Novel Bidentate Amine Ligand and the Interplay between Pd(II) and Pt(II) Coordination and Biological Activity.

Pt(II) and Pd(II) coordinating N-donor ligands have been extensively studied as anticancer agents after the success of cisplatin. In this work, a novel bidentate N-donor ligand, the N-[[4-(phenylmethoxy)phenyl]methyl]-2-pyridinemethanamine, was designed to explore the antiparasitic, antiviral and antitumor activity of its Pt(II) and Pd(II) complexes. Chemical and spectroscopic characterization confirm the formation of [MLCl2 ] complexes, where M=Pt(II) and Pd(II). Single crystal X-ray diffraction confirmed a square-planar geometry for the Pd(II) complex. Spectroscopic characterization of the Pt(II) complex suggests a similar structure. 1 H NMR, 195 Pt NMR and HR-ESI-MS(+) analysis of DMSO solution of complexes indicated that both compounds exchange the chloride trans to the pyridine for a solvent molecule with different reaction rates. The ligand and the two complexes were tested for in vitro antitumoral, antileishmanial, and antiviral activity. The Pt(II) complex resulted in a GI50 of 10.5 µM against the NCI/ADR-RES (multidrug-resistant ovarian carcinoma) cell line. The ligand and the Pd(II) complex showed good anti-SARS-CoV-2 activity with around 65 % reduction in viral replication at a concentration of 50 µM.

Dissection of phospholipases A2 reveals multifaceted peptides targeting cancer cells, Leishmania and bacteria.

Cationic peptides bio-inspired by natural toxins have been recognized as an efficient strategy for the treatment of different health problems. Due to the specific interaction with substrates from biological membranes, snake venom phospholipases (PLA2s) represent valuable scaffolds for the research and development of short peptides targeting parasites, bacteria, and cancer cells. Considering this, we evaluated the in vitro therapeutic potential of three biomimetic peptides (pCergo, pBmTxJ and pBmje) based on three different amino acid sequences from Asp49 PLA2s. First, short amino acid sequences (12-17 in length) derived from these membranolytic toxins were selected using a combination of bioinformatics tools, including AntiCP, AMPA, PepDraw, ToxinPred, and HemoPI. The peptide, from each polypeptide sequence, with the greatest average antimicrobial index, no toxicity, and no hemolysis predicted was synthesized, purified, and characterized. According to in vitro assays performed, pBmje showed moderate cytotoxicity specifically against MCF-7 (breast cancer cells) with an EC50 of 464.85 µM, whereas pBmTxJ showed an antimicrobial effect against Staphylococcus aureus (ATCC 25923) with an MIC of 37.5 µM, and pCergo against E. coli (ATCC 25922) with an MIC of 75 µM. In addition, pCergo showed antileishmanial activity with an EC50 of 93.69 µM and 110.40 µM against promastigotes of Leishmania braziliensis and L. amazonensis, respectively. Altogether, these results confirmed the versatility of PLA2-derived synthetic peptides, highlighting the relevance of the use of these membrane-interacting toxins as specific archetypes for drug design focused on public health problems.

Dihydroartemisinin, an active metabolite of artemisinin, interferes with Leishmania braziliensis mitochondrial bioenergetics and survival.

Leishmaniasis is one of the most neglected parasitic infections of the world and current therapeutic options show several limitations. In the search for more effective drugs, plant compounds represent a powerful natural source. Artemisinin is a sesquiterpene lactone extracted from Artemisia annua L. leaves, from which dihydroartemisinin (DQHS) and artesunic acid (AA)/artesunate are examples of active derivatives. These lactones have been applied successfully on malaria therapy for decades. Herein, we investigated the sensitivity of Leishmania braziliensis, one of the most prevalent Leishmania species that cause cutaneous manifestations in the New World, to artemisinin, DQHS, and AA. L. braziliensis promastigotes and the stage that is targeted for therapy, intracelular amastigotes, were more sensitive to DQHS, showing EC50 of 62.3 ± 1.8 and 8.9 ± 0.9 µM, respectively. Cytotoxicity assays showed that 50% of bone marrow-derived macrophages cultures were inhibited with 292.8 ± 3.8 µM of artemisinin, 236.2 ± 4.0 µM of DQHS, and 396.8 ± 6.7 µM of AA. The control of intracellular infection may not be essentially attributed to the production of nitric oxide. However, direct effects on mitochondrial bioenergetics and H2O2 production appear to be associated with the leishmanicidal effect of DQHS. Our data provide support for further studies of artemisinin and derivatives repositioning for experimental leishmaniasis.

