New Insights on Heme Uptake in Leishmania spp.

The protozoan parasite Leishmania, responsible for leishmaniasis, is one of the few aerobic organisms that cannot synthesize the essential molecule heme. Therefore, it has developed specialized pathways to scavenge it from its host. In recent years, some proteins involved in the import of heme, such as LHR1 and LFLVCRB, have been identified, but relevant aspects regarding the process remain unknown. Here, we characterized the kinetics of the uptake of the heme analogue Zn(II) Mesoporphyrin IX (ZnMP) in Leishmania major promastigotes as a model of a parasite causing cutaneous leishmaniasis with special focus on the force that drives the process. We found that ZnMP uptake is an active, inducible, and pH-dependent process that does not require a plasma membrane proton gradient but requires the presence of the monovalent cations Na+ and/or K+. In addition, we demonstrated that this parasite can efflux this porphyrin against a concentration gradient. We also found that ZnMP uptake differs among different dermotropic or viscerotropic Leishmania species and does not correlate with LHR1 or LFLVCRB expression levels. Finally, we showed that these transporters have only partially overlapping functions. Altogether, these findings contribute to a deeper understanding of an important process in the biology of this parasite.

Synthesis of sp2-Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties.

sp2-Iminosugar glycolipids (sp2-IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp2-hybridized N-atom imparts chemical and enzymatic stability to sp2-IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of O-, N-, C- and S-pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp2-IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5N,6O-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure-activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp2-IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C12 vs. C8), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.

Thiol-ene "Click" Synthesis and Pharmacological Evaluation of C-Glycoside sp2-Iminosugar Glycolipids.

The unique stereoelectronic properties of sp2-iminosugars enable their participation in glycosylation reactions, thereby behaving as true carbohydrate chemical mimics. Among sp2-iminosugar conjugates, the sp2-iminosugar glycolipids (sp2-IGLs) have shown a variety of interesting pharmacological properties ranging from glycosidase inhibition to antiproliferative, antiparasitic, and anti-inflammatory activities. Developing strategies compatible with molecular diversity-oriented strategies for structure-activity relationship studies was therefore highly wanted. Here we show that a reaction sequence consisting in stereoselective C-allylation followed by thiol-ene "click" coupling provides a very convenient access to α-C-glycoside sp2-IGLs. Both the glycone moiety and the aglycone tail can be modified by using sp2-iminosugar precursors with different configurational profiles (d-gluco or d-galacto in this work) and varied thiols, as well as by oxidation of the sulfide adducts (to the corresponding sulfones in this work). A series of derivatives was prepared in this manner and their glycosidase inhibitory, antiproliferative and antileishmanial activities were evaluated in different settings. The results confirm that the inhibition of glycosidases, particularly α-glucosidase, and the antitumor/leishmanicidal activities are unrelated. The data are also consistent with the two later activities arising from the ability of the sp2-IGLs to interfere in the immune system response in a cell line and cell context dependent manner.

Novel 1,2-dihydroquinazolin-2-ones: Design, synthesis, and biological evaluation against Trypanosoma brucei.

In 2014, a published report of the high-throughput screen of>42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.

Experimental resistance to drug combinations in Leishmania donovani: metabolic and phenotypic adaptations.

Together with vector control, chemotherapy is an essential tool for the control of visceral leishmaniasis (VL), but its efficacy is jeopardized by growing resistance and treatment failure against first-line drugs. To delay the emergence of resistance, the use of drug combinations of existing antileishmanial agents has been tested systematically in clinical trials for the treatment of visceral leishmaniasis (VL). In vitro, Leishmania donovani promastigotes are able to develop experimental resistance to several combinations of different antileishmanial drugs after 10 weeks of drug pressure. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we identified metabolic changes in lines that were experimentally resistant to drug combinations and their respective single-resistant lines. This highlighted both collective metabolic changes (found in all combination therapy-resistant [CTR] lines) and specific ones (found in certain CTR lines). We demonstrated that single-resistant and CTR parasite cell lines show distinct metabolic adaptations, which all converge on the same defensive mechanisms that were experimentally validated: protection against drug-induced and external oxidative stress and changes in membrane fluidity. The membrane fluidity changes were accompanied by changes in drug uptake only in the lines that were resistant against drug combinations with antimonials, and surprisingly, drug accumulation was higher in these lines. Together, these results highlight the importance and the central role of protection against oxidative stress in the different resistant lines. Ultimately, these phenotypic changes might interfere with the mode of action of all drugs that are currently used for the treatment of VL and should be taken into account in drug development.

