Natural endoperoxides as promising anti-leishmanials.

BACKGROUND: The discovery of artemisinin, an endoperoxide, encouraged the scientific community to explore endoperoxides as potential anti-parasitic molecules. Although artemisinin derivatives are rapidly evolving as potent anti-malarials, their potential as anti-leishmanials is emerging gradually. The treatment of leishmaniasis, a group of neglected tropical diseases is handicapped by lack of effective vaccines, drug toxicities and drug resistance. The weak antioxidant defense mechanism of the Leishmania parasites due to lack of catalase and a selenium dependent glutathione peroxidase system makes them vulnerable to oxidative stress, and this has been successful exploited by endoperoxides. PURPOSE: The study aimed to review the available literature on the anti-leishmanial efficacy of natural endoperoxides with a view to achieve insights into their mode of actions. METHODS: We reviewed more around 110 research and review articles restricted to the English language, sourced from electronic bibliographic databases including PubMed, Google, Web of Science, Google scholar etc. RESULTS: Natural endoperoxides could potentially augment the anti-leishmanial drug library, with artemisinin and ascaridole emerging as potential anti-leishmanial agents. Due to higher reactivity of the cyclic peroxide moiety, and exploiting the compromised antioxidant defense of Leishmania, endoperoxides like artemisinin and ascaridole potentiate their leishmanicidal efficacy by creating a redox imbalance. Furthermore, these molecules minimally impair oxidative phosphorylation; instead inhibit glycolytic functions, culminating in depolarization of the mitochondrial membrane and depletion of ATP. Additionally, the carbon-centered free radicals generated from endoperoxides, participate in chain reactions that can generate even more reactive organic radicals that are toxic to macromolecules, including lipids, proteins and DNA, leading to cell cycle arrest and apoptosis of Leishmania parasites. However, the precise target(s) of the toxic free radicals remains open-ended. CONCLUSION: In this overview, the spectrum of natural endoperoxide molecules as major anti-leishmanials and their mechanism of action has been delineated. In view of the substantial evidence that natural endoperoxides (e.g., artemisinin, ascaridole) exert a noxious effect on different species of Leishmania, identification and characterization of other natural endoperoxides is a promising therapeutic option worthy of further pharmacological consideration.

Compounds with potentialities as novel chemotherapeutic agents in leishmaniasis at preclinical level.

Leishmaniasis are neglected infectious diseases caused by kinetoplastid protozoan parasites from the genus Leishmania. These sicknesses are present mainly in tropical regions and almost 1 million new cases are reported each year. The absence of vaccines, as well as the high cost, toxicity or resistance to the current drugs determines the necessity of new treatments against these pathologies. In this review, several compounds with potentialities as new antileishmanial drugs are presented. The discussion is restricted to the preclinical level and molecules are organized according to their chemical nature, source and molecular targets. In this manner, we present antimicrobial peptides, flavonoids, withanolides, 8-aminoquinolines, compounds from Leish-Box, pyrazolopyrimidines, and inhibitors of tubulin polymerization/depolymerization, topoisomerase IB, proteases, pteridine reductase, N-myristoyltransferase, as well as enzymes involved in polyamine metabolism, response against oxidative stress, signaling pathways, and sterol biosynthesis. This work is a contribution to the general knowledge of these compounds as antileishmanial agents.

Larvicidal and Adulticidal Activity of Essential Oils from Four Cuban Plants against Three Mosquito Vector Species.

Mosquitoes are one of the main vectors of many important diseases and their degree of resistance to chemical insecticides has increased. Nowadays, it has become crucial to identify novel plant larvicides with an eco-friendly impact. The components of essential oils from Croton linearis Jacq. (EO-Cl), Lantana involucrata L. (EO-Li), Ocimum sanctum var. cubensis M. Gómez. (EO-Os), and Zanthoxylum pistaciifolium Griseb. (syn. Zanthoxylum flavum subsp. pistaciifolium (Griseb.) Reynel (EO-Zp) were determined using gas chromatography-mass spectrometry (GC-MS) analysis. Larvicidal and adulticidal bioassays against Aedes aegypti, Anopheles albitarsis and Culex quinquefasciatus, were performed according to the World Health Organization standard methods. A high diversity of compounds was identified in the four oils, with a total of 152 compounds (33-70 components). EO-Cl, EO-Li, and EO-Os were classified as active against both insect forms, larvae and adults. Lantana involucrata showed the best results, with LC50 values from 33.8 to 41.7 mg/L. In most of the cases, it was not possible to associate the main compounds with the measured activity, supporting the hypothesis about probable synergistic interactions among major and minor compounds. The results indicate EO-Cl, EO-Os, and EO-Li as good eco-friendly insecticides with potential.