Water-Soluble Glutamic Acid Derivatives Produced in Culture by Penicillium solitum IS1-A from King George Island, Maritime Antarctica.

A new method of screening was developed to generate 770 organic and water-soluble fractions from extracts of nine species of marine sponges, from the growth media of 18 species of marine-derived fungi, and from the growth media of 13 species of endophytic fungi. The screening results indicated that water-soluble fractions displayed significant bioactivity in cytotoxic, antibiotic, anti-Leishmania, anti-Trypanosoma cruzi, and inhibition of proteasome assays. Purification of water-soluble fractions from the growth medium of Penicillium solitum IS1-A provided the new glutamic acid derivatives solitumine A (1), solitumine B (2), and solitumidines A-D (3-6). The structures of compounds 1-6 have been established by analysis of spectroscopic data, chemical derivatizations, and vibrational circular dichroism calculations. Although no biological activity could be observed for compounds 1-6, the new structures reported for 1-6 indicate that the investigation of water-soluble natural products represents a relevant strategy in finding new secondary metabolites.

Correction to: In vitro schistosomicidal activity of tamoxifen and its effectiveness in a murine model of schistosomiasis at a single dose.

The original published version of this article contains error in Tables 1 and 2. Correct tables are presented here.

In vitro schistosomicidal activity of tamoxifen and its effectiveness in a murine model of schistosomiasis at a single dose.

Schistosomiasis is a neglected tropical disease affecting 220 million people worldwide. Praziquantel has proven to be effective against this parasitic disease, though there are increasing concerns regarding tolerance/resistance that calls for new drugs. Repurposing already existing and well-known drugs has been a desirable approach since it reduces time, costs, and ethical concerns. The anti-cancer drug tamoxifen (TAM) has been used worldwide for several decades to treat and prevent breast cancer. Previous reports stated that TAM affects Schistosoma hormonal physiology; however, no controlled schistosomicidal in vivo assays have been conducted. In this work, we evaluated the effect of TAM on female and male Schistosoma mansoni morphology, motility, and egg production. We further assessed worm survival and egg production in S. mansoni-infected mice. TAM induced morphological alterations in male and female parasites, as well as in eggs in vitro. Furthermore, in our in vivo experiments, one single dose of intraperitoneal TAM citrate reduced the total worm burden by 73% and led to a decrease in the amount of eggs in feces and low percentages of immature eggs in the small intestine wall. Eggs obtained from TAM citrate-treated mice were reduced in size and presented hyper-vacuolated structures. Our results suggest that TAM may be repurposed as a therapeutic alternative against S. mansoni infections.

Topical tamoxifen in the therapy of cutaneous leishmaniasis.

The aims of the present work were to test the effect of tamoxifen administered topically and the therapeutic efficacy of tamoxifen and pentavalent antimonial combinations in an experimental model of cutaneous leishmaniasis. BALB/c mice infected with a luciferase expressing line of Leishmania amazonensis were treated with topical tamoxifen in two different formulations (ethanol or oil-free cream) as monotherapy or in co-administration with pentavalent antimonial. Treatment efficacy was evaluated by lesion size and parasite burden, quantified through luminescence, at the end of treatment and 4 weeks later. Topical tamoxifen, formulated in ethanol or as a cream, was shown to be effective. The interaction between tamoxifen and pentavalent antimonial was additive in vitro. Treatment with combined schemes containing tamoxifen and pentavalent antimonial was effective in reducing lesion size and parasite burden. Co-administration of tamoxifen and pentavalent antimonial was superior to monotherapy with antimonial.

Isolation, Derivative Synthesis, and Structure-Activity Relationships of Antiparasitic Bromopyrrole Alkaloids from the Marine Sponge Tedania brasiliensis.

