Impact of the zinc complexation of polytopic polyaza ligands on the interaction with double and single stranded DNA/RNA and antimicrobial activity.

Metal complexes have gained a huge interest in the biomedical research in the last decade because of the access to unexplored chemical space with regards to organic molecules and to present additional functionalities to act simultaneously as diagnostic and therapeutic agents. Herein, we evaluated the interaction of two polytopic polyaza ligands and their zinc complexes with DNA and RNA by UV thermal denaturation, fluorescence and circular dichroism spectroscopic assays. The zinc coordination was investigated by X-ray diffraction and afforded the structure of the binuclear zinc complex of PYPOD. Thermal denaturation of DNA and RNA and fluorimetry analysis revealed preferential binding of the zinc-PHENPOD complexes towards GC-containing DNA in contrast to the free ligands. On the other hand, PYPOD metal complexes, compared to the free ligand, stabilized AT-based DNA (B-form) better than AU-RNA (A-form). With regards to single stranded RNA, the binuclear complex of PHENPOD and the free ligand can efficiently identify polyadenylic acid (poly A) among other RNA sequences by circular dichroism spectroscopy. The antimicrobial activity in S. aureus and E. coli bacteria showed the highest activity for the free ligands and their trinuclear zinc complexes. This work can provide valuable insights into the impact of the nuclearity of polytopic polyaza ligands in the binding to DNA/RNA and the antimicrobial effect.

Flavonoids from Piper Species as Promising Antiprotozoal Agents against Giardia intestinalis. Structure-Activity Relationship and Drug-Likeness Studies.

Diarrhea diseases caused by the intestinal protozoan parasite Giardia intestinalis are a major global health burden. Moreover, there is an ongoing need for novel anti-Giardia drugs due to drawbacks with currently available treatments. This paper reports on the isolation and structural elucidation of six new flavonoids (1-6), along with twenty-three known ones (7-29) from the Piper species. Their structures were established by spectroscopic and spectrometric techniques. Flavonoids were tested for in vitro antiprotozoal activity against Giardia intestinalis trophozoites. In addition, structure-activity relationship (SAR) and in silico ADME studies were performed to understand the pharmacophore and pharmacokinetic properties of these natural compounds. Eight flavonoids from this series exhibited remarkable activity in the micromolar range. Moreover, compound 4 was identified as having a 40-fold greater antiparasitic effect (IC50 61.0 nM) than the clinical reference drug, metronidazole (IC50 2.5 µM). This antiprotozoal potency was coupled with an excellent selectivity index (SI 233) on murine macrophages and in silico drug-likeness. SAR studies revealed that the substitution patterns, type of functional group, and flavonoid skeleton played an essential role in the activity. These findings highlight flavonoid 4 as a promising candidate to develop new drugs for the treatment of Giardia infections.

Potential Risk of Three Zoonotic Protozoa (Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii) Transmission from Fish Consumption.

In recent decades, worldwide fish consumption has increased notably worldwide. Despite the health benefits of fish consumption, it also can suppose a risk because of fishborne diseases, including parasitic infections. Global changes are leading to the emergence of parasites in new locations and to the appearance of new sources of transmission. That is the case of the zoonotic protozoa Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii; all of them reach aquatic environments and have been found in shellfish. Similarly, these protozoa can be present in other aquatic animals, such as fish. The present review gives an overview on these three zoonotic protozoa in order to understand their potential presence in fish and to comprehensively revise all the evidences of fish as a new potential source of Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii transmission. All of them have been found in both marine and freshwater fishes. Until now, it has not been possible to demonstrate that fish are natural hosts for these protozoa; otherwise, they would merely act as mechanical transporters. Nevertheless, even if fish only accumulate and transport these protozoa, they could be a "new" source of infection for people.

(E)-Piplartine Isolated from Piper pseudoarboreum, a Lead Compound against Leishmaniasis.