Mechanisms of action of substituted ß-amino alkanols on Leishmania donovani.

Leishmaniasis is the protozoan disease second in importance for human health, superseded only by malaria; however, the options for chemotherapeutic treatment are increasingly limited due to drug resistance and toxicity. Under this perspective, a quest for new chemical compounds is urgently needed. An N-substituted 2-aminoalkan-1-ol scaffold has been shown to be a versatile scaffold for antiparasitic activity. Knowledge about its mechanism of action is still rather limited. In this work, we endeavored to define the leishmanicidal profile of such ß-amino alkanol derivatives using a set of 15 N-mono- and disubstituted surrogates, tested on Leishmania donovani promastigotes and intracellular amastigotes. The best compound (compound 5), 2-ethylaminododecan-1-ol, had a 50% effective concentration (EC50) of 0.3 µM and a selectivity index of 72 for infected THP-1 cells and was selected for further elucidation of its leishmanicidal mechanism. It induced fast depletion of intracellular ATP content in promastigotes in the absence of vital dye intracellular entry, ruling out plasma membrane permeabilization as its origin. Confocal and transmission electron microscopy analyses showed that compound 5 induced severe mitochondrial swelling and vesiculation. Polarographic analysis using an oxygen electrode demonstrated that complex II of the respiratory chain (succinate reductase) was strongly inhibited by compound 5, identifying this complex as one of the primary targets. Furthermore, for other ß-amino alkanols whose structures differed subtly from that of compound 5, plasma membrane permeabilization or interference with membrane traffic was also observed. In all, N-substituted ß-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration.

4-amino bis-pyridinium derivatives as novel antileishmanial agents.

The antileishmanial activity of a series of bis-pyridinium derivatives that are analogues of pentamidine have been investigated, and all compounds assayed were found to display activity against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania major, with 50% effective concentrations (EC50s) lower than 1 µM in most cases. The majority of compounds showed similar behavior in both Leishmania species, being slightly more active against L. major amastigotes. However, compound VGP-106 {1,1'-(biphenyl-4,4'-diylmethylene)bis[4-(4-bromo-N-methylanilino)pyridinium] dibromide} exhibited significantly higher activity against L. donovani amastigotes (EC50, 0.86 ± 0.46 µM) with a lower toxicity in THP-1 cells (EC50, 206.54 ± 9.89 µM). As such, VGP-106 was chosen as a representative compound to further elucidate the mode of action of this family of inhibitors in promastigote forms of L. donovani. We have determined that uptake of VGP-106 in Leishmania is a temperature-independent process, suggesting that the compound crosses the parasite membrane by diffusion. Transmission electron microscopy analysis showed a severe mitochondrial swelling in parasites treated with compound VGP-106, which induces hyperpolarization of the mitochondrial membrane potential and a significant decrease of intracellular free ATP levels due to the inhibition of ATP synthesis. Additionally, we have confirmed that VGP-106 induces mitochondrial ROS production and an increase in intracellular Ca(2+) levels. All these molecular events can activate the apoptotic process in Leishmania; however, propidium iodide assays gave no indication of DNA fragmentation. These results underline the potency of compound VGP-106, which may represent a new avenue for the development of novel antileishmanial compounds.

