Anti-COVID Drugs (MMV COVID Box) as Leishmanicidal Agents: Unveiling New Therapeutic Horizons.

Leishmaniasis, a neglected tropical disease, poses a significant global health challenge, necessitating the urgent development of innovative therapies. In this study, we aimed to identify compounds from the COVID Box with potential efficacy against two Leishmania species, laying the foundation for future chemical development. Four promising molecules were discovered, demonstrating notable inhibitory effects against L. amazonensis and L. donovani. Our study revealed that bortezomib, almitrine, and terconazole induced a significant decrease in mitochondrial membrane potential, while the above compounds and ABT239 induced plasma permeability alterations, chromatin condensation, and reactive oxygen species accumulation, indicating early apoptosis in Leishmania amazonensis promastigotes, preventing inflammatory responses and tissue damage, thereby improving patient outcomes. Furthermore, ADME predictions revealed favorable pharmacokinetic profiles for all compounds, with bortezomib and ABT239 standing out as potential candidates. These compounds exhibited intestinal absorption, blood-brain barrier penetration (excluding bortezomib), and good drug-likeness for bortezomib and ABT239. Toxicity predictions for CYP-inhibition enzymes favored bortezomib as the safest candidate. In conclusion, our study identifies bortezomib as a promising aspirant for leishmaniasis treatment, demonstrating potent antiparasitic activity, favorable pharmacokinetics, and low toxicity. These findings emphasize the potential repurposing of existing drugs for neglected diseases and highlight the importance of the COVID Box in drug discovery against tropical diseases.

Acrylonitrile derivatives: In vitro activity and mechanism of cell death induction against Trypanosoma cruzi and Leishmania amazonensis.

Leishmaniasis and Chagas disease are parasitic infections that affect millions of people worldwide, producing thousands of deaths per year. The current treatments against these pathologies are not totally effective and produce some side effects in the patients. Acrylonitrile derivatives are a group of compounds that have shown activity against these two diseases. In this work, four novels synthetic acrylonitriles were evaluated against the intracellular form and extracellular forms of L. amazonensis and T. cruzi. The compounds 2 and 3 demonstrate to have good selectivity indexes against both parasites, specifically the compound 3 against the amastigote form (SI = 6 against L. amazonensis and SI = 7.4 against T. cruzi). In addition, the parasites treated with these two compounds demonstrate to produce a programmed cell death, since they were positive for the events studied related to this type of death, including chromatin condensation, accumulation of reactive oxygen species and alteration of the mitochondrial membrane potential. In conclusion, this work confirms that acrylonitriles is a source of possible new compounds against kinetoplastids, however, more studies are needed to corroborate this activity.

Salvadoran Celastraceae Species as a Source of Antikinetoplastid Quinonemethide Triterpenoids.

Chagas disease and leishmaniasis are among the most widespread neglected tropical diseases, and their current therapies have limited efficacy and several toxic side effects. The present study reports the chemical and antikinetoplastid profiles of extracts from five Salvadoran Celastraceae species against the Trypanosoma cruzi epimastigotes stage and Leishmania amazonensis and Leishmania donovani promastigote forms. The phytochemical profile evinced the presence of flavonoids, tannins, sterols, and triterpenes as the main components in all plant species, whereas quinonemethide triterpenoids (QMTs) were restricted to the root bark of the studied species. Antikinetoplastid evaluation highlights the root bark extracts from Zinowewia integerrima, Maytenus segoviarum, and Quetzalia ilicina as the most promising ones, exhibiting higher potency against T. cruzi (IC50 0.71-1.58 µg/mL) and L. amazonensis (IC50 0.38-2.05 µg/mL) than the reference drugs, benznidazole (IC50 1.81 µg/mL) and miltefosine (IC50 2.64 µg/mL), respectively. This potent activity was connected with an excellent selectivity index on the murine macrophage J774A.1 cell line. These findings reinforce the potential of QMTs as antikinetoplastid agents for the development of innovative phytopharmaceuticals and the plant species under study as a source of these promising lead compounds.

Laurequinone, a Lead Compound against Leishmania.