The current therapies of leishmaniasis, the second most widespread neglected tropical disease, have limited effectiveness and toxic side effects. In this regard, natural products play an important role in overcoming the current need for new leishmanicidal agents. The present study reports a bioassay-guided fractionation of the ethanolic extract of leaves of Piper pseudoarboreum against four species of Leishmania spp. promastigote forms, which afforded six known alkamides (1-6). Their structures were established on the basis of spectroscopic and spectrometric analysis. Compounds 2 and 3 were identified as the most promising ones, displaying higher potency against Leishmania spp. promastigotes (IC50 values ranging from 1.6 to 3.8 µM) and amastigotes of L. amazonensis (IC50 values ranging from 8.2 to 9.1 µM) than the reference drug, miltefosine. The efficacy of (E)-piplartine (3) against L. amazonensis infection in an in vivo model for cutaneous leishmaniasis was evidenced by a significant reduction of the lesion size footpad and spleen parasite burden, similar to those of glucantime used as the reference drug. This study reinforces the therapeutic potential of (E)-piplartine as a promising lead compound against neglected infectious diseases caused by Leishmania parasites.

An unprecedented chlorine-containing piperamide from Piper pseudoarboreum as potential leishmanicidal agent.

A phytochemical investigation of the ethanolic extract of leaves from Piper pseudoarboreum led to the isolation of 3-chlorosintenpyridone 1, an unprecedented chlorinated piperamide, together with the known compounds 2-12. Their structures were established based on 1D and 2D (COSY, ROESY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The proposed biosynthetic pathway of compound 1 is discussed. Compounds 1-12 were tested in vitro for their leishmanicidal potential against promastigote stages of Leishmania amazonensis, L braziliensis, L. guyanensis and L. infantum. Two compounds from this series, the alkamide 1 (IC50 3.4-5.2 µM) and the fatty acid 9 (IC50 18.7-29.6 µM) displayed higher or similar potency to Miltefosine, used as the reference drug.

Synthesis and Biological Studies of (+)-Liquiditerpenoic Acid A (Abietopinoic Acid) and Representative Analogues: SAR Studies.

The first semisynthesis and biological profiling of the new abietane diterpenoid (+)-liquiditerpenoic acid A (abietopinoic acid) (7) along with several analogues are reported. The compounds were obtained from readily available methyl dehydroabietate (8), which was derived from (-)-abietic acid (1). Biological comparison was conducted according to the different functional groups, leading to some basic structure-activity relationships (SAR). In particular, the ferruginol and sugiol analogues 7 and 10-16 were characterized by the presence of an acetylated phenolic moiety, an oxidized C-7 as a carbonyl, and a different functional group at C-18 (methoxycarbonyl, carboxylic acid, and hydroxymethyl). The biological properties of these compounds were investigated against a panel of six representative human tumor solid cells (A549, HBL-100, HeLa, SW1573, T-47D, and WiDr), five leukemia cellular models (NALM-06, KOPN-8, SUP-B15, UoCB1, and BCR-ABL), and four Leishmania species ( L. infantum, L. donovani, L. amazonensis, and L. guyanensis). A molecular docking study pointed out some targets in these Leishmania species. In addition, the ability of the compounds to modulate GABAA receptors (α1ß2γ2s) is also reported. The combined findings indicate that these abietane diterpenoids offer a source of novel bioactive molecules with promising pharmacological properties from cheap chiral-pool building blocks.

Applying Loop-mediated Isothermal Amplification (LAMP) in the Diagnosis of Malaria, Leishmaniasis and Trypanosomiasis as Point-of-Care Tests (POCTs).

One of the main objectives of the WHO is controlling transmission of parasitic protozoa vector- borne diseases. A quick and precise diagnosis is critical in selecting the optimal therapeutic regime that avoids unnecessary treatments and the emergence of resistance. Molecular assays based on Loop- Mediated Isothermal Amplification (LAMP) techniques are a good alternative to light microscopy and antigen-based rapid diagnostic tests in developing countries, since they allow for a large amount of genetic material generated from a few copies of DNA, and use primers that lead to high sensitivity and specificity, while the amplification process can be performed in isothermal conditions without the need of sophisticated equipment to interpret the results. In this review, the main advances in the development of LAMP assays for the diagnosis of malaria, leishmaniasis and Chagas' disease are discussed as well as the feasibility of their implementation in developing countries and use as point- of-care diagnostic tests.

