Rafael Balaña-Fouce

Visceral leishmaniasis (VL) is a life-threatening, vector-borne systemic disease caused by the parasitic protozoans Leishmania donovani and Leishmania infantum. Among the affected organs, the liver represents a unique immunological environment in which, in commonly used experimental murine models, infection is often controlled through the coordinated formation of granulomas around infected Kupffer cells. However, effective hepatic parasite control does not necessarily result in complete parasite elimination, and the course of infection may vary across host species and experimental models. Understanding the molecular mechanisms governing parasite clearance and persistence has been greatly advanced by transcriptomic technologies. The transition from bulk RNA sequencing to single-cell RNA sequencing and, more recently, spatial transcriptomics has enabled increasingly detailed characterization of hepatic immune responses, revealing cellular heterogeneity, spatial organization, and regulatory networks underlying host–parasite interactions. In parallel, the development of advanced in vitro models, including multicellular co-cultures and organoid-based systems, has provided complementary platforms for investigating these mechanisms under controlled experimental conditions. This review provides an integrated analysis of transcriptomic studies investigating hepatic host responses to Leishmania infection across different experimental models and transcriptomic approaches. We examine the molecular pathways underlying host defense, granuloma formation, and parasite persistence, compare the complementary insights provided by distinct transcriptomic approaches, and critically evaluate the extent to which in vitro models reproduce the hepatic transcriptional landscape observed in vivo. By integrating findings across experimental platforms and transcriptomic resolutions, we identify conserved immune signatures, model-specific limitations, and key biological and technological knowledge gaps that should guide the development of more physiologically relevant experimental models and future transcriptomic studies of the liver during VL.

Leishmaniasis requires safer and more effective therapies, and the trypanothione-dependent redox metabolism of trypanosomatids, particularly trypanothione reductase (TryR), represents an attractive druggable target. An alkaloid library comprising 432 compounds was screened to identify inhibitors of Leishmania donovani TryR (LdTryR). Six compounds displayed both LdTryR inhibitory activity and antileishmanial effects against intramacrophage amastigotes: (−)-alkannin (EC50: 8.98 ± 1.96 μM; Ki: 2.83 ± 1.99 μM), chelerythrine (EC50: 1.61 ± 0.05 μM; Ki: 27.80 ± 9.43 μM), dehydronuciferine (EC50: > 50 μM; Ki: 15.77 ± 8.34 μM), ethoxysanguinarine (EC50: 0.84 ± 0.03 μM; Ki: 21.22 ± 7.32 μM), sanguinarine chloride (EC50: 0.85 ± 0.03 μM; Ki: 28.57 ± 9.62 μM) and tryptanthrin (EC50: 1.91 ± 0.21 μM; Ki: 16.80 ± 10.61 μM). Kinetic analyses revealed competitive inhibition patterns for benzophenanthridines, whereas (−)-alkannin, tryptanthrin, and dehydronuciferine exhibited uncompetitive inhibition behavior. Cytotoxicity analysis revealed that tryptanthrin was the only compound that fulfilled the hit criteria commonly applied in visceral leishmaniasis. Benzophenanthridine alkaloids induced rapid reactive oxygen species (ROS) accumulation, whereas (−)-alkannin produced delayed oxidative stress. Compounds inducing the highest parasite ROS levels also stimulated nitrite accumulation in macrophages. Molecular docking and molecular dynamics simulations indicated that the six compounds engaged a common region of the trypanothione-binding site, although binding was weak and transient and did not rank with their measured antileishmanial potency. (−)-Alkannin was a notable exception, reproducibly forming a ternary complex with the bound substrate, in line with its uncompetitive inhibition. Overall, these findings support LdTryR as a relevant antileishmanial target.

Plant-derived secondary metabolites are a promising source of new anti-infective agents owing to their remarkable chemical diversity and broad spectrum of biological activities. Among them, halophytes are particularly promising because adaptation to saline environments promotes the accumulation of structurally diverse secondary metabolites. In the present study, organic extracts from four Tunisian halophytic species collected across different seasons were evaluated for their activity against Leishmania infantum axenic amastigotes, the etiological agent of visceral leishmaniasis in the Mediterranean Basin. Among the species tested, Suaeda vera was the only one to exhibit significant antileishmanial activity. Mass spectrometry-based phytochemical profiling of the bioactive extracts resulted in the putative annotation of 53 secondary metabolites and revealed qualitative differences in their annotated chemical composition among the seasonal samples. Three compounds putatively annotated in the active extracts—demethylzeylasteral, caffeic acid, and protocatechuic aldehyde—were subsequently evaluated individually using commercially available reference compounds and exhibited EC₅₀ values of 0.20, 1.09, and 14.56 μM, respectively. Caffeic acid displayed the most favorable overall profile, combining potent antileishmanial activity with a high selectivity index (SI > 91.7) in mouse intestinal organoids. In vitro assays revealed that none of the three compounds inhibited trypanothione reductase-associated activity. Furthermore, in silico drug-likeness and ADMET analyses suggested that caffeic acid may have a comparatively favorable predicted pharmacokinetic and toxicity profile among the three compounds evaluated. Collectively, these findings highlight S. vera as a promising source of antileishmanial metabolites and establish caffeic acid as the most promising hit compound for further investigation.

