lines, Base J functions have been targeted and shone a light on that matter, showing genus-specific features. This review aims to explore Base J's reported participation as a regulator of RNA polymerase II transcription termination and to summarize the functional and structural characteristics and similarities of the remarkable JBP proteins in pathogenic trypanosomatids.

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Acetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2 (AF2)-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MORF4 related gene (MRG) domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes. The results identified a structurally conserved complex, TcTINTIN, which is orthologous to human and yeast trimer independent of NuA4 for transcription interaction (TINTIN) complexes; and another trimeric complex involving an MRG domain, only seen in trypanosomatids. The identification of a key component of TcTINTIN, TcMRGBP, would not have been possible through traditional homology-based methods. We also conducted molecular dynamics simulations, revealing a conformational change that potentially affects its affinity for TcBDF6. The study also revealed a novel way in which an MRG domain participates in simultaneous interactions with two MRG binding proteins binding two different surfaces, a phenomenon not previously reported. Overall, this study demonstrates the potential of using AF2-processed interactomic datasets to identify protein complexes in deeply branched eukaryotes, which can be challenging to study based on sequence similarity. The findings provide new insights into the acetylation signaling pathways in trypanosomatids, specifically highlighting the importance of MRG domain-containing proteins in forming complexes, which may have important implications for understanding the biology of these organisms and developing new therapeutics. On the other hand, our validation of AF2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology.

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BACKGROUND: Trypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution. METHODS: We used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses. RESULTS: One hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia. CONCLUSIONS: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.

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The endoplasmic reticulum (ER) of higher eukaryotic cells forms an intricate membranous network that serves as the main processing facility for folding and assembling of secreted and membrane proteins. The ER is a highly dynamic organelle that interacts with other intracellular structures, as well as endosymbiotic pathogenic and non-pathogenic microorganisms. A strict ER quality control (ERQC) must work to ensure that proteins entering the ER are folded and processed correctly. Unfolded or misfolded proteins are usually identified, selected, and addressed to Endoplasmic Reticulum-Associated Degradation (ERAD) complex. Conversely, when there is a large demand for secreted proteins or ER imbalance, the accumulation of unfolded or misfolded proteins activates the Unfold Protein Response (UPR) to restore the ER homeostasis or, in the case of persistent ER stress, induces the cell death. Pathogenic trypanosomatids, such as Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp are the etiological agents of important neglected diseases. These protozoans have a complex life cycle alternating between vertebrate and invertebrate hosts. The ER of trypanosomatids, like those found in higher eukaryotes, is also specialized for secretion, and depends on the ERAD and non-canonical UPR to deal with the ER stress. Here, we reviewed the basic aspects of ER biology, organization, and quality control in trypanosomatids. We also focused on the unusual way by which T. cruzi, T. brucei, and Leishmania spp. respond to ER stress, emphasizing how these parasites' ER-unrevealed roads might be an attractive target for chemotherapy.

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Rodents are one of the most relevant groups of mammals involved in the process of zoonotic disease transmission. Their ability to adapt to anthropized environments allows them to come into contact with humans with often negative consequences for the latter. The present study designed to detect the presence of Trypanosoma cruzi and Leishmania spp. in rodents living in the peri-urban area of Queretaro in central Mexico. This research was carried out during two seasons of collection of wild and domestic rodents, in three localities within the peri-urban area of the state of Queretaro. These collections were carried out during the dry season of February-May 2017 and in the rainy season of August-November 2017. Samples were obtained from the tail tip, from which DNA was purified using the DNeasy Blood & Tissue Kit. End-point PCR was used for the identification of Trypanosoma cruzi and Leishmania spp. A total of 82 rodents were caught, represented in three families, six genera and seven species, of which 29 (35.3%) were positive for Trypanosoma cruzi; 13 (15.8%) for Leishmania spp.; and 12 individuals presented co-infection with both parasites (14.6%). This study confirmed the presence of Trypanosoma cruzi and Leishmania spp. in synanthropic rodents in the peri-urban area of Queretaro, where Chagas and Leishmaniosis diseases are not considered endemic. It is necessary to continue researching for the presence of vectors, as well as for the detection of diseases caused by parasites in humans and thus be able to confirm the transmission cycle of Trypanosoma cruzi and Leishmania spp. in this central Mexican city.

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Trypanosomatids belong to a remarkable group of unicellular, parasitic organisms of the order Kinetoplastida, an early diverging branch of the phylogenetic tree of eukaryotes, exhibiting intriguing biological characteristics affecting gene expression (intronless polycistronic transcription, trans-splicing, and RNA editing), metabolism, surface molecules, and organelles (compartmentalization of glycolysis, variation of the surface molecules, and unique mitochondrial DNA), cell biology and life cycle (phagocytic vacuoles evasion and intricate patterns of cell morphogenesis). With numerous genomic-scale data of several trypanosomatids becoming available since 2005 (genomes, transcriptomes, and proteomes), the scientific community can further investigate the mechanisms underlying these unusual features and address other unexplored phenomena possibly revealing biological aspects of the early evolution of eukaryotes. One fundamental aspect comprises the processes and mechanisms involved in the acquisition and loss of genes throughout the evolutionary history of these primitive microorganisms. Here, we present a comprehensive in silico analysis of pseudogenes in three major representatives of this group: Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Pseudogenes, DNA segments originating from altered genes that lost their original function, are genomic relics that can offer an essential record of the evolutionary history of functional genes, as well as clues about the dynamics and evolution of hosting genomes. Scanning these genomes with functional proteins as proxies to reveal intergenic regions with protein-coding features, relying on a customized threshold to distinguish statistically and biologically significant sequence similarities, and reassembling remnant sequences from their debris, we found thousands of pseudogenes and hundreds of open reading frames, with particular characteristics in each trypanosomatid: mutation profile, number, content, density, codon bias, average size, single- or multi-copy gene origin, number and type of mutations, putative primitive function, and transcriptional activity. These features suggest a common process of pseudogene formation, different patterns of pseudogene evolution and extant biological functions, and/or distinct genome organization undertaken by those parasites during evolution, as well as different evolutionary and/or selective pressures acting on distinct lineages.

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Leishmaniasis is a neglected tropical disease caused by Leishmania species. Available therapeutic options have several limitations. The drive to develop new, more potent, and selective antileishmanial agents is thus a major goal. Herein we report the synthesis and the biological activity evaluation against promastigote and amastigote forms of Leishmania amazonensis of nine 4,8-dimethoxynaphthalenyl chalcones. Compound ((E)-1-(4,8-dimethoxynaphthalen-1-yl)-3-(4-nitrophenyl)prop-2-en-1-one), 4f, was the most promising with an IC50 = 3.3 ± 0.34 µM (promastigotes), a low cytotoxicity profile (CC50 = 372.9 ± 0.04 µM), and a high selectivity index (SI = 112.6). Furthermore, 4f induced several morphological and ultrastructural changes in the free promastigote forms, loss of plasma membrane integrity, and increased reactive oxygen species (ROS). An in silico analysis of drug-likeness and ADME parameters suggested high oral bioavailability and intestinal absorption. Compound 4f reduced the number of infected macrophages and the number of amastigotes per macrophage, with an IC50 value of 18.5 ± 1.19 µM. Molecular docking studies with targets, ARG and TR, showed that compound 4f had more hydrogen bond interactions with the ARG enzyme, indicating a more stable protein-ligand binding. These results suggest that 4,8-dimethoxynaphthalenyl chalcones are worthy of further study as potential antileishmanial drugs.