The isolation and identification of a series of new pseudoceratidine (1) derivatives from the sponge Tedania brasiliensis enabled the evaluation of their antiparasitic activity against Plasmodium falciparum, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) infantum, and Trypanosoma cruzi, the causative agents of malaria, cutaneous leishmaniasis, visceral leishmaniasis, and Chagas disease, respectively. The new 3-debromopseudoceratidine (4), 20-debromopseudoceratidine (5), 4-bromopseudoceratidine (6), 19-bromopseudoceratidine (7), and 4,19-dibromopseudoceratidine (8) are reported. New tedamides A-D (9-12), with an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxamide moiety, are also described. Compounds 4 and 5, 6 and 7, 9 and 10, and 11 and 12 have been isolated as pairs of inseparable structural isomers differing in their sites of bromination or oxidation. Tedamides 9+10 and 11+12 were obtained as optically active pairs, indicating an enzymatic formation rather than an artifactual origin. N12-Acetylpseudoceratidine (2) and N12-formylpseudoceratidine (3) were obtained by derivatization of pseudoceratidine (1). The antiparasitic activity of pseudoceratidine (1) led us to synthesize 23 derivatives (16, 17, 20, 21, 23, 25, 27-29, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51) with variations in the polyamine chain and aromatic moiety in sufficient amounts for biological evaluation in antiparasitic assays. The measured antimalarial activity of pseudoceratidine (1) and derivatives 4, 5, 16, 23, 25, 31, and 50 provided an initial SAR evaluation of these compounds as potential leads for antiparasitics against Leishmania amastigotes and against P. falciparum. The results obtained indicate that pseudoceratidine represents a promising scaffold for the development of new antimalarial drugs.

The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds.

The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca(2+)-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca(2+)-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery.

Leishmania-mediated inhibition of iron export promotes parasite replication in macrophages.

Leishmania parasites infect macrophages, cells that play an important role in organismal iron homeostasis. By expressing ferroportin, a membrane protein specialized in iron export, macrophages release iron stored intracellularly into the circulation. Iron is essential for the intracellular replication of Leishmania, but how the parasites compete with the iron export function of their host cell is unknown. Here, we show that infection with Leishmania amazonensis inhibits ferroportin expression in macrophages. In a TLR4-dependent manner, infected macrophages upregulated transcription of hepcidin, a peptide hormone that triggers ferroportin degradation. Parasite replication was inhibited in hepcidin-deficient macrophages and in wild type macrophages overexpressing mutant ferroportin that is resistant to hepcidin-induced degradation. Conversely, intracellular growth was enhanced by exogenously added hepcidin, or by expression of dominant-negative ferroportin. Importantly, dominant-negative ferroportin and macrophages from flatiron mice, a mouse model for human type IV hereditary hemochromatosis, restored the infectivity of mutant parasite strains defective in iron acquisition. Thus, inhibition of ferroportin expression is a specific strategy used by L. amazonensis to inhibit iron export and promote their own intracellular growth.

Combination therapy with tamoxifen and amphotericin B in experimental cutaneous leishmaniasis.

Leishmaniasis chemotherapy remains very challenging. The high cost of active drugs, along with the severity of their side effects and the increasing failure rate of the current therapeutic schemes, calls for the discovery of new active drugs and schemes of treatment. The use of combination therapy has gained much attention in recent years as a possible strategy for overcoming the various shortcomings in the present arsenal. We recently described the effectiveness of tamoxifen in murine models of leishmaniasis, and here, we investigated the interactions between tamoxifen and amphotericin B, one of the most potent drugs used in leishmaniasis treatment. The in vitro interactions were indifferent for the association of tamoxifen and amphotericin B. The association was also assayed in vivo in Leishmania amazonensis-infected BALB/c mice and was found to yield at least additive effects at low doses of both drugs.

Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.

Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.

Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence.