Among neglected tropical diseases, leishmaniasis is one of the leading causes, not only of deaths but also of disability-adjusted life years. This disease, caused by protozoan parasites of the genus Leishmania, triggers different clinical manifestations, with cutaneous, mucocutaneous, and visceral forms. As existing treatments for this parasitosis are not sufficiently effective or safe for the patient, in this work, different sesquiterpenes isolated from the red alga Laurencia johnstonii have been studied for this purpose. The different compounds were tested in vitro against the promastigote and amastigote forms of Leishmania amazonensis. Different assays were also performed, including the measurement of mitochondrial potential, determination of ROS accumulation, and chromatin condensation, among others, focused on the detection of the cell death process known in this type of organism as apoptosis-like. Five compounds were identified that displayed leishmanicidal activity: laurequinone, laurinterol, debromolaurinterol, isolaurinterol, and aplysin, showing IC50 values against promastigotes of 1.87, 34.45, 12.48, 10.09, and 54.13 µM, respectively. Laurequinone was the most potent compound tested and was shown to be more effective than the reference drug miltefosine against promastigotes. Different death mechanism studies carried out showed that laurequinone appears to induce programmed cell death or apoptosis in the parasite studied. The obtained results underline the potential of this sesquiterpene as a novel anti-kinetoplastid therapeutic agent.

In vitro activity and mechanism of cell death induction of cyanomethyl vinyl ethers derivatives against Trypanosoma cruzi.

Chagas disease causes a problematic pathology that can lead to megacolon and heart disease, and can even cause the death of the patient. Current therapies for this disease are the same as they were 50 years ago, are not fully effective and have strong side effects. The lack of a safe and effective therapy makes it necessary to search for new, less toxic and totally effective compounds against this parasite. In this work, the antichagasic activity of 46 novel cyanomethyl vinyl ether derivatives was studied. In addition, to elucidate the type of cell death that these compounds produce in parasites, several events related to programmed cell death were studied. The results highlight four more selective compounds, E63, E64, E74 and E83, which also appear to trigger programmed cell death, and are therefore postulated as good candidates to use in future therapeutics for Chagas disease.

Multi-target withaferin-A analogues as promising anti-kinetoplastid agents through the programmed cell death.

Leishmaniasis and Chagas disease, two of the most prevalent neglected tropical diseases, are a world health problem. The harsh reality of these infective diseases is the absence of effective and safe therapies. In this framework, natural products play an important role in overcoming the current need to development new antiparasitic agents. The present study reports the synthesis, antikinetoplastid screening, mechanism study of fourteen withaferin A derivatives (2-15). Nine of them (2-6, 8-10 and 12) showed a potent dose-dependent inhibitory effect on the proliferation of Leishmania amazonensis and L. donovani promastigotes and Trypanosoma cruzi epimastigotes with IC50 values ranging from 0.19 to 24.01 µM. Outstandingly, the fully acetylated derivative 10 (4,27-diacetylwithaferin A) was the most potent compound showing IC50 values of 0.36, 2.82 and 0.19 µM against L. amazonensis, L. donovani and T. cruzi, respectively. Furthermore, analogue 10 exhibited approximately 18 and 36-fold greater antikinetoplastid activity, on L. amazonensis and T. cruzi, than the reference drugs. The activity was accompanied by significantly lower cytotoxicity on the murine macrophage cell line. Moreover, compounds 2, 3, 5-7, 9 and 10 showed more potent activity than the reference drug against the intracellular amastigotes forms of L. amazonensis and T.cruzi, with a good selectivity index on a mammalian cell line. In addition, withaferin A analogues 3, 5-7, 9 and 10 induce programmed cell death through a process of apoptosis-like and autophagy. These results strengthen the anti-parasitic potential of withaferin A-related steroids against neglected tropical diseases caused by Leishmania spp. and T. cruzi parasites.

Meroterpenoids from Gongolaria abies-marina against Kinetoplastids: In Vitro Activity and Programmed Cell Death Study.