Heteroctenus junceus Scorpion Venom Modulates the Concentration of Pro-Inflammatory Cytokines in F3II Tumor Cells.

The ability of Heteroctenus junceus scorpion venom to modulate the concentration of cytokines related to its antitumoral effect is unknown. F3II cells were treated with » IC50, ½ IC50 and the IC50 of H. junceus scorpion venom. Tumor growth kinetics in F3II-bearing mice were evaluated after 24 days of oral administration of venom doses. The effect of tumor lysates on F3II cell viability was evaluated by MTT assay, while cytokines present in each sample were determined by ELISA. In supernatant, H. junceus scorpion venom decreased the concentration of IL-6 (p < 0.001), IFN-γ (p < 0.001), IL-1ß (p < 0.01); meanwhile IL-12 (p < 0.001) and TNF-α (p < 0.001) levels increased significantly, according to the concentration and the time of incubation. Heteroctenus junceus scorpion venom effectively inhibits in vivo tumor progression. In the sera, a significant decrease was observed in TNF-α levels (p < 0.05). In tumor lysates, IL-6 decreased significantly in the groups treated with 12.5 mg/kg (p < 0.001) and 25 mg/kg (p < 0.05). Heteroctenus junceus scorpion venom is capable of modulating other proinflammatory and protumoral cytokines involved in the inflammation associated with cancer.

Neglected Zoonotic Diseases: Advances in the Development of Cell-Penetrating and Antimicrobial Peptides against Leishmaniosis and Chagas Disease.

In 2020, the WHO established the road map for neglected tropical diseases 2021-2030, which aims to control and eradicate 20 diseases, including leishmaniosis and Chagas disease. In addition, since 2015, the WHO has been developing a Global Action Plan on Antimicrobial Resistance. In this context, the achievement of innovative strategies as an alternative to replace conventional therapies is a first-order socio-sanitary priority, especially regarding endemic zoonoses in poor regions, such as those caused by Trypanosoma cruzi and Leishmania spp. infections. In this scenario, it is worth highlighting a group of natural peptide molecules (AMPs and CPPs) that are promising strategies for improving therapeutic efficacy against these neglected zoonoses, as they avoid the development of toxicity and resistance of conventional treatments. This review presents the novelties of these peptide molecules and their ability to cross a whole system of cell membranes as well as stimulate host immune defenses or even serve as vectors of molecules. The efforts of the biotechnological sector will make it possible to overcome the limitations of antimicrobial peptides through encapsulation and functionalization methods to obtain approval for these treatments to be used in clinical programs for the eradication of leishmaniosis and Chagas disease.

A review of nemorosone: Chemistry and biological properties.

Nemorosone is a bicyclic polyprenylated acylphloroglucinol derivative originally isolated from Clusia spp. and it can be obtained through chemical synthesis employing different synthetic strategies. Since its discovery, it has attracted great attention both from a biological and chemical viewpoint. In the present article, we attempted to review various chemical and biological topics around nemorosone, with an emphasis on its antiproliferative activities. For this purpose, relevant data was collected from different scientific databases including Google Scholar, PubMed, Scopus and ISI Web of Knowledge. This natural compound has shown activity against several types of malignancies such as leukemia, human colorectal, pancreatic, and breast cancer because it modulates multiple molecular pathways. Nemorosone has both cytostatic and cytotoxic activity and it also seems to induce apoptosis and ferroptosis. Additionally, it has antimicrobial capabilities against Gram-positive bacteria and parasites belonging to genus Leishmania. Its promising antiproliferative pre-clinical effects deserve further attention for anticancer and anti-parasitic drug development and translation to the clinic.

Eugenol-Rich Essential Oil from Pimenta dioica: In Vitro and In Vivo Potentialities against Leishmania amazonensis.