The emergence of drug resistance and the limited availability of effective therapeutics continue to hinder the treatment of protozoal diseases, highlighting the need for new chemotypes with broad-spectrum antiparasitic potential. In the present study, a series of nitro and trifluoromethyl-substituted thiochromene carboxamides, previously identified as antileishmanial agents, were further explored for their broad-spectrum antiparasitic potential. Biological screening identified several compounds with promising activity and favorable selectivity against Plasmodium falciparum (D10 and W2 strains) and Trypanosoma brucei (AnTat1.1E strain). As the synthesized analogues exhibited a more favorable antitrypanosomal activity and selectivity profile, representative analogues were subsequently evaluated for trypanothione reductase (TryR) inhibition to investigate their potential mechanism of action, as TryR is a validated and highly conserved drug target across kinetoplastid parasites. Among the synthesized molecules, compound 5p showed the highest antiplasmodial potency (D10 EC50 = 2.1 μM), whereas compounds 5c and 5r were the most potent antitrypanosomal analogues (EC50 = 1.8 μM). Structure–activity relationship analysis demonstrated that piperidine and piperazine-containing carboxamide is a key pharmacophoric feature for broad-spectrum activity, whereas incorporation of a nitro substituent enhanced the activity against T. brucei. To investigate the potential mode of killing, representative compounds were evaluated for inhibition of TryR, identifying compound 5v as the most potent enzyme inhibitor. Enzyme kinetic studies established an uncompetitive mode of inhibition (ki ≈ 29.5 μM), suggesting interaction at an allosteric site rather than the active catalytic site. Consistent with these findings, SiteMap analysis identified a druggable allosteric pocket at the dimer interface of substrate-bound TryR, where the active compounds exhibited favorable binding affinities and interaction profiles. Collectively, these results demonstrate that thiochromene carboxamides constitute a promising scaffold for the development of broad-spectrum antiprotozoal agents and support allosteric inhibition of TryR as a viable strategy for antitrypanosomal drug discovery.

In medicinal chemistry, nitrogen-containing heterocyclic scaffolds and their synthetic approaches are fundamental. They play a vital role as they greatly enhance the diversity of drug-like molecules available, due to their remarkable chemical versatility. These compounds interact efficiently with a wide range of biological targets, making them crucial in the discovery and development of new drugs. Among various heterocycles, quinoline and isoquinoline derivatives have garnered significant attention due to their diverse neuroprotective potential in addressing various neurodegenerative disorders. These scaffolds are central to the design of small molecules, which aim to slow, delay, or reverse the course of neurodegenerative disorders. This chapter presents a detailed overview of recent progress in the general synthesis, structural modifications, and pharmacological insights, as well as drawing on structure–activity relationship (SAR) analysis and computational modelling underpinning neuroprotection against a spectrum of neurodegenerative disorders, including Alzheimer's, Parkinson's, Huntington's, and related diseases. The unique planar aromatic structure of quinoline and isoquinoline derivatives facilitates neuroprotective mechanisms, including antioxidant activity, inhibition of monoamine oxidase, modulation of cholinesterase enzymes, regulation of neurotransmitter systems, and suppression of neuroinflammatory cascades. Furthermore, this chapter explores how quinoline and isoquinoline scaffolds can be integrated into hybrids and prodrugs, with enhanced blood–brain barrier (BBB) permeability and reduced cytotoxicity, highlighting creative ways to overcome pharmacokinetic limitations and challenges in translating pre-clinical findings into clinical success. By detailing recent advancements in this field, this chapter identifies quinoline and isoquinoline scaffolds as promising templates for the development of next-generation neuroprotective drugs, combining chemistry, pharmacology, and molecular modelling approaches.

Visceral leishmaniasis, caused by Leishmania donovani, is characterized by profound alterations in hepatic immune responses that promote parasite persistence. However, the lack of physiologically relevant in vitro systems limits investigation of tissue-level host–pathogen interactions. An immunocompetent three-dimensional hepatic organoid model incorporating bone marrow-derived macrophages was established to investigate liver-stage immune responses to L. donovani infection. Integrated cytokine profiling and transcriptomic analyses were benchmarked against infected mouse liver tissue. Macrophages acquired functional characteristics consistent with a Kupffer cell-like phenotype and contributed to immune homeostasis within the co-culture. Infection induced a conserved immune response characterized by inflammatory and chemotactic activation. This response was accompanied by coordinated suppression of antimicrobial pathways, including the IL-12–IFN-γ axis and interferon-stimulated genes. These immune alterations were largely shared between the organoid model and infected liver tissue, although systemic metabolic reprogramming and multicellular complexity were not fully recapitulated. Our findings identify a conserved pattern of hepatic immune dysregulation in which inflammatory activation is uncoupled from effective antimicrobial signaling, potentially facilitating parasite persistence. This immunocompetent hepatic organoid platform provides a physiologically relevant system for investigating host–pathogen interactions and evaluating host-directed therapeutic strategies for visceral leishmaniasis.