Leishmaniasis and Chagas disease affect millions of people worldwide. The available treatments against these parasitic diseases are limited and display multiple undesired effects. The brown alga belonging to the genus Gongolaria has been previously reported as a source of compounds with different biological activities. In a recent study from our group, Gongolaria abies-marine was proven to present antiamebic activity. Hence, this brown alga could be a promising source of interesting molecules for the development of new antiprotozoal drugs. In this study, four meroterpenoids were isolated and purified from a dichloromethane/ethyl acetate crude extract through a bioguided fractionation process targeting kinetoplastids. Moreover, the in vitro activity and toxicity were evaluated, and the induction of programmed cell death was checked in the most active and less toxic compounds, namely gongolarone B (2), 6Z-1'-methoxyamentadione (3) and 1'-methoxyamentadione (4). These meroterpenoids triggered mitochondrial malfunction, oxidative stress, chromatin condensation and alterations of the tubulin network. Furthermore, a transmission electron microscopy (TEM) image analysis showed that meroterpenoids (2-4) induced the formation of autophagy vacuoles and ER and Golgi complex disorganization. The obtained results demonstrated that the mechanisms of action at the cellular level of these compounds were able to induce autophagy as well as an apoptosis-like process in the treated parasites.

Withaferin A-silyl ether analogs as potential anti-kinetoplastid agents targeting the programmed cell death.

Current therapies of leishmaniasis and Chagas disease, two of the most widespread neglected tropical diseases, have limited efficacy and toxic side effects. In this regard, natural products play an important role in overcoming the current need for new antiparasitic agents. The present study reports the leishmanicidal and trypanocidal activities of twenty-four known silyl-ether derivatives of withaferin A. Eleven compounds from this series (4, 7, 8, 10, 12, 15, 17, 18, 20, 22 and 25) showed a potent dose-dependent inhibitory effect on the proliferation of Leishmania amazonensis promastigotes and Trypanosoma cruzi epimastigotes respectively, even higher than the references drugs, miltefosine and benznidazole. Among them, the most promising compound, derivative 10, exhibited approximately 34-fold higher leishmanicidal activity and 49-fold higher trypanocidal activity compared to the reference drugs, as well as lower cytotoxicity. Moreover, compounds 4, 7, 10, 12 and 15 were more active than the reference drugs against the amastigote forms of L. amazonensis, presenting a high selectivity index. Assays performed to study the ATP levels, mitochondrial membrane potential, plasma membrane permeability, chromatin condensation, reactive oxygen species and autophagy indicated that these withaferin A-silyl analogs appear to induce events characteristic of apoptosis-like and also autophagy leading to programmed cell death. These findings support the therapeutic potential of withaferin A-related steroids as anti-Leishmania and Trypanosoma agents.

Ozone treatment effectively eliminates SARS-CoV-2 from infected face masks.

The current COVID-19 pandemic is causing profound health, economic, and social problems worldwide. The global shortage of medical and personal protective equipment (PPE) in specialized centers during the outbreak demonstrated the need for efficient methods to disinfect and recycle them in times of emergency. We have previously described that high ozone concentrations destroyed viral RNA in an inactivated SARS-CoV-2 strain within a few minutes. However, the efficient ozone dosages for active SARS-CoV-2 are still unknown. The present study aimed to evaluate the systematic effects of ozone exposure on face masks from hospitalized patients infected with SARS-CoV-2. Face masks from COVID-19 patients were collected and treated with a clinical ozone generator at high ozone concentrations in small volumes for short periods. The study focused on SARS-CoV-2 gene detection (assessed by real-time quantitative polymerase chain reaction (RT-qPCR)) and on the virus inactivation by in vitro studies. We assessed the effects of different high ozone concentrations and exposure times on decontamination efficiency. We showed that high ozone concentrations (10,000, 2,000, and 4,000 ppm) and short exposure times (10, 10, and 2 minutes, respectively), inactivated both the original strain and the B.1.1.7 strain of SARS-CoV-2 from 24 contaminated face masks from COVID-19 patients. The validation results showed that the best condition for SARS-CoV-2 inactivation was a treatment of 4,000 ppm of ozone for 2 minutes. Further studies are in progress to advance the potential applications of these findings.

In vitro activity and cell death mechanism induced by acrylonitrile derivatives against Leishmania amazonensis.