Leishmaniasis in the major endemic region of Plurinational State of Bolivia: Species identification, phylogeography and drug susceptibility implications.

The Plurinational State of Bolivia is one of the Latin American countries with the highest prevalence of leishmaniasis, highlighting the lowlands of the Department of La Paz where about 50% of the total cases were reported. The control of the disease can be seriously compromised by the intrinsic variability of the circulating species that may limit the efficacy of treatment while favoring the emergence of resistance. Fifty-five isolates of Leishmania from cutaneous and mucocutaneous lesions from patients living in different provinces of the Department of La Paz were tested. Molecular characterization of isolates was carried out by 3 classical markers: the rRNA internal transcribed spacer 1 (ITS-1), the heat shock protein 70 (HSP70) and the mitochondrial cytochrome b (Cyt-b). These markers were amplified by PCR and their products digested by the restriction endonuclease enzymes AseI and HaeIII followed by subsequent sequencing of Cyt-b gene and ITS-1 region for subsequent phylogenetic analysis. The combined use of these 3 markers allowed us to assign 36 isolates (65.5%) to the complex Leishmania (Viannia) braziliensis, 4 isolates (7, 27%) to L. (Viannia) lainsoni. and the remaining 15 isolates (23.7%) to a local variant of L. (Leishmania) mexicana. Concerning in vitro drug susceptibility the amastigotes from all isolates where highly sensitive to Fungizone® (mean IC50 between 0.23 and 0.5µg/mL) whereas against Glucantime® the sensitivity was moderate (mean IC50 ranging from 50.84µg/mL for L. (V.) braziliensis to 18.23µg/mL for L. (L.) mexicana. L. (V.) lainsoni was not sensitive to Glucantime®. The susceptibility to miltefosine was highly variable among species isolates, being L. (L.) mexicana the most sensitive, followed by L. (V.) braziliensis and L. (V.) lainsoni (mean IC50 of 8.24µg/mL, 17.85µg/mL and 23.28µg/mL, respectively).

Engineering Synergistically Active and Bioavailable Cost-effective Medicines for Neglected Tropical Diseases; The Role of Excipients.

Leishmaniasis is a neglected tropical disease responsible for the ninth largest disease burden in the world threatening 350 million people mostly in developing countries. The lack of efficacy, severe adverse effects, long duration, high cost and parenteral administration of the current therapies result in poor patient compliance and emergence of resistance. Leishmaniasis' unmet need for safer, affordable and more effective treatments is only partly addressed by today's global health product pipeline that focuses on products amenable to rapid clinical development, mainly by reformulating or repurposing existing drugs for new uses. Excipients are necessary for ensuring the stability and bioavailability of currently available antileishmaniasis drugs which in their majority are poorly soluble or have severe side-effects. Thus, selection of excipients that can ensure bioavailability and safety as well as elicit a synergistic effect against the Leishmania parasites without compromising safety will result in a more efficacious, safe and fast to market medicine. We have evaluated the in vitro activity of 30 commercially available generally regarded as safe (GRAS) excipients against different Leishmania spp., their cytotoxicity and potential use for inclusion in novel formulations. Amongst the tested excipients, the compounds with higher selectivity index were Eudragit E100 (cationic triblock copolymer of dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate), CTAB (cetyltrimethylammonium bromide, cationic), lauric acid, Labrasol(non-ionic, caprylocaproyl polyoxyl- 8 glycerides) and sodium deoxycholate. An ideal excipient need to possess amphiphilic nature with ionic/polar groups and possess a short or medium fatty acid chain such as lauric (C12), capric C10) or caprylicacid (C8). Inclusion of these excipients and identification of the optimal combination of drug and excipients would lead to a more effective and safer antileishmanial therapies.

Synthesis and antileishmanial activity of C7- and C12-functionalized dehydroabietylamine derivatives.

