Publications by authors named "Danilo C Miguel"

34 Publications

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

Bioorg Chem 2021 May 31;114:105041. Epub 2021 May 31.

Biomolecules Discovery Group, Universidad Regional Amazónica Ikiam, Km 7 Via Muyuna, Tena, Napo, Ecuador. Electronic address:

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 (PLAs) 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 PLAs. 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 EC 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 EC of 93.69 µM and 110.40 µM against promastigotes of Leishmania braziliensis and L. amazonensis, respectively. Altogether, these results confirmed the versatility of PLA-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.
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http://dx.doi.org/10.1016/j.bioorg.2021.105041DOI Listing
May 2021

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

Trends Parasitol 2021 08 14;37(8):694-697. Epub 2021 May 14.

Department of Animal Biology, Parasitology Section, Biology Institute, University of Campinas - UNICAMP, Campinas, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1016/j.pt.2021.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8120483PMC
August 2021

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

ChemMedChem 2021 Jun 22;16(11):1681-1695. Epub 2021 Mar 22.

Institute of Chemistry, University of Campinas, PO Box 6154, 13083-970, Campinas, SP, Brazil).

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 Au and Au 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.
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http://dx.doi.org/10.1002/cmdc.202100022DOI Listing
June 2021

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

Parasitol Res 2021 Feb 8;120(2):705-713. Epub 2021 Jan 8.

Departamento de Biologia Animal - Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP, Campinas, São Paulo, Brazil.

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 EC 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 HO 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.
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http://dx.doi.org/10.1007/s00436-020-07019-1DOI Listing
February 2021

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

Acta Trop 2021 Mar 26;215:105803. Epub 2020 Dec 26.

Instituto de Biologia. Departamento de Biologia Animal. Universidade Estadual de Campinas (UNICAMP), Brasil; Programa de Pós-Graduação em Biodiversidade e Evolução (PPGBioEvo), Instituto de Biologia, Universidade Federal da Bahia (UFBA), Salvador, Brasil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.actatropica.2020.105803DOI Listing
March 2021

Cruzioseptins, antibacterial peptides from Cruziohyla calcarifer skin, as promising leishmanicidal agents.

Pathog Dis 2020 09;78(6)

Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo, Brasil. CEP 13083-862.

Screenings of natural products have significantly contributed to the discovery of novel leishmanicidal agents. In this study, three known cruzioseptins-antibacterial peptides from Cruziohyla calcarifer skin-were synthesized and evaluated against promastigotes and amastigotes stages of Leishmania (L.) amazonensis and L. (V.) braziliensis. EC50 ranged from 9.17 to 74.82 μM, being cruzioseptin-1 the most active and selective compound, with selectivity index > 10 for both promastigotes and amastigotes of L. (V.) braziliensis. In vitro infections incubated with cruzioseptins at 50 μM showed up to ∼86% reduction in the amastigote number. Cruzioseptins were able to destabilize the parasite's cell membrane, allowing the incorporation of a DNA-fluorescent dye. Our data also demonstrated that hydrophobicity and charge appear to be advantageous features for enhancing parasiticidal activity. Antimicrobial cruzioseptins are suitable candidates and alternative molecules that deserve further in vivo investigation focusing on the development of novel antileishmanial therapies.
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http://dx.doi.org/10.1093/femspd/ftaa053DOI Listing
September 2020

Comparing the Antileishmanial Activity of Gold(I) and Gold(III) Compounds in L. amazonensis and L. braziliensis in Vitro.

ChemMedChem 2020 Nov 28;15(22):2146-2150. Epub 2020 Sep 28.

Department of Animal Biology, Biology Institute, University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, 13083-862., Campinas, SP, Brazil.

