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1.
PLoS Negl Trop Dis ; 14(2): e0007991, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023254

RESUMO

BACKGROUND: During infections involving intracellular pathogens, iron performs a double-edged function by providing the pathogen with nutrients, but also boosts the host's antimicrobial arsenal. Although the role of iron has been described in visceral leishmaniasis, information regarding its status in the dermal sequel, Post Kala-azar Dermal Leishmaniasis (PKDL) remains limited. Accordingly, this study aimed to establish the status of iron within monocytes/macrophages of PKDL cases. METHODOLOGY/PRINCIPAL FINDINGS: The intramonocytic labile iron pool (LIP), status of CD163 (hemoglobin-haptoglobin scavenging receptor) and CD71 (transferrin receptor, Tfr) were evaluated within CD14+ monocytes by flow cytometry, and soluble CD163 by ELISA. At the lesional sites, Fe3+ status was evaluated by Prussian blue staining, parasite load by qPCR, while the mRNA expression of Tfr (TfR1/CD71), CD163, divalent metal transporter-1 (DMT-1), Lipocalin-2 (Lcn-2), Heme-oxygenase-1 (HO-1), Ferritin, Natural resistance-associated macrophage protein (NRAMP-1) and Ferroportin (Fpn-1) was evaluated by droplet digital PCR. Circulating monocytes demonstrated elevated levels of CD71, CD163 and soluble CD163, which corroborated with an enhanced lesional mRNA expression of TfR, CD163, DMT1 and Lcn-2. Additionally, the LIP was raised along with an elevated mRNA expression of ferritin and HO-1, as also iron exporters NRAMP-1 and Fpn-1. CONCLUSIONS/SIGNIFICANCE: In monocytes/macrophages of PKDL cases, enhancement of the iron influx gateways (TfR, CD163, DMT-1 and Lcn-2) possibly accounted for the enhanced LIP. However, enhancement of the iron exporters (NRAMP-1 and Fpn-1) defied the classical Ferritinlow/Ferroportinhigh phenotype of alternatively activated macrophages. The creation of such a pro-parasitic environment suggests incorporation of chemotherapeutic strategies wherein the availability of iron to the parasite can be restricted.


Assuntos
Ferro/metabolismo , Leishmaniose Cutânea/metabolismo , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Humanos , Índia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Leishmaniose Cutânea/parasitologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Adulto Jovem
2.
J Enzyme Inhib Med Chem ; 35(1): 432-459, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31899980

RESUMO

A series of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline, and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives was designed, synthesised, and evaluated in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiprotozoal activity with IC50 values in the µM range. In addition, the in vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The quinoline 1c was identified as the most potent antimalarial candidate with a ratio of cytotoxic to antiparasitic activities of 97 against the P. falciparum CQ-sensitive strain 3D7. The quinazoline 3h was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 43 on T. brucei brucei strain. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma are possible targets of this kind of nitrogen heterocyclic compounds, we have also investigated stabilisation of the Plasmodium and Trypanosoma telomeric G-quadruplexes by our best compounds through FRET melting assays.


Assuntos
Antiprotozoários/química , Antiprotozoários/farmacologia , Desenho de Fármacos , Quinolinas/química , Quinolinas/farmacologia , Antiprotozoários/síntese química , Células Hep G2 , Humanos , Leishmania donovani/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Quinolinas/síntese química , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos
3.
Parasitol Res ; 119(2): 649-657, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31897791

RESUMO

Leishmania donovani is the causative agent of visceral leishmaniasis. Annually, 500 million new cases of infection are reported mainly in poor communities, decreasing the interest of the pharmaceutical industries. Therefore, the repositioning of new drugs is an ideal strategy to fight against these parasites. SQ109, a compound in phase IIb/III of clinical trials to treat resistant Mycobacterium tuberculosis, has a potent effect against Trypanosoma cruzi, responsible for Chagas' disease, and on Leishmania mexicana, the causative agent of cutaneous and muco-cutaneous leishmaniasis. In the latter, the toxic dose against intramacrophagic amastigotes is very low (IC50 ~ 11 nM). The proposed mechanism of action on L. mexicana involves the disruption of the parasite intracellular Ca2+ homeostasis through the collapse of the mitochondrial electrochemical potential (ΔΨm). In the present work, we show a potent effect of SQ109 on L. donovani, the parasite responsible for visceral leishmaniasis, the more severe and uniquely lethal form of these infections, obtaining a toxic effect on amastigotes inside macrophages even lower to that obtained in L. mexicana (IC50 of 7.17 ± 0.09 nM) and with a selectivity index > 800, even higher than in L. mexicana. We also demonstrated for first time that SQ109, besides collapsing ΔΨm of the parasite, induced a very rapid damage to the parasite acidocalcisomes, essential organelles involved in the bioenergetics and many other important functions, including Ca2+ homeostasis. Both effects of the drug on these organelles generated a dramatic increase in the intracellular Ca2+ concentration, causing parasite death.