Leishmaniasis produces approximately-one million of new cases annually, making it one of the most important tropical diseases. As current treatments are not fully effective and are toxic, it is necessary to develop new therapies that are more effective and less toxic, and cause a controlled cell death, with which we can avoid the immunological problems caused by necrosis. In this work 32 acrylonitriles were studied in vitro against Leishmania amazonensis. Three compounds Q20 (12.41), Q29 (11.2) and Q31 (11.56) had better selectivity than the reference compound, miltefosine (11.14) against promastigotes of these parasites, for this reason they were selected to determine their mechanism of action to know the cell death type of they produce. The results of the mechanisms of action show that these three acrylonitriles tested produce chromatin condensation, decreased mitochondrial membrane potential, altered plasma permeability and production of reactive oxygen species. All these characteristic events seem to indicate programmed cell death. Therefore, this study demonstrates the activity of acrylonitriles derivatives as possible leishmanicidal agents.

Discovery of New Chemical Tools against Leishmania amazonensis via the MMV Pathogen Box.

The protozoan parasite Leishmania causes a spectrum of diseases and there are over 1 million infections each year. Current treatments are toxic, expensive, and difficult to administer, and resistance to them is emerging. In this study, we screened the antileishmanial activity of the Pathogen Box compounds from the Medicine for Malaria Venture against Leishmania amazonensis, and compared their structures and cytotoxicity. The compounds MMV676388 (3), MMV690103 (5), MMV022029 (7), MMV022478 (9) and MMV021013 (10) exerted a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and intracellular amastigotes. Moreover, studies on the mechanism of cell death showed that compounds 3 and 5 induced an apoptotic process while the compounds 7, 9 and 10 seem to induce an autophagic mechanism. The present findings underline the potential of these five molecules as novel therapeutic leishmanicidal agents.

Antikinetoplastid Activity of Sesquiterpenes Isolated from the Zoanthid Palythoa aff. clavata.

Leishmaniasis and Chagas disease are neglected tropical diseases that cause problems in developing countries. The causative agents, Leishmania spp. and Trypanosoma cruzi, produce a clinical picture that can be fatal for the patient, such as Chagas heart disease, visceral leishmaniasis and megacolon, among others. Current treatments for these diseases are not very effective and highly toxic, since they require very prolonged treatments. The development of innovative, effective and safe drugs to fight infections caused by these parasites remains a challenge. For this reason, in recent years, there has been an increase in the search for new therapies. In this study, the antikinetoplastid activity of 13 sesquiterpene lactones obtained from Palythoa aff. clavata was screened against L. amazonensis, L. donovani and T. cruzi. The results revealed that the sesquiterpene lactones anhydroartemorin (2), cis,trans-costunolide-14-acetate (3) and 4-hydroxyarbusculin A (11) were the most selective against the kinetoplastid species studied. These molecules seem to induce the mechanisms involved in an apoptotic-like death or programmed cell death (PCD) in the kinetoplastids, and since they do not cause necrosis, the inflammatory events associated with this type of cell death will not be triggered.

Acrylonitrile Derivatives against Trypanosoma cruzi: In Vitro Activity and Programmed Cell Death Study.

The neglected infection known as Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, results in more than 7000 deaths per year, with an increasing number of cases in non-endemic areas such as Europe or the United States. Moreover, with the current available therapy, only two compounds which are active against the acute phase of the disease are readily available. In addition, these therapeutic agents display multiple undesired side effects such as high toxicity, they are expensive, the treatment is lengthy and the resistant strain has emerged. Therefore, there is a need to find new compounds against Chagas disease which should be active against the parasite but also cause low toxicity to the patients. In the present work, the activity of novel acrylonitriles against Trypanosoma cruzi was evaluated as well as the analysis of the physiological events induced in the treated parasites related to the cell death process. Hence, the characteristic features of an apoptosis-like process such as chromatin condensation and mitochondrial membrane potential, among others, were studied. From the 32 compounds tested against the epimastigote stage of T. cruzi, 11 were selected based on their selectivity index to determine if these compounds were able to induce programmed cell death (PCD) in the treated parasites. Furthermore, acrylonitriles Q5, Q7, Q19, Q27 and Q29 were shown to trigger physiological events related in the PCD. Therefore, this study highlights the therapeutic potential of acrylonitriles as novel trypanocidal agents.

In Vitro Susceptibility of Kinetoplastids to Celastroloids from Maytenus chiapensis.