Pimenta dioica L. is one the most recognized species with diverse biological activities. In this study, in vitro activity and in vivo efficacy of essential oil from P. dioica (EO-Pd) was evaluated. The main compound was also included in the animal studies and its in silico prediction related to biological activities, molecular ligands, drug likeness, and ADME (absorption, distribution, metabolism, and excretion) properties are listed. The chemical composition analyzed by GC-MS retrieved 45 components, which the most abundant compound was the eugenol (80.1%). The EO-Pd was able to inhibit the growth of L. amazonensis (IC50 = 9.7 ± 0.7 and 11.3 ± 2.1 µg/mL, promastigotes and amastigotes, respectively). The cytotoxicity assay showed a CC50 of 104.5 ± 0.9 µg/mL and a selectivity index of 9. In the model of cutaneous leishmaniasis in BALB/c mice, the effect of EO-Pd and eugenol was observed after treatment at 30 mg/kg by intralesional route with 5 administrations every 4 days. In the in silico predictions, some targets that justified the antileishmanial activity of eugenol and good drug like properties for this compound, were obtained. This study showed for first time the potential of EO-Pd to inhibit L. amazonensis, which could be linked to the activity of major compound eugenol.

Chemical Composition and In Vitro and In Silico Antileishmanial Evaluation of the Essential Oil from Croton linearis Jacq. Stems.

Croton linearis Jacq. is an aromatic shrub that has been utilized in traditional medicine in the Bahamas, Jamaica, and Cuba. Recent studies have revealed the antiprotozoal potential of its leaves. The present work is aimed to identify the volatile constituents of essential oil from the stems of C. linearis (CLS-EO) and evaluate its in vitro antileishmanial activity. In addition, an in silico study of the molecular interactions was performed using molecular docking. A gas chromatographic-mass spectrometric analysis of CLS-EO identified 1,8-cineole (27.8%), α-pinene (11.1%), cis-sabinene (8.1%), p-cymene (5.7%), α-terpineol (4.4%), epi-γ-eudesmol (4.2%), linalool (3.9%), and terpinen-4-ol (2.6%) as major constituents. The evaluation of antileishmanial activity showed that CLS-EO has good activity on both parasite forms (IC50Promastigote = 21.4 ± 0.1 µg/mL; IC50Amastigote = 18.9 ± 0.3 µg/mL), with a CC50 of 49.0 ± 5.0 µg/mL on peritoneal macrophages from BALB/c mice (selectivity index = 2 and 3 using the promastigote and amastigote results). Molecular docking showed good binding of epi-γ-eudesmol with different target enzymes of Leishmania. This study is the first report of the chemical composition and anti-Leishmania evaluation of CLS-EO. These findings provide support for further studies of the antileishmanial effect of this product.

Antileishmanial Anthracene Endoperoxides: Efficacy In Vitro, Mechanisms and Structure-Activity Relationships.

Leishmaniasis is a vector-borne disease caused by protozoal Leishmania parasites. Previous studies have shown that endoperoxides (EP) can selectively kill Leishmania in host cells. Therefore, we studied in this work a set of new anthracene-derived EP (AcEP) together with their non-endoperoxidic analogs in model systems of Leishmania tarentolae promastigotes (LtP) and J774 macrophages for their antileishmanial activity and selectivity. The mechanism of effective compounds was explored by studying their reaction with iron (II) in chemical systems and in Leishmania. The correlation of structural parameters with activity demonstrated that in this compound set, active compounds had a LogPOW larger than 3.5 and a polar surface area smaller than 100 Å2. The most effective compounds (IC50 in LtP < 2 µM) with the highest selectivity (SI > 30) were pyridyl-/tert-butyl-substituted AcEP. Interestingly, also their analogs demonstrated activity and selectivity. In mechanistic studies, it was shown that EP were activated by iron in chemical systems and in LtP due to their EP group. However, the molecular structure beyond the EP group significantly contributed to their differential mitochondrial inhibition in Leishmania. The identified compound pairs are a good starting point for subsequent experiments in pathogenic Leishmania in vitro and in animal models.

Inhibition of Bacterial Adhesion and Biofilm Formation by Seed-Derived Ethanol Extracts from Persea americana Mill.