A series of mononuclear coordination or organometallic Au /Au complexes (1-9) have been comparatively studied in vitro for their antileishmanial activity against promastigotes and amastigotes, the clinically relevant parasite form, of Leishmania amazonensis and Leishmania braziliensis. One of the cationic Au bis-N-heterocyclic carbenes (3) has low EC values (ca. 4 μM) in promastigotes cells and no toxicity in host macrophages. Together with two other Au complexes (6 and 7), the compound is also extremely effective in intracellular amastigotes from L. amazonensis. Initial mechanistic studies include an evaluation of the gold complexes' effect on L. amazonensis' plasma membrane integrity.
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http://dx.doi.org/10.1002/cmdc.202000536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756297PMC
November 2020

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

J Nat Prod 2020 01 2;83(1):55-65. Epub 2020 Jan 2.

Instituto de Química de São Carlos , Universidade de São Paulo , CP 780, CEP 13560-970 , São Carlos , SP , Brazil.

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-, anti-, and inhibition of proteasome assays. Purification of water-soluble fractions from the growth medium of IS1-A provided the new glutamic acid derivatives solitumine A (), solitumine B (), and solitumidines A-D (-). The structures of compounds - have been established by analysis of spectroscopic data, chemical derivatizations, and vibrational circular dichroism calculations. Although no biological activity could be observed for compounds -, the new structures reported for - indicate that the investigation of water-soluble natural products represents a relevant strategy in finding new secondary metabolites.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00635DOI Listing
January 2020

Potential use of 13-mer peptides based on phospholipase and oligoarginine as leishmanicidal agents.

Comp Biochem Physiol C Toxicol Pharmacol 2019 Dec 24;226:108612. Epub 2019 Aug 24.

Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo, Brazil. Electronic address:

Phospholipase A toxins present in snake venoms interact with biological membranes and serve as structural models for the design of small peptides with anticancer, antibacterial and antiparasitic properties. Oligoarginine peptides are capable of increasing cell membrane permeability (cell penetrating peptides), and for this reason are interesting delivery systems for compounds of pharmacological interest. Inspired by these two families of bioactive molecules, we have synthesized two 13-mer peptides as potential antileishmanial leads gaining insights into structural features useful for the future design of more potent peptides. The peptides included p-Acl, reproducing a natural segment of a Lys49 PLA from Agkistrodon contortrix laticinctus snake venom, and its p-AclR7 analogue where all seven lysine residues were replaced by arginines. Both peptides were active against promastigote and amastigote forms of Leishmania (L.) amazonensis and L. (L.) infantum, while displaying low cytotoxicity for primary murine macrophages. Spectrofluorimetric studies suggest that permeabilization of the parasite's cell membrane is the probable mechanism of action of these biomolecules. Relevantly, the engineered peptide p-AclR7 was more active in both life stages of Leishmania and induced higher rates of ethidium bromide incorporation than its native template p-Acl. Taken together, the results suggest that short peptides based on phospholipase toxins are potential scaffolds for development of antileishmanial candidates. Moreover, specific amino acid substitutions, such those herein employed, may enhance the antiparasitic action of these cationic peptides, encouraging their future biomedical applications.
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http://dx.doi.org/10.1016/j.cbpc.2019.108612DOI Listing
December 2019

Unveiling the Kinomes of and Empowers the Discovery of New Kinase Targets and Antileishmanial Compounds.

Comput Struct Biotechnol J 2019 8;17:352-361. Epub 2019 Feb 8.

Labmol - Laboratory for Molecular Modeling and Drug Design, Faculdade de Farmácia, Universidade Federal de Goiás - UFG, Goiânia, GO, 74605-510, Brazil.

Leishmaniasis is a neglected tropical disease caused by parasites of the genus (NTD) endemic in 98 countries. Although some drugs are available, current treatments deal with issues such as toxicity, low efficacy, and emergence of resistance. Therefore, there is an urgent need to identify new targets for the development of new antileishmanial drugs Protein kinases (PKs), which play an essential role in many biological processes, have become potential drug targets for many parasitic diseases. A refined bioinformatics pipeline was applied in order to define and compare the kinomes of L. and L. species that cause cutaneous and visceral manifestations of leishmaniasis in the Americas, the latter being potentially fatal if untreated. Respectively, 224 and 221 PKs were identified in L. and L. overall. Almost all unclassified eukaryotic PKs were assigned to six of nine major kinase groups and, consequently, most have been classified into family and subfamily. Furthermore, revealing the kinomes for both species allowed for the prioritization of potential drug targets that could be explored for discovering new drugs against leishmaniasis. Finally, we used a drug repurposing approach and prioritized seven approved drugs and investigational compounds to be experimentally tested against . Trametinib and NMS-1286937 inhibited the growth of L. and L. promastigotes and amastigotes and therefore might be good candidates for the drug repurposing pipeline.
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http://dx.doi.org/10.1016/j.csbj.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429582PMC
February 2019