The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire this essential factor from the environment. Previous studies showed that L. amazonensis incorporates heme through the transmembrane protein LHR1 (Leishmania Heme Response 1). LHR1-null promastigotes were not viable, suggesting that the transporter is essential for survival. Here, we compared the growth, differentiation, and infectivity for macrophages and mice of wild-type, LHR1-single-knockout (LHR1/Δlhr1), and LHR1-complemented (LHR1/Δlhr1 plus LHR1) L. amazonensis strains. LHR1/Δlhr1 promastigotes replicated poorly in heme-deficient media and had lower intracellular heme content than wild-type parasites. LHR1/Δlhr1 promastigotes were also less effective in reducing ferric iron to ferrous iron, a reaction mediated by the heme-containing parasite enzyme LFR1 (Leishmania Ferric Reductase 1). LHR1/Δlhr1 parasites differentiated normally into aflagellated forms expressing amastigote-specific markers but were not able to replicate intracellularly after infecting macrophages. Importantly, the intracellular growth of LHR1/Δlhr1 amastigotes was fully restored when macrophages were allowed to phagocytose red blood cells prior to infection. LHR1/Δlhr1 parasites were also severely defective in the development of cutaneous lesions in mice. All phenotypes observed in LHR1/Δlhr1 L. amazonensis were rescued by expression of episomal LHR1. Our results reveal the importance of efficient heme uptake for L. amazonensis replication and vertebrate host infectivity, reinforcing the potential usefulness of LHR1 as a target for new antileishmanial drugs.

Unlocking the potential of snake venom-based molecules against the malaria, Chagas disease, and leishmaniasis triad.

Malaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.

Models for cytotoxicity screening of antileishmanial drugs: what has been done so far?

A growing number of studies have demonstrated the in vitro potential of an impressive number of antileishmanial candidates in the past years. However, the lack of uniformity regarding the choice of cell types for cytotoxicity assays may lead to uncomparable and inconclusive data. In vitro assays relying solely on non-phagocytic cell models may not represent a realistic result as the effect of an antileishmanial agent should ideally be presented based on its cytotoxicity profile against reticuloendothelial system cells. In the present review, we have assembled studies published in the scientific literature from 2015 to 2021 that explored leishmanicidal candidates, emphasising the main host cell models used for cytotoxicity assays. The pros and cons of different host cell types as well as primary cells and cell lines are discussed in order to draw attention to the need to establish standardised protocols for preclinical testing when assessing new antileishmanial candidates.

Causative Agents of American Tegumentary Leishmaniasis Are Able to Infect 3T3-L1 Adipocytes In Vitro.

Although macrophages have long been considered key players in the course of Leishmania infections, other non-professional phagocytes have lately been shown to maintain low levels of the parasite in safe intracellular niches. Recently, it was demonstrated that the adipose tissue is capable of harboring Old World L. (L.) infantum in mice. However, there is no evidence of experimental adipocyte infection with New World Leishmania species so far. In addition, it was not known whether adipocytes would be permissive for formation of the unique, large and communal parasitophorous vacuoles that are typical of L. (L.) amazonensis in macrophages. Here we evaluated the ability of L. (L.) amazonensis and L. (V.) braziliensis promastigotes and amastigotes to infect 3T3-L1 fibroblast-derived adipocytes (3T3-Ad) using light and transmission electron microscopy. Our results indicate that amastigotes and promastigotes of both species were capable of infecting and surviving inside pre- and fully differentiated 3T3-Ad for up to 144 h. Importantly, L. (L.) amazonensis amastigotes resided in large communal parasitophorous vacuoles in pre-adipocytes, which appeared to be compressed between large lipid droplets in mature adipocytes. In parallel, individual L. (V.) braziliensis amastigotes were detected in single vacuoles 144 h post-infection. We conclude that 3T3-Ad may constitute an environment that supports low loads of viable parasites perhaps contributing to parasite maintenance, since amastigotes of both species recovered from these cells differentiated into replicative promastigotes. Our findings shed light on the potential of a new host cell model that can be relevant to the persistence of New World Leishmania species.

Antileishmanial activity and insights into the mechanisms of action of symmetric Au(I) benzyl and aryl-N-heterocyclic carbenes.