Abietane-type diterpenoids, either naturally occurring or synthetic, have shown a wide range of pharmacological actions, including antiprotozoal properties. In this study, we report on the antileishmanial evaluation of a series of (+)-dehydroabietylamine derivatives functionalized at C7 and/or C12. Thus, the activity in vitro against Leishmania infantum, Leishmania donovani, Leishmania amazonensis and Leishmania guyanensis, was studied. Most of the benzamide derivatives showed activities at low micromolar concentration against cultured promastigotes of Leishmania spp. (IC50 = 2.2-46.8 µM), without cytotoxicity on J774 macrophage cells. Compound 15, an acetamide, was found to be the most active leishmanicidal agent, though it presented some cytotoxicity on J774 cells. Among the benzamide derivatives, compounds 8 and 10, were also active against L. infantum intracellular amastigotes, being 18- and 23-fold more potent than the reference compound miltefosine, respectively. Some structure-activity relationships have been identified for the antileishmanial activity in these dehydroabietylamine derivatives.

Triazolopyridopyrimidines: an emerging family of effective DNA photocleavers. DNA binding. Antileishmanial activity.

Triazolopyridopyrimidines 3-phenyl-6,8-di(2-pyridyl)-[1,2,3]triazolo[5',1':6,1]pyrido[2,3-d]pyrimidine (1a), 6,8-di(pyridin-2-yl)-[1,2,3]triazolo[1',5':1,6]pyrido[2,3-d]pyrimidine (1b) and 3-methyl-6,8-di(2-pyridyl)-[1,2,3]triazolo[5',1':6,1]pyrido[2,3-d]pyrimidine (1c) were prepared and their electrochemical and luminescence properties were studied in depth. The DNA binding ability of this series of compounds has been investigated by means of UV-vis absorption and fluorescence titrations, steady-state emission quenching with ferrocyanide as well as viscosity measurements. Results have shown that triazolopyridopyrimidine 1a interacts strongly at DNA grooves. This compound also displays preferential binding to GC-rich sequences and the ability to photooxidize guanine. Moreover, these studies have revealed the key role of the phenyl substituent at the triazole ring in the binding affinity of 1a-c. Compounds 1b and 1c did not show appreciable propensity for DNA binding, however these triazolopyridopyrimidines demonstrated to present photoinduced DNA cleavage activity, 1b being more active than 1c. DNA photocleavage mediated by these compounds takes place mainly through single strand scission events and, in a minor extent, through double strand cuts. Mechanistic investigations using radical scavengers showed that both 1b and 1c generate reactive oxygen species (singlet oxygen, superoxide and hydroxyl radicals) upon irradiation. Both type I and type II mechanisms are involved in the photocleavage process. Furthermore, compounds 1a-c were tested for their antiprotozoal activity against four different Leishmania spp. (L. infantum, L. braziliensis, L. guyanensis and L. amazonensis). Triazolopyridopyrimidines 1a and 1c resulted to be more active and selective than the reference drug (miltefosine) in vitro against L. infantum amastigotes. Compound 1a exhibited high leishmanicidal activity against L. infantum spleen forms in the in vivo test.

Oral particle uptake and organ targeting drives the activity of amphotericin B nanoparticles.

There are very few drug delivery systems that target key organs via the oral route, as oral delivery advances normally address gastrointestinal drug dissolution, permeation, and stability. Here we introduce a nanomedicine in which nanoparticles, while also protecting the drug from gastric degradation, are taken up by the gastrointestinal epithelia and transported to the lung, liver, and spleen, thus selectively enhancing drug bioavailability in these target organs and diminishing kidney exposure (relevant to nephrotoxic drugs). Our work demonstrates, for the first time, that oral particle uptake and translocation to specific organs may be used to achieve a beneficial therapeutic response. We have illustrated this using amphotericin B, a nephrotoxic drug encapsulated within N-palmitoyl-N-methyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan (GCPQ) nanoparticles, and have evidenced our approach in three separate disease states (visceral leishmaniasis, candidiasis, and aspergillosis) using industry standard models of the disease in small animals. The oral bioavailability of AmB-GCPQ nanoparticles is 24%. In all disease models, AmB-GCPQ nanoparticles show comparable efficacy to parenteral liposomal AmB (AmBisome). Our work thus paves the way for others to use nanoparticles to achieve a specific targeted delivery of drug to key organs via the oral route. This is especially important for drugs with a narrow therapeutic index.