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

Parasitol Res 2019 05;118(5):1685-1686

Biology Institute, Department of Animal Biology - Parasitology, University of Campinas - UNICAMP, Campinas, São Paulo, Brazil.

The original published version of this article contains error in Tables 1 and 2. Correct tables are presented here.
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http://dx.doi.org/10.1007/s00436-019-06290-1DOI Listing
May 2019

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

Parasitol Res 2019 May 24;118(5):1625-1631. Epub 2019 Feb 24.

Biology Institute, Department of Animal Biology - Parasitology, University of Campinas - UNICAMP, Campinas, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1007/s00436-019-06259-0DOI Listing
May 2019

Peptide R18H from BRN2 Transcription Factor POU Domain Displays Antitumor Activity In Vitro and In Vivo and Induces Apoptosis in B16F10-Nex2 Cells.

Anticancer Agents Med Chem 2019 ;19(3):389-401

Nucleo Integrado de Biotecnologia (NIB), Universidade de Mogi das Cruzes, UMC, Mogi das Cruzes, SP, Brazil.

Background: BRN2 transcription factor is associated with the development of malignant melanoma. The cytotoxic activities and cell death mechanism against B16F10-Nex2 cells were determined with synthetic peptide R18H derived from the POU domain of the BRN2 transcription factor.

Objective: To determine the cell death mechanisms and in vivo activity of peptide R18H derived from the POU domain of the BRN2 transcription factor against B16F10-Nex2 cells.

Methods: Cell viability was determined by the MTT method. C57Bl/6 mice were challenged with B16F10-Nex2 cells and treated with R18H. To identify the type of cell death, we used TUNEL assay, Annexin V and PI, Hoechst, DHE, and determination of caspase activation and cytochrome c release. Transmission electron microscopy was performed to verify morphological alterations after peptide treatment.

Results: Peptide R18H displayed antitumor activity in the first hours of treatment and the EC50% was calculated for 2 and 24h, being 0.76 ± 0.045 mM and 0.559 ± 0.053 mM, respectively. After 24h apoptosis was evident, based on DNA degradation, chromatin condensation, increase of superoxide anion production, phosphatidylserine translocation, activation of caspases 3 and 8, and release of extracellular cytochrome c in B16F10-Nex2 cells. The peptide cytotoxic activity was not affected by necroptosis inhibitors and treated cells did not release LDH in the extracellular medium. Moreover, in vivo antitumor activity was observed following treatment with peptide R18H.

Conclusion: Peptide R18H from BRN2 transcription factor induced apoptosis in B16F10-Nex2 and displayed antitumor activity in vivo.
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http://dx.doi.org/10.2174/1871520618666181109164246DOI Listing
December 2019

Tamoxifen inhibits the biosynthesis of inositolphosphorylceramide in Leishmania.

Int J Parasitol Drugs Drug Resist 2018 12 24;8(3):475-487. Epub 2018 Oct 24.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, São Paulo, SP, 05508-000, Brazil. Electronic address:

Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [H]-sphingosine and myo-[H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C)alkyl -2-O-(C)acylglycerol-3-HPO-inositol and sn-1-O-(C)acyl-2-O-(C)acylglycerol-3-HPO-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC value of 8.48 μM (95% CI 7.68-9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites.
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http://dx.doi.org/10.1016/j.ijpddr.2018.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216108PMC
December 2018

Challenges in drug discovery targeting TriTryp diseases with an emphasis on leishmaniasis.

Int J Parasitol Drugs Drug Resist 2018 12 28;8(3):430-439. Epub 2018 Sep 28.