Leishmaniasis and Chagas are among the most significant neglected tropical diseases. Due to several drawbacks with the current chemotherapy, developing new antikinetoplastid drugs has become an urgent issue. In the present work, a bioassay-guided investigation of the root bark of Maytenus chiapensis on Leishmania amazonensis and Trypanosoma cruzi led to the identification of two D:A-friedo-nor-oleanane triterpenoids (celastroloids), 20ß-hydroxy-tingenone (celastroloid 5) and 3-O-methyl-6-oxo-tingenol (celastroloid 8), as promising antikinetoplastid leads. They displayed higher potency on L. amazonensis promastigotes (50% inhibitory concentrations [IC50s], 0.44 and 1.12 µM, respectively), intracellular amastigotes (IC50s, 0.83 and 1.91 µM, respectively), and T. cruzi epimastigote stage (IC50s, 2.61 and 3.41 µM, respectively) than reference drugs miltefosine and benznidazole. This potency was coupled with an excellent selectivity index on murine macrophages. Mechanism of action studies, including mitochondrial membrane potential (Δψm) and ATP-level analysis, revealed that celastroloids could induce apoptotic cell death in L. amazonensis triggered by the mitochondria. In addition, the structure-activity relationship is discussed. These findings strongly underline the potential of celastroloids as lead compounds to develop novel antikinetoplastid drugs.

New phenalenone analogues with improved activity against Leishmania species.

The in vitro activity against Leishmania spp. of five novel designed compounds, phenalenone derivatives, is described in this study. Previous works have shown that some phenalenones present leishmanicidal activity, some of which could induce programmed cell death events in L. amazonensis parasites. In this research, we focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure and mitochondrial membrane potential, among others. The results showed that the new derivatives have comparable or better activity and selectivity than the commonly prescribed anti-leishmanial drug. This result was obtained by inducing stronger mitochondrial depolarization or more intense phosphatidylserine exposure than miltefosine, highlighting compound 8 with moreover 9-times better selectivity index. In addition, the new five molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing.

Sesquiterpenoids and flavonoids from Inula viscosa induce programmed cell death in kinetoplastids.

Neglected tropical diseases such as leishmaniasis and American trypanosomiasis represent an increasing health problem. Current treatments are not satisfactory which remains an urgent need for novel, cheap and safe chemotherapies. In the course of our ongoing search for new potential anti-protozoal agents, this study aimed to perform a bio-guided fractionation of Inula viscosa (Asteraceae) using in vitro assays against three strains of Leishmania and Trypanosma genus. Eight known compounds were identified from the ethanolic extract of leaves, sesquiterpenoids (3 and 4) and flavonoids (5 and 6) were characterized as the main bioactive constituents. Sesquiterpene lactones 3 and 4 (IC50 values between 4.99 and 14.26 µM) showed promising antiparasitic activity against promastigotes of L. donovani, L. amazonensis and epimastigotes of T. cruzi. Their structures were successfully characterized by spectroscopic techniques including 1D and 2D NMR experiments. Furthermore, the main bioactive compounds 4, 5 and 6 displayed higher potency (IC50 values between 0.64 and 2.13 µM) against amastigotes of L. amazonensis than miltefosine (IC50 3.11 µM), and a low toxicity on macrophages cell line (SI > 45). The analysis of structure-activity relationship (SAR) of the anti-protozoal activity revealed that lactonization or oxidation enhanced the biological profile, suggesting that the hydrophobic moiety was presumably involved in the activity by increasing the affinity and/or cell membrane permeability. In order to get an insight into the mechanism of action of these compounds, programmed cell death (PCD) experiments were performed, and the obtained results suggest that the reported compounds induced PCD in the treated parasites. These results highlight that sesquiterpenoids and flavonoids from I. viscosa could constitute an interesting scaffold for the development of novel antikinetoplastid agents.

Silver Nanoparticles as a Novel Potential Preventive Agent against Acanthamoeba Keratitis.