Leishmania amazonensis and L. braziliensis are the main etiological agents of the American Tegumentary Leishmaniasis (ATL). Taking into account the limited effectiveness and high toxicity of the current drug arsenal to treat ATL, novel options are urgently needed. Inspired by the fact that gold-based compounds are promising candidates for antileishmanial drugs, we studied the biological action of a systematic series of six (1)-(6) symmetric Au(I) benzyl and aryl-N-heterocyclic carbenes. All compounds were active at low micromolar concentrations with 50% effective concentrations ranging from 1.57 to 8.30 µM against Leishmania promastigotes. The mesityl derivative (3) proved to be the best candidate from this series, with a selectivity index ~13 against both species. The results suggest an effect of the steric and electronic parameters of the N-substituent in the activity. Intracellular infections were drastically reduced after 24h of (2)-(5) incubation in terms of infection rate and amastigote burden. Further investigations showed that our compounds induced significant parasites' morphological alterations and membrane permeability. Also, (3) and (6) were able to reduce the residual activity of three Leishmania recombinant cysteine proteases, known as possible targets for Au(I) complexes. Our promising results open the possibility of exploring gold complexes as leishmanicidal molecules to be further screened in in vivo models of infection.

The impact of COVID-19 on neglected parasitic diseases: what to expect?

Here we highlight coinfections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with ectoparasites, helminths, and protozoa, described in the literature, and the urgent need to understand the conditions of these associated pathologies. We emphasize the notion that such information is crucial for the continuity of measures that have been used for decades to control neglected parasitic diseases.

A "Golden Age" for the discovery of new antileishmanial agents: Current status of leishmanicidal gold complexes and prospective targets beyond the trypanothione system.

Leishmaniasis is one of the most neglected diseases worldwide and is considered a serious public health issue. The current therapeutic options have several disadvantages that make the search for new therapeutics urgent. Gold compounds are emerging as promising candidates based on encouraging in vitro and limited in vivo results for several AuI and AuIII complexes. The antiparasitic mechanisms of these molecules remain only partially understood. However, a few studies have proposed the trypanothione redox system as a target, similar to the mammalian thioredoxin system, pointed out as the main target for several gold compounds with significant antitumor activity. In this review, we present the current status of the investigation and design of gold compounds directed at treating leishmaniasis. In addition, we explore potential targets in Leishmania parasites beyond the trypanothione system, taking into account previous studies and structure modulation performed for gold-based compounds.

The risk of oral transmission in an area of a Chagas disease outbreak in the Brazilian northeast evaluated through entomological, socioeconomic and schooling indicators.

Chagas disease is a neglected tropical disease strongly associated with low socioeconomic status, affecting nearly 8 million people - mainly Latin Americans. The current infection risk is based on acute case reports, most of which are typically associated with oral transmissions. In the semi-arid region of Northeastern Brazil, serious outbreaks of this transmission type have surged in the last years. One of those occurred in 2016 in the state of Rio Grande do Norte. Rural residents of four municipalities surrounding Marcelino Vieira ingested sugar cane juice - which was probably ground with Trypanosoma cruzi-infected insects. Eighteen cases of Chagas disease were confirmed serologically, with two deaths reported. Socioeconomic information, schooling of residents and the structure of peridomestic and domestic environments in the rural area of Marcelino Vieira, along with entomological indicators, were investigated to understand better the factors related to the outbreaks in this region. We found triatomines (mainly Triatoma brasiliensis) in 35% (24/67) of domiciliary units and all rocky outcrops inspected (n = 7). Overall, 25% (91/357) of examined T. brasiliensis were infected by T. cruzi in artificial ecotopes, with almost the same prevalence in the sylvatic environment (22%; 35/154). Among all ecotopes investigated, wood/tile/brick piles were the ones linked to high insect infestations and triatomine T. cruzi infection prevalence. Ninety-five percent of people interviewed recognized the triatomines and knew the classic route of transmission of disease - triatomine bite-dependent. However, only 7.5% admitted knowledge that Chagas disease can also be acquired orally - which poses a risk this transmission route currently recognized. Here, we highlight the physical proximity between humans and triatomine populations with high T. cruzi infection prevalence as an additional risk factor to oral/vector contaminations. In sum, residents have low income, low level of education, and/or a willful disregard for the routes of Chagas disease transmission (specifically oral transmission), a combination of factors that may have favored the Chagas disease outbreak. We here provide recommendations to avoid further outbreaks.