Biology Institute, University of Campinas - UNICAMP, Campinas, São Paulo, Brazil. Electronic address:

Tritryps diseases are devastating parasitic neglected infections caused by Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei subspecies. Together, these parasites affect more than 30 million people worldwide and cause high mortality and morbidity. Leishmaniasis comprises a complex group of diseases with clinical manifestation ranging from cutaneous lesions to systemic visceral damage. Antimonials, the first-choice drugs used to treat leishmaniasis, lead to high toxicity and carry significant contraindications limiting its use. Drug-resistant parasite strains are also a matter for increasing concern, especially in areas with very limited resources. The current scenario calls for novel and/or improvement of existing therapeutics as key research priorities in the field. Although several studies have shown advances in drug discovery towards leishmaniasis in recent years, key knowledge gaps in drug discovery pipelines still need to be addressed. In this review we discuss not only scientific and non-scientific bottlenecks in drug development, but also the central role of public-private partnerships for a successful campaign for novel treatment options against this devastating disease.
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http://dx.doi.org/10.1016/j.ijpddr.2018.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195035PMC
December 2018

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

J Nat Prod 2018 01 3;81(1):188-202. Epub 2018 Jan 3.

Instituto de Química de São Carlos, Universidade de São Paulo , CP 780, CEP 13560-970, São Carlos, SP, Brazil.

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. N-Acetylpseudoceratidine (2) and N-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.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989537PMC
January 2018

Topical tamoxifen in the therapy of cutaneous leishmaniasis.

Parasitology 2018 04 9;145(4):490-496. Epub 2017 Mar 9.

Departamento de Parasitologia,Instituto de Ciências Biomédicas, Universidade de São Paulo,Av. Prof. Lineu Prestes 1374,05508-000 São Paulo,Brazil.

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.
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http://dx.doi.org/10.1017/S0031182017000130DOI Listing
April 2018

The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis.

PLoS Negl Trop Dis 2015 May 22;9(5):e0003804. Epub 2015 May 22.

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America; PathSensors, Inc., Baltimore, Maryland, United States of America.

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.
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http://dx.doi.org/10.1371/journal.pntd.0003804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441390PMC
May 2015

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

J Cell Sci 2015 Jan 6;128(1):27-32. Epub 2014 Nov 6.

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742-5815, USA

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.
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http://dx.doi.org/10.1242/jcs.150573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282046PMC
January 2015

Antileishmanial activity of the estrogen receptor modulator raloxifene.

PLoS Negl Trop Dis 2014 May 8;8(5):e2842. Epub 2014 May 8.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.

Background: The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis.

Methodology/principal Findings: Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden.

Conclusions/significance: The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.
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http://dx.doi.org/10.1371/journal.pntd.0002842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014391PMC
May 2014

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

Antimicrob Agents Chemother 2014 May 18;58(5):2608-13. Epub 2014 Feb 18.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1128/AAC.01315-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993234PMC
May 2014

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

PLoS Pathog 2014 Jan 30;10(1):e1003901. Epub 2014 Jan 30.

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1003901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907422PMC
January 2014

Discovery of synthetic Leishmania inhibitors by screening of a 2-arylbenzothiophene library.

Chem Biol Drug Des 2014 Mar 26;83(3):289-96. Epub 2013 Dec 26.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, 05508-900, Brazil.

Tamoxifen has been shown to be active in vitro against Leishmania and effective in the treatment for leishmaniasis in murine models. Through the screening of a compound library of estrogen receptor modulator analogs, we identified the major characteristics required for antileishmanial activity. To overcome the difficulties presented by tamoxifen's propensity for E/Z isomerization, we used the 2-arylbenzothiophene compound BTP as a more stable alternative. Directed screening of a small compound library based on BTP led to active compounds against Leishmania. Subsequent structure-activity data for the synthetic 2-arylbenzothiophenes evaluated in this study indicate that optimal antileishmanial potency is dependent on the presence of two basic side chains. In addition, the primary structural features required for estrogen receptor binding, the phenols, are not required for inhibiting parasitic growth. Significantly, the most active antileishmanial benzothiophenes lack the pharmacophore for estrogen receptor activity and therefore address potential concerns about the undesirable effects of using selective estrogen receptor modulators in women and children with leishmaniasis. Three compounds selected from the screening have shown consistent activity against all species and stages of Leishmania in vitro although improvements in selectivity are needed. These compounds represent viable starting points for further optimization as antileishmanial agents.
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http://dx.doi.org/10.1111/cbdd.12239DOI Listing
March 2014

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

Infect Immun 2013 Oct 22;81(10):3620-6. Epub 2013 Jul 22.