Free living, cosmopolitan amoebae from Acanthamoeba genus present a serious risk to human health. As facultative human parasites, these amoebae may cause Acanthamoeba keratitis (AK). Acanthamoeba keratitis is a severe, vision-threatening corneal infection with non-specific symptoms. The number of reported AK cases worldwide has been increasing every year. Moreover, 90% of Acanthamoeba keratitis cases are related to contact lens use. Wearing and storage contact lenses not in accordance with the physicians and manufacturers recommendations are the primary key risk factors of this disease. Amoebae can easily adhere to the contact lens surface and transmit to the corneal epithelium. Preventing amoebae adhesion to the contact lens surface could significantly decrease the number of AK infections. Until now, the effective therapy against AK is still under development. Currently proposed therapies are mainly limited to the chlorhexidine digluconate combined with propamidine isethionate or hexamidine applications, which are insufficient and very toxic to the eye. Due to lack of effective treatment, looking for new potential preventive agents is crucial to decrease the number of Acanthamoeba keratitis infections, especially among contact lens users. Nanoparticles have been already included in several novel therapies against bacteria, viruses, fungi, and protist. However, their anti-amoebic potential has not been fully tested yet. The aim of this study was to assess silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) anti-amoebic activity and influence on the amoebae adhesion to the surface of four different groups of contact lenses-classified according to the Food and Drugs Administration (FDA) guidelines. The obtained results show that both tested nanoparticles were effective against Acanthamoeba trophozoites and decreased the amoebae adhesion to the contact lens surface. AgNPs showed better anti-amoebic activity to cytotoxicity dependence and reduced amoebae adhesion in a wider spectrum of the tested contact lenses. Our studies also confirmed that ionization next to hydration of the contact lens material is a crucial parameter influencing the Acanthamoeba adhesion to the contact lens surface. In conclusion, silver nanoparticles might be considered as a novel preventive agent against Acanthamoeba keratitis infection.

Antikinetoplastid Activity of Indolocarbazoles from Streptomyces sanyensis.

Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of Trypanosoma and Leishmania genera that affect poor and remote populations in developing countries. These parasites share similar complex life cycles and modes of infection. It has been demonstrated that the particular group of phosphorylating enzymes, protein kinases (PKs), are essential for the infective mechanisms and for parasite survival. The natural indolocarbazole staurosporine (STS, 1) has been extensively used as a PKC inhibitor and its antiparasitic effects described. In this research, we analyze the antikinetoplastid activities of three indolocarbazole (ICZs) alkaloids of the family of staurosporine STS, 2-4, and the commercial ICZs rebeccamycin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) in order to establish a plausive approach to the mode of action and to provide a preliminary qualitative structure-activity analysis. The most active compound was 7-oxostaurosporine (7OSTS, 2) that showed IC50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, and a Selectivity Index (CC50/IC50) of 52 against amastigotes of L. amazonensis compared to the J774A.1 cell line of mouse macrophages.

In vitro activity of 1H-phenalen-1-one derivatives against Leishmania spp. and evidence of programmed cell death.

BACKGROUND: The in vitro activity against Leishmania spp. of a novel group of compounds, phenalenone derivatives, is described in this study. Previous studies have shown that some phenalenones present leishmanicidal activity, and induce a decrease in the mitochondrial membrane potential in L. amazonensis parasites, so in order to elucidate the evidence of programmed cell death occurring inside the promastigote stage, different assays were performed in two different species of Leishmania. METHODS: We focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure, mitochondrial membrane potential, and chromatin condensation among others. RESULTS: The results showed that four molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing. CONCLUSIONS: The present results highlight the potential use of phenalenone derivatives against Leishmania species and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.

Withanolides from Withania aristata as Antikinetoplastid Agents through Induction of Programmed Cell Death.

Leishmaniasis and American trypanosomiasis are parasitic diseases that cause significant clinical, social and economic impact on the population of tropical and subtropical countries. Their current treatment is limited and presents multiple drawbacks, including high toxicity, high cost, lengthy treatment plans, as well as the emergence of resistant species. Therefore, there is a need to find new lead compounds with high potency against parasites and low toxicity in patients. In the present work, the bioguided fractionation of an endemic plant from the Canary Islands, Withania aristata, led to the identification of withanolide-type metabolites (1-3) with leishmanicidal and trypanocidal activities. Compounds 1 and 3 showed a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and T. cruzi epimastigotes, higher than the reference drugs, miltefosine and benznidazole, respectively. Moreover, compounds 1-3 were more potent (IC50 0.055-0.663 µM) than the reference drug against the intracellular amastigote stage of L. amazonensis, with a high selectivity index on murine macrophage cells (SI 58.66-216.73). Studies on the mechanism of death showed that the compounds induced programmed cell death or that which was apoptosis-like. The present findings underline the potential of withanolides as novel therapeutic antikinetoplastid agents.