The increase in antibiotic resistance demands innovative strategies to combat microorganisms. The current study evaluated the antibacterial and antivirulence effects of ethanol extracts from Persea americana seeds obtained by the Soxhlet (SE) and maceration (MaE) methods. The UHPLC-DAD-QTOF analysis showed mainly the presence of polyphenols and neolignan. Ethanol extracts were not cytotoxic to mammalian cells (CC50 > 500 µg/mL) and displayed a moderate antibacterial activity against Pseudomonas aeruginosa (IC50 = 87 and 187 µg/mL) and Staphylococcus aureus (IC50 = 144 and 159 µg/mL). Interestingly, no antibacterial activity was found against Escherichia coli. SE and MaE extracts were also able to significantly reduce the bacterial adhesion to A549 lung epithelial cells. Additionally, both extracts inhibited the biofilm growth at 24 h and facilitated the release of internal cell components in P. aeruginosa, which might be associated with cell membrane destabilization. Real-time PCR and agarose electrophoresis gel analysis indicated that avocado seed ethanol extracts (64 µg/mL) downregulated virulence-related factors such as mexT and lasA genes. Our results support the potential of bioproducts from P. americana seeds as anti-adhesive and anti-biofilm agents.

Imported cases of cutaneous leishmaniasis in Cuba, 2017: role of human movement.

BACKGROUND: Leishmaniasis is a vector-borne disease caused by several species from genus Leishmania. An increase in the number of cases related to human movement has been informed in the last years. Due to the increase of suspicious leishmaniasis cases arriving in Cuba during 2017, a general analysis is presented herein. METHODS: Clinical samples were collected from 5 patients suspicious of leishmaniasis, received from January to December 2017 at the Institute of Tropical Medicine Pedro Kourí, Cuba. Skin lesion samples were analyzed using different diagnostic assays: direct smear, histological examination, and molecular analysis for species identification. Epidemiological and demographic data were requested from each case and analyzed. Treatment and follow up of patient was also performed. RESULTS: Five cases were confirmed as Leishmania infection according to microscopic observation and molecular methods results. PCR-18S, PCR-N/RFLP and PCR-F/RFLP identified the following species: L. panamensis (2 cases), L. braziliensis (1 case), L.panamensis/L.guyanensis (1 case), L. mexicana complex (1 case). In treated patients, drugs were well tolerated, cure were documented and no relapse have been currently reported (3 years later). CONCLUSIONS: Clinical characteristics, demographic data, and epidemiological features of infection for each case evidence the potential risk related with travel to endemic areas of leishmaniasis. KEYWORKS: Cutaneous leishmaniasis, Epidemiology, Imported cases.

In Vitro Pharmacological Screening of Essential Oils from Baccharis parvidentata and Lippia origanoides Growing in Brazil.

In this study, the in vitro antimicrobial, antiparasitic, antiproliferative and cytotoxic activities of essential oil from Baccharis parvidentata Malag. (EO-Bp) and Lippia origanoides Kunth (EO-Lo) were explored. The relevant effects were observed against the parasitic protozoans Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei and Leishmania amazonensis (ranging 0.6 to 39.7 µg/mL) and malignant MCF-7, MCF-7/HT, 22Rv1, and A431 cell lines (ranging 6.1 to 31.5 µg/mL). In parallel, EO-Bp showed better selective indexes in comparison with EO-Lo against peritoneal macrophages from BALB/c mice and MRC-5 cell line. In conclusion, EO-Lo is known to show a wide range of health benefits that could be added as another potential use of this oil with the current study. In the case of EO-Bp, the wide spectrum of its activities against protozoal parasites and malignant cells, as well as its selectivity in comparison with non-malignant cells, could suggest an interesting candidate for further tests as a new therapeutic alternative.

Machine Learning Analysis of Essential Oils from Cuban Plants: Potential Activity against Protozoa Parasites.

Essential oils (EOs) are a mixture of chemical compounds with a long history of use in food, cosmetics, perfumes, agricultural and pharmaceuticals industries. The main object of this study was to find chemical patterns between 45 EOs and antiprotozoal activity (antiplasmodial, antileishmanial and antitrypanosomal), using different machine learning algorithms. In the analyses, 45 samples of EOs were included, using unsupervised Self-Organizing Maps (SOM) and supervised Random Forest (RF) methodologies. In the generated map, the hit rate was higher than 70% and the results demonstrate that it is possible find chemical patterns using a supervised and unsupervised machine learning approach. A total of 20 compounds were identified (19 are terpenes and one sulfur-containing compound), which was compared with literature reports. These models can be used to investigate and screen for bioactivity of EOs that have antiprotozoal activity more effectively and with less time and financial cost.