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.

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.
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http://dx.doi.org/10.1128/IAI.00687-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811768PMC
October 2013

Parasite burden in Leishmania (Leishmania) amazonensis-infected mice: validation of luciferase as a quantitative tool.

J Microbiol Methods 2013 May 4;93(2):95-101. Epub 2013 Mar 4.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-000 São Paulo, Brazil.

Given the lack of effective and safe alternatives to the drugs already in use, considerable efforts are being applied to the search of new therapeutic options to treat leishmaniasis. A necessary step in the discovery of antileishmanial drugs is the validation of drug candidates in mouse models. The standard methods to quantify the parasite burden in animal models, mainly culture-based, are time consuming and expensive. In recent years, in vivo imaging systems have been proposed as a tool to overcome these problems, allowing parasite detection in living organisms. Here we compared different treatment efficacy evaluation approaches. Recombinant Leishmania (L.) amazonensis lines expressing the luciferase gene (La-LUC) were obtained and characterized for biological properties as compared with the wild type (WT) parental line. Bioluminescence generated by La-LUC was shown to correlate with the number of promastigotes in vitro. La-LUC promastigotes and intracellular amastigotes were equally sensitive to amphotericin B (AmB) as the WT parasites. The clinical pattern of lesion development upon infection with the transgenic lines was similar to lesions observed after infection with the WT strain. The half maximal effective dose (ED50) of AmB was determined in La-LUC infected mice through quantification of bioluminescence in vivo and ex vivo, by limiting dilution and using clinical parameters. There was agreement in the ED50 determined by all methods. Quantification of bioluminescence in vivo and/or ex vivo was elected as the best tool for determining parasite burden to assess drug efficacy in infected mice. Furthermore, the detailed analysis of AmB effectiveness in this model generated useful data to be used in drug combination experiments.
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http://dx.doi.org/10.1016/j.mimet.2013.02.007DOI Listing
May 2013

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

PLoS Pathog 2012 12;8(7):e1002795. Epub 2012 Jul 12.

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1002795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395602PMC
January 2013

A dysflagellar mutant of Leishmania (Viannia) braziliensis isolated from a cutaneous leishmaniasis patient.

Parasit Vectors 2012 Jan 11;5:11. Epub 2012 Jan 11.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.

Background: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features.

Methods: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo.

Results: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis.

Conclusions: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.
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http://dx.doi.org/10.1186/1756-3305-5-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271977PMC
January 2012

Synthesis and in vitro activity of limonene derivatives against Leishmania and Trypanosoma.

Eur J Med Chem 2010 Apr 14;45(4):1524-8. Epub 2010 Jan 14.

Lab. de Síntese Orgânica Medicinal (LaSOM), Faculdade de Farmácia, UFRGS, Avenida Ipiranga, 2752, sala 705, 90610-000 Porto Alegre, RS, Brazil.

The synthesis and in vitro activity of R(+)-Limonene derivatives against Leishmania and Trypanosoma cruzi strains are reported. Seven compounds have shown better in vitro activity against Leishmania (V.)braziliensis than the standard drug pentamidine. Additionally, we have identified two promising new anti-T. cruzi limonene derivatives.
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http://dx.doi.org/10.1016/j.ejmech.2009.12.061DOI Listing
April 2010

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B.

Trop Med Int Health 2010 Jan 26;15(1):68-76. Epub 2009 Oct 26.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC(50), EC(90) and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.
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http://dx.doi.org/10.1111/j.1365-3156.2009.02414.xDOI Listing
January 2010
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