Antileishmanial and Immunomodulatory Activity of Paclitaxel and Docetaxel Combined with Miltefosine and Paromomycin Estela Melcón-Fernández, Rafael Balaña-Fouce, Carlos García-Estrada, Rosa M. Reguera, Celia Fernández-Rubio, et al. International Journal of Molecular Sciences, 2026 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.
Discovery of Potential Antileishmanial Compounds Through Phenotypic Screening of an Alkaloid Library Cathy Soh-Kamdjo, María-Cristina González-Montero, Carlos García-Estrada, Estela Melcón-Fernández, Celia Fernández-Rubio, et al. Molecules, 2025 Visceral leishmaniasis caused by Leishmania donovani is one of the major neglected tropical diseases attributable to parasitic protozoa. In the absence of an effective vaccine, chemotherapy remains the only available therapeutic option. However, current treatments rely on a limited number of drugs that are largely obsolete, highly toxic or require intravenous administration, and their extensive use has led to the emergence of drug resistance. Consequently, the discovery of new antileishmanial agents is an urgent priority. In this study, a commercial library of 449 alkaloids in a high-throughput screening format was evaluated against both axenic bone marrow-derived amastigotes and intramacrophagic amastigotes from mice infected with L. donovani IRFP, a strain engineered to emit infrared fluorescence in its viable form. Six isoquinoline-type alkaloids showed the best antileishmanial efficacy against intramacrophagic amastigotes while exhibiting minimal cytotoxicity toward RAW 264.7 and HepG2 cell lines, with a promising selective index higher than four, and good mouse intestinal tolerance in mouse organoids. Among these compounds, the protoberberine scaffold emerged as the most promising candidate for further drug development.
Development of Sheep Duodenum Intestinal Organoids and Implementation of High-Throughput Screening Platform for Veterinary Applications Giulio Galli, Estela Melcón-Fernández, María Gracia de Garnica García, Beatriz Martínez-Fernández, Mahsa Dehnavi, et al. International Journal of Molecular Sciences, 2025 New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry, like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals, with consequent ethical and economic issues, the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however, the characterization and application of such organoids remain limited. In this study, ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs, and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types, and distinct apical–basal polarity, while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications, with Z’-factor tests indicating robust assay performance. Dose–response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology, offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health.
High-Throughput Screening of Five Compound Libraries for Anthelmintic Activity and Toxicity Leads to the Discovery of Two Flavonoid Compounds Giulio Galli, Marta Ruiz-Somacarrera, Laura González del Palacio, Estela Melcón-Fernández, Rubén González-Pérez, et al. International Journal of Molecular Sciences, 2025 Gastrointestinal nematode infections (GINs) in ruminants are a major constraint to efficient livestock production worldwide. Currently, only a limited number of anthelmintic drugs are available for the control of these infections, but their widespread use in preventive deworming campaigns and the incorrect administration of the drugs are responsible for the emergence of resistance. Therefore, new anthelmintic drugs are urgently needed. However, drug discovery methods for new anthelmintics based on GINs isolated from ruminants often have low throughput. In this study, a screening of five commercial collections of chemical compounds, including one collection of anti-infective drugs, three plant-based natural product collections, and one collection from the FDA-approved Chinese Pharmacopoeia, with a total of 2228 molecules, have been carried out in a high-throughput format. In the single slot screen, 32 compounds (1.44% success rate) achieved a >70% motility inhibition rate. Of these, 10 are known anthelmintic drugs, while the remaining 22 were tested against Haemonchus contortus and a resistant strain of Teladorsagia circumcincta. Four compounds (two flavonoids, chalcone and trans-chalcone), and two anti-infectives (octenidine and tolfenpyrad), showed anthelmintic activity with EC50 values below 20 µM, and were further tested for their safety against HepG2 spheroids and mouse intestinal organoids. Trans-chalcone and chalcone emerged as promising candidates for future development, showing selective indexes > 5, while tolfenpyrad and octenidine require careful evaluation due to their toxicity profiles.
Feed restriction during the suckling period of ewe Assaf lambs (F0) modifies milk quality and milk exosomal miRNAome of the filial generation (F1) Alba Martín, Carlos García-Estrada, Estela Melcón-Fernández, Raffaele A. Calogero, Maddalena Arigoni, et al. Animal Feed Science and Technology, 2024 Feed restriction during the early life of ewe lambs (F0) triggers the transfer of epigenetic marks to the next generation, thus impairing the reproductive performance of F1. However, the effects of this factor on milk production and composition, including its abundance in regulatory miRNA (many of which are contained in exosomes, small vesicles of endocytic origin that play a role in the modulation of immune response of the offspring) has not been tested so far. Therefore, in this study, the replacement ewe lambs (F0) obtained in a previous project (a group of ewes fed milk replacer ad libitum –ADL– vs. a group of ewes restricted –RES– to 62.5% the intake level of milk replacer during the suckling period) were raised under similar post-weaning conditions and mated to obtain the progeny (F1). The F1 female lambs were also mated to obtain F2. Milk production was recorded during the peak lactation period of F1, and milk samples were obtained for each gland (udder half) separately to measure chemical composition, somatic cell counts (SCC), and bacteria counts. Moreover, exosomes were also isolated from the milk of each gland separately to obtain the miRNAome following a Next Generation Sequencing approach. No significant differences were found in either milk production of F1, chemical composition of milk (fat, protein, lactase), or bacteriology (colony forming units, CFU). However, SCC was reduced significantly in milk samples of F1-RES dairy sheep, whereas the abundance of five miRNAs was also modified. Thus, oar-miR-150, oar-miR-221, oar-miR-23a, oar-miR-27a, oar-miR-376c were all down modulated in F1-RES when compared to F1-ADL. Most of these miRNAs have been found to play a role in biological functions such as development, apoptosis, muscle differentiation, reproduction, or milk production. However, the exosomes extracted from the milk of these sheep (F1-RES) did not affect the production of IL-9 and IL-2 cytokines after in vitro culture with CaCo-2 cells. This study reveals that nutritional programming events such as feed restriction may drive the abundance of not only SCC but also some milk's bioactive components such as miRNAs, although it is not clear if these changes may modulate the immune response at the intestinal level of the offspring.