LC-MS Characterization and Biological Activities of Cuban Cultivars of Plectranthus neochilus Schltr.

Plectranthus neochilus Schltr. (Lamiaceae) is a plant recently introduced in Cuba. Worldwide, it is an ethnomedicinal alternative for its use against microbial infections, but the Cuban population use the extracts to treat sleep disorders. To address this apparent incongruity, four collections (from different seasonal conditions in the year) of Cuban P. neochilus cultivars were analyzed in terms of their pharmacognostic characteristics. Three extracts using fresh and dried leaves were chemically and biologically characterized. UPLC-DAD-MS/MS analysis was performed to determine their chemical composition, while a panel of nine microorganisms was used to evaluate their antimicrobial activity. Finally, cytotoxic effects of different fractions were measured in three cell lines by the resazurin viability assay. In contrast to previously reported micro and macromorphological properties of P. neochilus, the leaves from the Cuban cultivars did not present glandular trichomes, nor did they produce quantifiable levels of essential oils. Moreover, aqueous extracts used by the population revealed no significant antimicrobial activity and were not cytotoxic. The three extracts showed a similar phytochemical composition, i.e., eight flavonoids, seven abietane diterpenes, and rosmarinic acid as the major constituent, most of them reported for the first time in this species. The low yield of essential oil, the absence of glandular trichomes, compounds with a high level of oxidation, and a moderate antimicrobial activity detected were the most distinctive pharmacognostic and biological characteristics of P. neochilus grown in Cuba. These aspects could explain its non-use as an antimicrobial.

Bioactive Essential Oils from Cuban Plants: An Inspiration to Drug Development.

Aromatic plants and essential oils are important agents as complementary and alternative medicines in many cultures and geographical locations. In this review, a literature search on essential oils from Cuba, their chemical compositions, and their pharmacological properties was carried out. Out of 171 published scientific articles on essential oils of Cuban plants, a total of 31 documents, focused on both chemical composition and pharmacological properties, were considered for this review. In general, an increase in articles published in the last decade was noted, particularly in recognized international journals in English. Myrtaceae and Piperaceae were the most representative families collected in the occidental area of the country. Leaves and aerial parts were predominantly used, while a wide and variable number of components were identified, including terpenes, aliphatic derivatives, sulfur-containing compounds, phenylpropanoids, alkaloids and amine-type compounds. Finally, different biological activities were reported such as antiprotozoal, antibacterial, antifungal, cytotoxic, anthelmintic, larvicidal and insecticidal. In conclusion, we encourage further studies that would promote the use of essential oils from Cuban plants in new pharmaceutical products.

Synthesis of 7-amino-6-halogeno-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides: a way forward for targeting hypoxia and drug resistance of cancer cells.

To establish a new approach for the synthesis of quinoxaline 1,4-dioxides as hypoxia-selective cytotoxic agents, an original multi-step preparation of derivatives possessing the diamine moiety at position 7 was evaluated. Herein, we present the synthesis of a series of novel 7-amino-6-halogeno-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 13a-h, 14a,b,g based on the regioselective Beirut reaction. Comparison of antitumor properties of derivatives possessing the diamine moiety at position 7 with structurally close congeners possessing the corresponding amino groups at position 6 revealed key differences in the cytotoxicity profiles and HIF-1α inhibition. All the synthesized 7-amino-6-halogeno derivatives 13a-h, 14a,b,g demonstrated significant cytotoxic activities against breast cancer cell lines (MCF7, MDA-MB-231) in normoxia and hypoxia with IC50 values ranging from 0.1 to 7.6 µM. Most of these novel derivatives can circumvent the multidrug resistance of tumor cells caused by P-glycoprotein over expression. The lead compounds 13a, 14a and 14b can suppress the expression of HIF-1α at low micromolar concentrations and induce apoptosis in breast cancer MCF7 cells. In addition, compound 14b effectively inhibits BCL2 and ERα expression in MCF7 cells. The current research opens a new direction for targeting hypoxia and drug resistance of cancer cells.