Clinical features and predictors of delayed neurological syndrome in carbon monoxide poisoning: the AMICO study. / Caracterización clínica y factores predictivos de desarrollo de síndrome neurológico tardío en la intoxicación por monóxido de carbono: estudio AMICO.

OBJECTIVES: To identify predictors for developing delayed neurological syndrome (DNS) after an initial episode of carbon monoxide (CO) poisoning in the interest of detecting patients most likely to develop DNS so that they can be followed. MATERIAL AND METHODS: Retrospective review of cases of CO poisoning treated in the past 10 years in the emergency departments of 4 hospitals in the AMICO study (Spanish acronym for the multicenter analysis of CO poisoning). We analyzed demographic characteristics of the patients and the clinical characteristics of the initial episode. The records of the cohort of patients with available follow-up information were reviewed to find cases of DNS. Data were analyzed by multivariant analysis to determine the relationship to characteristics of the initial exposure to CO. RESULTS: A total of 240 cases were identified. The median (interquartile range) age of the patients was 36.2 years (17.6-49.6 years); 108 patients (45.0%) were men, and the poisoning was accidental in 223 cases (92.9%). The median carboxyhemoglobin concentration on presentation was 12.7% (6.2%-18.7%). Follow-up details were available for 44 patients (18.3%). Eleven of those patients (25%) developed DNS. A low initial Glasgow Coma Scale score predicted the development of DNS with an odds ratio (OR) of 0.61 (95% CI, 0.41-0.92) and an area under the receiver operating characteristic curve of 0.876 (95% CI, 0.761-0.990) (P .001). CONCLUSION: The initial Glasgow Coma Scale score seems to be a clinical predictor of DNS after CO poisoning. We consider it important to establish follow-up protocols for patients with CO poisoning treated in hospital EDs.

OBJETIVO: Identificar factores pronósticos de desarrollo de síndrome neurológico tardío (SNT) después de un episodio inicial de intoxicación por monóxido de carbono (ICO), con el fin detectar precozmente a la población más susceptible y facilitar su acceso a un seguimiento específico. METODO: Revisión retrospectiva de todos los casos de ICO que acudieron a los servicios de urgencias (SU) de 4 hospitales durante los últimos 10 años. Se analizaron datos demográficos y características clínicas en el momento del episodio. En la cohorte de pacientes con datos de seguimiento disponibles, se evaluó la aparición de SNT y su relación con diferentes variables en la exposición inicial al CO a través de técnicas de análisis multivariante. RESULTADOS: Se identificaron 240 pacientes. La mediana de edad fue de 36,2 años (17,6-49,6). De ellos 108 (45,0%) eran hombres y 223 casos (92,9%) fueron accidentales. El nivel medio de COHb fue del 12,7% (6,2-18,7). En 44 (18,3%) episodios se disponía de datos de un seguimiento específico. En esta cohorte, 11 (25%) pacientes desarrollaron SNT. Una puntuación inicial más baja en la Escala Coma de Glasgow (GCS) (OR: 0,61, IC 95%: 0,41-0,92) fue predictor independiente del desarrollo del SNT, con un ABC en la curva COR de 0,876 (IC 95%: 0,761-0,990, p 0,001). CONCLUSIONES: Una puntuación inicial baja en la GCS parece ser un predictor clínico de desarrollo de SNT en la ICO. Dada la incidencia de SNT, consideramos fundamental establecer protocolos de seguimiento específico de estos pacientes tras su asistencia inicial en los SU.

Label-Free Mass Spectrometry Proteomics Reveals Different Pathways Modulated in THP-1 Cells Infected with Therapeutic Failure and Drug Resistance Leishmania infantum Clinical Isolates.