In Vitro and Ex Vivo Synergistic Effect of Pyrvinium Pamoate Combined with Miltefosine and Paromomycin against Leishmania Estela Melcón-Fernández, Giulio Galli, Rafael Balaña-Fouce, Nerea García-Fernández, María Martínez-Valladares, et al. Tropical Medicine and Infectious Disease, 2024 One of the major drawbacks of current treatments for neglected tropical diseases is the low safety of the drugs used and the emergence of resistance. Leishmaniasis is a group of neglected diseases caused by protozoa of the trypanosomatidae family that lacks preventive vaccines and whose pharmacological treatments are scarce and unsafe. Combination therapy is a strategy that could solve the above-mentioned problems, due to the participation of several mechanisms of action and the reduction in the amount of drug necessary to obtain the therapeutic effect. In addition, this approach also increases the odds of finding an effective drug following the repurposing strategy. From the previous screening of two collections of repositioning drugs, we found that pyrvinium pamoate had a potent leishmanicidal effect. For this reason, we decided to combine it separately with two clinically used leishmanicidal drugs, miltefosine and paromomycin. These combinations were tested in axenic amastigotes of Leishmania infantum obtained from bone marrow cells and in intramacrophagic amastigotes obtained from primary cultures of splenic cells, both cell types coming from experimentally infected mice. Some of the combinations showed synergistic behavior, especially in the case of the combination of pyrvinium pamoate with paromomycin, and exhibited low cytotoxicity and good tolerability on intestinal murine organoids, which reveal the potential of these combinations for the treatment of leishmaniasis.
Antileishmanial Effect of 1,5- and 1,8-Substituted Fused Naphthyridines Estela Melcón-Fernandez, Endika Martín-Encinas, Francisco Palacios, Gulio Galli, Rosa M. Reguera, et al. Molecules, 2024 In the absence of a vaccine, there is a need to find new drugs for the treatment of neglected tropical diseases, such as leishmaniasis, that can overcome the many drawbacks of those currently used. These disadvantages include cost, the need to maintain a cold chain, the route of administration, the associated adverse effects and the generation of resistance. In this work we have evaluated the antileishmanial effect of 1,5- and 1,8-substituted fused naphthyridines through in vitro and ex vivo assays, using genetically modified axenic and intramacrophagic Leishmania infantum amastigotes. The toxicity of these compounds has been tested in the mammalian host cell using murine splenic macrophages, as well as in murine intestinal organoids (miniguts) in order to assess their potential for oral administration. The 1,8- derivatives showed greater leishmanicidal activity and the presence of a nitrogen atom in the fused ring to the naphthyridine was important to increase the activity of both types of molecules. The aromatization of the pyridine ring also had marked differences in the activity of the compounds.
Miltefosine and Nifuratel Combination: A Promising Therapy for the Treatment of Leishmania donovani Visceral Leishmaniasis Estela Melcon-Fernandez, Giulio Galli, Carlos García-Estrada, Rafael Balaña-Fouce, Rosa M. Reguera, et al. International Journal of Molecular Sciences, 2023 Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel—a nitrofurantoin used against vaginal infections—as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine—the only oral drug currently used against leishmaniasis—with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.
Design, synthesis and evaluation of novel phenanthridine triazole analogs as potential antileishmanial agents Adinarayana Nandikolla, Srinivasarao Singireddi, Karan Kumar Banoth, Sankaranarayanan Murugesan, Himanshu Aggarwal, et al. Future Medicinal Chemistry, 2022 <b>Aim:</b> To synthesize and screen phenanthridine and 1,2,3-triazole derivatives for antileishmanial activity. <b>Methodology:</b> Synthesized analogs were tested for antileishmanial activity against transgenic strain of <i>Leishmania infantum</i> promastigotes and <i>ex vivo</i> infections. <b>Results:</b> Compounds <b>T01, T08</b> and <b>T11</b> revealed significant activity with EC<sub>50</sub> <30 μm and lacked toxicity in mouse spleen and HepG2 cells. <b>T01</b> with EC<sub>50</sub> 3.07 μm is fourfold more potent than the drug miltefosine (EC<sub>50</sub> 12.6 μM) against <i>L. infantum</i> promastigotes. <i>In silico</i> studies indicate that the analogs are nontoxic. A molecular docking analysis was also carried out on the <b>T01</b> and <b>T08</b> to investigate the binding pattern at the active site of the chosen target trypanothione reductase. <b>Conclusion:</b> The results of this study reveal that phenanthridine triazoles exhibit antileishmanial activity.