Triazolopyridyl ketones as a novel class of antileishmanial agents. DNA binding and BSA interaction.

A new series of triazolopyridyl pyridyl ketones has been synthetized by regioselective lithiation of the corresponding [1,2,3]triazolo[1,5-a]pyridine at 7 position followed by reaction with different electrophiles. The in vitro antileishmanial activity of these compounds was evaluated against Leishmaniainfantum, Leishmaniabraziliensis, Leishmaniaguyanensis and Leishmaniaamazonensis. Compounds 6 and 7 were found to be the most active leishmanicidal agents. Both of them showed activities at micromolar concentration against cultured promastigotes of Leishmania spp. (IC50=99.8-26.8 µM), without cytotoxicity on J774 macrophage cells. These two compounds were also tested in vivo in a murine model of acute infection by L. infantum. The triazolopyridine derivative 6 was effective against both spleen and liver parasites forms, while 7 was inactive against liver parasites. Mechanistic aspects of the antileishmanial activity were investigated by means of DNA binding studies (UV-titration and viscosimetry). Results have revealed that these active ligands are able to interact strongly with DNA [Kb=1.14 × 10(5)M(-1) (6) and 3.26 × 10(5)M(-1) (7)]. Moreover, a DNA groove binding has been proposed for both 6 and 7. To provide more insight on the mode of action of compounds 6 and 7 under biological conditions, their interaction with bovine serum albumin (BSA) was monitored by fluorescence titrations and UV-visible spectroscopy. The quenching constants and binding parameters were determined. Triazolopyridine ketones 6 and 7 have exhibited significant affinity towards BSA [Kb=2.5 × 10(4)M(-1) (6) and 1.9 × 10(4)M(-1) (7)]. Finally, to identify the binding location of compounds 6 and 7 on the BSA, competitive binding experiments were carried out, using warfarin, a characteristic marker for site I, and ibuprofen as one for site II. Results derived from these studies have indicated that both compounds interact at BSA site I and, to a lesser extent, at site II.

In vitro and in vivo antileishmanial and trypanocidal studies of new N-benzene- and N-naphthalenesulfonamide derivatives.

We report in vivo and in vitro antileishmanial and trypanocidal activities of a new series of N-substituted benzene and naphthalenesulfonamides 1-15. Compounds 1-15 were screened in vitro against Leishmania infantum , Leishmania braziliensis , Leishmania guyanensis , Leishmania amazonensis , and Trypanosoma cruzi . Sulfonamides 6e, 10b, and 10d displayed remarkable activity and selectivity toward T. cruzi epimastigotes and amastigotes. 6e showed significant trypanocidal activity on parasitemia in a murine model of acute Chagas disease. Moreover, 6e, 8c, 9c, 12c, and 14d displayed interesting IC50 values against Leishmania spp promastigotes as well as L. amazonensis and L. infantum amastigotes. 9c showed excellent in vivo activity (up to 97% inhibition of the parasite growth) in a short-term treatment murine model for acute infection by L. infantum. In addition, the effect of compounds 9c and 14d on tubulin as potential target was assessed by confocal microscopy analysis applied to L. infantum promastigotes.

Nuclease activity and ultrastructural effects of new sulfonamides with anti-leishmanial and trypanocidal activities.