As the world is facing increasing difficulties to treat leishmaniasis with current therapies, deeper investigation into the molecular mechanisms responsible for both drug resistance and treatment failure (TF) is essential in drug discovery and development. So far, few available drugs cause severe side effects and have developed several resistance mechanisms. Drug resistance and TF parasite strains from clinical isolates may have acquired altered expression of proteins that characterize specific mechanisms leading to therapy inefficacy. This work aims to identify the biochemical pathways of THP-1 human monocytes infected by different Leishmania infantum clinical isolates from patients with either resistance or with TF outcome, using whole cell differential Mass Spectrometry proteomics. We have adopted network enrichment analysis to integrate the transcriptomics and the proteomic results of infected cells studies. Transferrin receptor C (TFRC) and nucleoside diphosphate kinase 3 (NDK3) were discovered as overexpressed proteins in THP-1 cells infected with paromomycin, antimony, and miltefosine resistant L. infantum lines. The overall achievements represent founding concepts to confirm new targets involved in the parasitic drug resistance and TF mechanisms, and to consider in perspective the importance of a dual host-guest pharmacological approach to treat the acute stage of the disease.

Strasseriolides display in vitro and in vivo activity against trypanosomal parasites and cause morphological and size defects in Trypanosoma cruzi.

Neglected diseases caused by kinetoplastid parasites are a health burden in tropical and subtropical countries. The need to create safe and effective medicines to improve treatment remains a priority. Microbial natural products are a source of chemical diversity that provides a valuable approach for identifying new drug candidates. We recently reported the discovery and bioassay-guided isolation of a novel family of macrolides with antiplasmodial activity. The novel family of four potent antimalarial macrolides, strasseriolides A-D, was isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. In the present study, we analyze these strasseriolides for activity against kinetoplastid protozoan parasites, namely, Trypanosoma brucei brucei, Leishmania donovani and Trypanosoma cruzi. Compounds exhibited mostly low activities against T. b. brucei, yet notable growth inhibition and selectivity were observed for strasseriolides C and D in the clinically relevant intracellular T. cruzi and L. donovani amastigotes with EC50 values in the low micromolar range. Compound C is fast-acting and active against both intracellular and trypomastigote forms of T. cruzi. While cell cycle defects were not identified, prominent morphological changes were visualized by differential interference contrast microscopy and smaller and rounded parasites were visualized upon exposure to strasseriolide C. Moreover, compound C lowers parasitaemia in vivo in acute models of infection of Chagas disease. Hence, strasseriolide C is a novel natural product active against different forms of T. cruzi in vitro and in vivo. The study provides an avenue for blocking infection of new cells, a strategy that could additionally contribute to avoid treatment failure.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].

Non-reducing trisaccharide fatty acid monoesters: novel detergents in membrane biochemistry.

Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to µM. The new compounds are capable of dissolving phospholipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed ß-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.

A new ATP-binding cassette protein is involved in intracellular haem trafficking in Leishmania.

The characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG half-transporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis.

Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 µM during growth and 60 µM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

New Insights on Drug-Resistant Clinical Isolates of Leishmania infantum-Infected Human Macrophages as Determined by Comparative Transcriptome Analyses.

Leishmaniasis is the second most important neglected tropical parasitic disease after malaria. This disease is distributed worldwide and can be present in a variety of clinical forms, depending on the parasite species and host's genetic background. As chemotherapy is the only effective weapon whose effectiveness is limited by the frequent appearance of drug resistance and therapeutic failure, new therapeutic strategies are required. To better understand the factors that contribute to therapeutic failure and drug resistance in leishmaniasis, we studied the transcriptomic changes in host THP-1 cells after infection with clinical Leishmania infantum isolates with different susceptibilities to antileishmanial drugs by RNA-seq. Analysis of the differentially expressed genes (DEGs) in infected host cells revealed variations in DEG numbers in the THP-1-infected cells depending on the Leishmania line. A key conclusion of this study is that the modulation of host cells is Leishmania line dependent. Gene ontology enrichment analyses of DEGs indicated that certain biological processes were modulated in the infected host cells, specifically related to cellular metabolism, immune response, defense response, signaling pathways, and cell proliferation and apoptosis. Furthermore, this study provides new potential therapeutic markers and insights into the THP-1 host transcriptomic changes that occur after late infection with drug-resistant L. infantum clinical isolates.