Leishmania amazonensis response to artemisinin and derivatives.

The worldwide presence of Leishmania parasites increases in the poorest regions. Current leishmaniasis treatments are unsatisfactory due to resistance development, side effects and cost. Herein, we describe the in vitro activity of artemisinin (ART), artemether (ATM), artesunate (ATS) and dihydroartemisinin (DHA) against Leishmania amazonensis. Selected compounds were assayed in the animal model of cutaneous leishmaniasis in BALB/c mice. On intracellular amastigotes, similar activity (p > 0.05) was observed for ART, ATM and ATS (IC50 = 15.0-19.2 µM), which were inferior (p < 0.05) respect to reference endoperoxide ascaridole (IC50 = 11.5 ±â€¯1.0 µM) and superior (p < 0.05) compared with reference drug Glucantime® (IC50 = 30.1 ±â€¯9.0 µM). In contrast, DHA (IC50 = 38.5 ±â€¯4.7 µM) showed higher IC50 values (p < 0.05) than other artemisinins and ascaridole, but similar (p > 0.05) than Glucantime®; while deoxyartemisinin caused smaller inhibition (IC50 = 88.9 ±â€¯5.2 µM). Selectivity indexes of >13, 6, 11 and 1 were obtained for ART, ATM, ATS and DHA, respectively. In addition, the potential effect of ART and ATS was also demonstrated in the murine model, causing a significant reduction (p < 0.05) of the lesion size and parasite load regarding untreated animals and treated with vehicle. Effects of both artemisinins were comparable (p > 0.05) with Glucantime® and ascaridole-treated mice. In particular, artemisinin is recommended to further studies, which could be an advantage over the ascaridole endoperoxide and could be useful in endemic areas of parasite resistance to antimonials.

Essential Oil from Melaleuca leucadendra: Antimicrobial, Antikinetoplastid, Antiproliferative and Cytotoxic Assessment.

Essential oils (EOs) are known for their use in cosmetics, food industries, and traditional medicine. This study presents the chemical composition and therapeutic properties against kinetoplastid and eukaryotic cells of the EO from Melaleucaleucadendra (L.) L. (Myrtaceae). Forty-five compounds were identified in the oil by GC-MS, containing a major component the 1,8-cineole (61%). The EO inhibits the growth of Leishmania amazonensis and Trypanosoma brucei at IC50 values <10 µg/mL. However, 1,8 cineole was not the main compound responsible for the activity. Against malignant (22Rv1, MCF-7, EFO-21, including resistant sublines MCF-7/Rap and MCF-7/4OHTAMO) and non-malignant (MCF-10A, J774A.1 and peritoneal macrophage) cells, IC50 values from 55 to 98 µg/mL and from 94 to 144 µg/mL were obtained, respectively. However, no activity was observed on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida parapsilosis, Microsporum canis, or Trypanosoma cruzi. The EO was able to control the lesion size and parasite burden in the model of cutaneous leishmaniasis in BALB/c mice caused by L. amazonensis compared to untreated animals (p < 0.05) and similar with those treated with Glucantime® (p > 0.05). This work constitutes the first evidence of antiproliferative potentialities of EO from M. leucadendra growing in Cuba and could promote further preclinical investigations to confirm the medical value of this plant, in particular for leishmaniasis treatment.

Therapeutic Potential of Quercetin: New Insights and Perspectives for Human Health.

Quercetin (Que) and its derivatives are naturally occurring phytochemicals with promising bioactive effects. The antidiabetic, anti-inflammatory, antioxidant, antimicrobial, anti-Alzheimer's, antiarthritic, cardiovascular, and wound-healing effects of Que have been extensively investigated, as well as its anticancer activity against different cancer cell lines has been recently reported. Que and its derivatives are found predominantly in the Western diet, and people might benefit from their protective effect just by taking them via diets or as a food supplement. Bioavailability-related drug-delivery systems of Que have also been markedly exploited, and Que nanoparticles appear as a promising platform to enhance their bioavailability. The present review aims to provide a brief overview of the therapeutic effects, new insights, and upcoming perspectives of Que.

Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link.

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.