Deoxynivalenol (DON) is a prevalent trichothecene mycotoxin in cereals that poses food and feed safety risks while causing important economic losses. Microbial biotransformation offers a selective, mild strategy for DON detoxification. Here, we screened aerobic soil-derived bacterial communities from diverse agricultural environments, using DON as the sole carbon source for this mycotoxin depletion. More than half of the tested enrichment samples showed a reduced DON signal, as observed by HPLC-UV. To assess the biological relevance, culture extracts were tested for cytotoxicity in HepG2 cells. Z13, a soil sample that depleted DON but produced no other detectable metabolites, showed reduced cytotoxicity, comparable to the negative control. In contrast, samples that depleted DON but produced 3-keto-DON remained toxic. High-resolution LC-MS analysis indicated the formation of metabolites putatively identified as 3-keto-DON in enrichment cultures and 3-epi-DON in a Devosia strain culture. Community composition was profiled with 16S rRNA gene amplicon sequencing, which showed that Z13 presented a remarkable drop in diversity upon microbial cultivation, and included genera such as Devosia, Nocardioides, and Pseudomonas. Together, these results provide integrated chemical, biological, and ecological evidence for aerobic DON biotransformation in soil-derived communities, identify pathway products, and highlight practical constraints related to community dependence and storage sensitivity.

Chalcones, a subclass of flavonoid-derived phenolic compounds, have demonstrated promising anthelmintic activity against parasitic nematodes. This study aimed to obtain insights into the biological effects a cis/trans mixture of chalcone and its geometric isomer, trans-chalcone, using RNA sequencing in the model organism Caenorhabditis elegans. Fourth-stage larvae (L4) were exposed to cis/trans-chalcone or trans-chalcone for 3 h, and total RNA was extracted for high-throughput sequencing. Transcriptomic analysis revealed that exposure to cis/trans-chalcone and trans-chalcone induced pronounced modulation of genes involved in lipid metabolism and repression of collagen and structural genes, potentially leading to defective extracellular matrix maintenance, thereby suggesting these combined effects as potential mechanisms underlying their anthelmintic activity. Also, metabolic and stress response pathways, with several genes implicated in detoxification and cellular defense, were markedly upregulated. These findings provide new insights into the molecular mechanisms affected by chalcones, advancing our understanding of their anthelmintic potential and supporting future drug development efforts.

A wide range of Leishmania species, transmitted by phlebotomine, cause leishmaniasis, which presents diverse clinical manifestations. Leishmaniasis has a high impact on vulnerable communities, primarily affecting people suffering from malnutrition or poor housing. Because leishmaniasis is associated with poverty, access to treatment is limited. In addition, high drug toxicity and therapeutic failure, related to drug resistance, remain major challenges. Therefore, there is a need to develop new therapeutic approaches that are safer and more effective. Drug combinations and repurposing are two strategies used in the development of treatments. The combination of drugs with different mechanisms of action can minimise resistance and allow dose reduction, increasing the likelihood of successful drug repurposing. This study evaluated the antileishmanial effects of combining antitumoral agents (paclitaxel and docetaxel) with standard drugs (miltefosine and paromomycin). Results demonstrated synergistic effects at higher doses. Furthermore, the antitumoral compounds enhanced the host immune response by promoting macrophage polarisation toward the M1 phenotype, essential for parasite control. These findings highlight a promising approach that could improve efficacy and reduce resistance.

A wide range of Leishmania species, transmitted by phlebotomine, cause a group of diseases known as leishmaniasis, which present diverse clinical manifestations. Leishmaniasis has a high impact on the most vulnerable communities, primarily affecting people suffering from malnutrition or living in poor housing conditions. Due to leishmaniasis are strongly associated with poverty the access to treatment is often limited. Besides, high drug toxicity and therapeutic failure, related to the emergence of drug resistance, remain major challenges associated with currently available treatments. Therefore, there is a need for developing new therapeutic approaches safer and more effective. Drug combinations and drug repurposing are two strategies currently used in the development of new treatments targeting multiple diseases. The combination of drugs with different mechanisms of action can minimize the emergence of resistance and allow drug dosage reduction, increasing the likelihood of successful drug repurposing. The research evaluated the leishmanicidal effects of combining antitumoral agents (paclitaxel and docetaxel) with standard drugs (miltefosine and paromomycin). Results demonstrated synergistic effects at higher doses. Furthermore, the antitumoral compounds boosted the host immune response by promoting macrophage polarization toward the M1 phenotype, which is essential for parasite control. These findings highlight a promising therapeutic approach that could improve treatment efficacy and reduce resistance.