Transcriptomic Analysis in Human Macrophages Infected with Therapeutic Failure Clinical Isolates of Leishmania infantum.

Leishmaniasis is one of the neglected tropical diseases with a worldwide distribution, affecting humans and animals. In the absence of an effective vaccine, current treatment is through the use of chemotherapy; however, existing treatments have frequent appearance of drug resistance and therapeutic failure (TF). The identification of factors that contribute to TF in leishmaniasis will provide the basis for a future therapeutic strategy more efficient for the control of this disease. In this article, we have evaluated the transcriptomic changes in the host cells THP-1 after infection with clinical Leishmania infantum isolates from leishmaniasis patients with TF. Our results show that distinct L. infantum isolates differentially modulate host cell response, inducing phenotypic changes that probably may account for parasite survival and TF of patients. Analysis of differential expression genes (DEGs), with a statistical significance threshold of a fold change ≥ 2 and a false discovery rate value ≤ 0.05, revealed a different number of DEGs according to the Leishmanialine. Globally, there was a similar number of genes up- and downregulated in all the infected host THP-1 cells, with exception of Hi-L2221, which showed a higher number of downregulated DEGs. We observed a total of 58 DEGs commonly modulated in all infected host cells, including upregulated (log2FC ≥ 1) and downregulated (log2FC ≤ -1) genes. Based on the results obtained from the analysis of RNA-seq, volcano plot, and GO enrichment analysis, we identified the most significant transcripts of relevance for their possible contribution to the TF observed in patients with leishmaniasis.

Mapping microglia and astrocyte activation in vivo using diffusion MRI.

While glia are increasingly implicated in the pathophysiology of psychiatric and neurodegenerative disorders, available methods for imaging these cells in vivo involve either invasive procedures or positron emission tomography radiotracers, which afford low resolution and specificity. Here, we present a noninvasive diffusion-weighted magnetic resonance imaging (MRI) method to image changes in glia morphology. Using rat models of neuroinflammation, degeneration, and demyelination, we demonstrate that diffusion-weighted MRI carries a fingerprint of microglia and astrocyte activation and that specific signatures from each population can be quantified noninvasively. The method is sensitive to changes in glia morphology and proliferation, providing a quantitative account of neuroinflammation, regardless of the existence of a concomitant neuronal loss or demyelinating injury. We prove the translational value of the approach showing significant associations between MRI and histological microglia markers in humans. This framework holds the potential to transform basic and clinical research by clarifying the role of inflammation in health and disease.

Transcriptome Analysis of Intracellular Amastigotes of Clinical Leishmania infantum Lines from Therapeutic Failure Patients after Infection of Human Macrophages.

Leishmaniasis is considered to be one of the most neglected tropical diseases affecting humans and animals around the world. Due to the absence of an effective vaccine, current treatment is based on chemotherapy. However, the continuous appearance of drug resistance and therapeutic failure (TF) lead to an early obsolescence of treatments. Identification of the factors that contribute to TF and drug resistance in leishmaniasis will constitute a useful tool for establishing future strategies to control this disease. In this manuscript, we evaluated the transcriptomic changes in the intracellular amastigotes of the Leishmania infantum parasites isolated from patients with leishmaniasis and TF at 96 h post-infection of THP-1 cells. The adaptation of the parasites to their new environment leads to expression alterations in the genes involved mainly in the transport through cell membranes, energy and redox metabolism, and detoxification. Specifically, the gene that codes for the prostaglandin f2α synthase seems to be relevant in the pathogenicity and TF since it appears substantially upregulated in all the L. infantum lines. Overall, our results show that at the late infection timepoint, the transcriptome of the parasites undergoes significant changes that probably improve the survival of the Leishmania lines in the host cells, contributing to the TF phenotype as well as drug therapy evasion.