Our aim was to evaluate the in vitro efficacy of a series of N-benzenesulfonamides of amine substituted aromatic rings, sulfonamides 1-6, against Trypanosoma cruzi and Leishmania spp. and to compare their trypanocidal and leishmanicidal profile. In order to elucidate the probable mechanism of action, the interaction of selected sulfonamides with pUC18 plasmid DNA was investigated by nuclease activity assays. In addition, the cellular targets of these sulfonamides in treated parasites were also searched by transmission and scanning electron microscopy. The most active compounds 4-nitro-N-pyrimidin-2-ylbenzenesulfonamide 1a and 4-chloro-N-5-methyl-thiazol-2-yl-benzenesulfonamide 2d displayed significant in vitro activity against Leishmania spp. promastigotes, without toxicity to J774 macrophages. Selected sulfonamides 1a, 4-nitro-N-pyrazin-2-yl-benzenesulfonamide 1n and 2d were also active against Leishmania infantum intracellular amastigotes. Compounds 1n and 2d showed nuclease activity in the presence of copper salt analogous to our previous results with sulfonamide 1a. Mechanistic data reveal the involvement of a redox process. Evidence for the formation of reactive oxygen species (ROS) responsible for DNA strand scission is provided for sulfonamides 1a, 1n and 2d. Transmission electron microscopic (TEM) analysis of L. infantum promastigotes treated with compounds 1a, 1n and 2d shows an overall cellular disorganization effects which are mainly addressed to DNA bearing structures such as the nucleus, mitochondria and kinetoplast. Disruption of double nuclear membrane and loss of cellular integrity along with accumulation of cytoplasmic electrodense bodies were also frequently observed.

Productos naturales con actividad leishmanicida y tripanocida / Natural products with leishmanicidal and trypanocidal activity

Las enfermedades parasitarias constituyen un importante problema de salud, y muchas de ellas están emergiendo en países donde se consideraban erradicadas. La leishmaniasis, la enfermedad del sueño y la enfermedad de Chagas, causadas por los parásitos Leishmania spp, Trypanosoma brucei y Trypanosoma cruzi, respectivamente, se encuentran entre las enfermedades parasitarias más prevalentes. La principal alternativa para tratarlas es la quimioterapia. Sin embargo, los tratamientos actuales se encuentran lejos de ser satisfactorios. La toxicidad de los fármacos, la vía de administración, la duración de los tratamientos y la aparición de resistencias hacen necesario el desarrollo de nuevas moléculas activas, más seguras y eficaces. Estudios recientes ponen de manifiesto la actividad leishmanicida y tripanocida in vivo de una amplia variedad de compuestos fenólicos, alcaloides y terpenos. En este artículo se revisan los productos naturales activos frente a leishmaniasis, enfermedad del sueño y enfermedad de Chagas (AU)

Diseases caused by protozoan parasites are still an important human health problem, since many of them are becoming “emerging” infectious sickness in geographical areas where they were considered eradicated. Leishmaniasis, African sleeping sickness and Chagas disease, caused by the parasites Leishmania spp, Trypanosoma brucei and Trypanosoma cruzi, respectively, are among the most important parasitic diseases. The main alternative to control such parasitosis is chemotherapy. Nevertheless, the current drug treatments are far from being satisfactory. Toxic side effects, route of administration, long-term treatments and the apparition of resistance, highlight the urgent need of developing new active molecules, more safe and effective. Recent studies report the leishmanicidal and trypanocidal activities of a wide variety of phenolic compounds, alkaloids and terpenes that have shown activity in vivo. This review outlines the current understanding of natural products against leishmaniasis, African sleeping sickness and Chagas disease (AU)

In vivo and in vitro anti-leishmanial activities of 4-nitro-N-pyrimidin- and N-pyrazin-2-ylbenzenesulfonamides, and N2-(4-nitrophenyl)-N1-propylglycinamide.

A series of compounds containing the nitrobenzene and sulfonamido moieties were synthesized and their leishmanicidal effect was assessed in vitro against Leishmaniainfantum promastigotes. Among the compounds evaluated, the p-nitrobenzenesulfonamides 4Aa and 4Ba, and the p-nitroaniline 5 showed significant activity with a good selectivity index. In a Balb/c mice model of L. Infantum, administration of compounds 4Aa, 4Ba or 5 (5mg/kg/day for 10 days, injected ip route) led to a clear-cut parasite burden reduction (ca. 99%). In an attempt to elucidate their mechanism of action, the DNA interaction of 4Aa and 5 was investigated by means of viscosity studies, thermal denaturation and nuclease activity assay. Both compounds showed nuclease activity in the presence of copper salt. The results suggest that compounds 4Aa, 4Ba and 5 represent possible candidates for drug development in the therapeutic control of leishmaniasis.