Assuntos
Adamantano/análogos & derivados , Etilenodiaminas/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Adamantano/farmacologia , Animais , Proliferação de Células , Doença de Chagas/tratamento farmacológico , Citoplasma , Humanos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Mitocôndrias , Trypanosoma cruzi/efeitos dos fármacos
4.
Eur J Med Chem ; 186: 111860, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31759728

RESUMO

Methionine aminopeptidase 1 of Leishmania donovani (LdMetAP1) is a novel antileishmanial target for its role in vital N-terminal methionine processing. After LdMetAP1 expression and purification, we employed a series of biochemical assays to determine optimal conditions for catalysis, metal dependence and substrate preferences for this ubiquitous enzyme. Screening of newly synthesized quinoline-carbaldehyde derivatives in inhibition assays led to the identification of HQ14 and HQ15 as novel and specific inhibitors for LdMetAP1 which compete with substrate for binding to the catalytic active site. Both leads bind LdMetAP1 with high affinity and possess druglikeness. Biochemical studies suggested HQ14 and HQ15 to be comparatively less effective against purified HsMetAP1 and showed no or less toxicity. We further show selectivity and inhibition of lead inhibitors is sensed through a non-catalytic Thr residue unique to LdMetAP1. Finally, structural studies highlight key differences in the binding modes of HQ14 and HQ15 to LdMetAP1 and HsMetAP1 providing structural basis for differences in inhibition. The study demonstrates the feasibility of deploying small drug like molecules to selectively target the catalytic activity of LdMetAP1 which may provide an effective treatment of leishmaniasis.


Assuntos
Aldeídos/farmacologia , Aminopeptidases/antagonistas & inibidores , Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Quinolinas/farmacologia , Aldeídos/síntese química , Aldeídos/química , Aminopeptidases/metabolismo , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Leishmania donovani/enzimologia , Estrutura Molecular , Testes de Sensibilidade Parasitária , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-Atividade
5.
Fitoterapia ; 140: 104420, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733344

RESUMO

We report for the first time the isolation of 2-furyl(phenyl)methanol (5) from the chloroform extracts of the Atractylis gummifera roots. A. gummifera is a thistle belonging to the Asteraceae family that produces the ent-kaurane diterpenoid glycoside atractyloside (ATR). ATR (1) was isolated and chemically modified to obtain its aglycone atractyligenin (2) and the methylated derivatives ATR-OMe (3) and genine-OMe (4). The compounds 1-5 were structurally characterised and evaluated against the intracellular amastigote, cultured within macrophages, and the extracellular promastigote of Leishmania donovani, the protozoan parasite responsible for the highly infective disease visceral leishmaniasis, which is fatal if untreated. The 2-furyl(phenyl)methanol 5 exhibited notable activity against the promastigote.


Assuntos
Antiprotozoários/farmacologia , Atractylis/química , Leishmania donovani/efeitos dos fármacos , Metanol/farmacologia , Animais , Antiprotozoários/isolamento & purificação , Itália , Macrófagos/parasitologia , Metanol/análogos & derivados , Metanol/isolamento & purificação , Camundongos Endogâmicos BALB C , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais , Rizoma/química
6.
J Enzyme Inhib Med Chem ; 35(1): 59-64, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31663383

RESUMO

A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from pathogens (bacteria, fungi, and protozoa) was shown to produce an impairment of the microorganism growth and virulence. As phosphonamidates have been recently validated as human α-CA inhibitors (CAIs) and no phosphorus-based zinc-binding group have been assessed to date against ß-class CAs, herein we report an inhibition study with this class of compounds against ß-CAs from pathogenic bacteria, fungi, and protozoa. Our data suggest that phosphonamidates are among the CAIs with the best selectivity for ß-class over human isozymes, making them interesting leads for the development of new anti-infectives.


Assuntos
Amidas/farmacologia , Anti-Infecciosos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Compostos Organometálicos/farmacologia , Ácidos Fosfóricos/farmacologia , Amidas/química , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Fungos/efeitos dos fármacos , Fungos/enzimologia , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/enzimologia , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Ácidos Fosfóricos/química , Fósforo/química , Fósforo/farmacologia , Relação Estrutura-Atividade , Zinco/química , Zinco/farmacologia
7.
J Ethnopharmacol ; 247: 112270, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31589965

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Primates forage on a variety of plant parts to balance their dietary intake to meet requirements of energy, nutrition and maintenance, however the reason(s) leading them to ingest some plants which have no nutritional value and/or contain bioactive or even toxic secondary metabolites is recently gaining closer attention. The growing literature suggests that primates consume plants for medicinal purposes (self-medication) as well, particularly when infected with parasites and pathogens (bacteria, viruses, microbes). Interestingly, some of the plants they consume are also used by humans for similar purposes or may have potential uses for humans. MATERIALS AND METHODS: As part of a 16-month study of the parasite ecology of a sub-species of Japanese macaques (Macaca fuscata yakui) on the island of Yakushima, we surveyed their feeding habits and collected a subset of plants and plant parts observed being ingested by macaques. The ethnomedicinal value of these plants was surveyed and methanolic extracts of 45 plant parts were tested in vitro against important parasites of humans, including four protozoan parasites Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, and the trematode flatworm Schistosoma mansoni. Potential toxicity of the extracts was also assessed on mammalian cells. RESULTS: A wide range of ethnomedicinal uses in Asia for these plants is noted, with 37% associated with the treatment of parasites, pathogens and related symptoms. Additionally, the 45 extracts tested showed broad and significant activity against our test organisms. All extracts were active against T. b. rhodesiense. The majority (over 80%) inhibited the growth of P. falciparum and L. donovani. Half of the extracts also displayed antiprotozoal potential against T. cruzi while only several extracts were active against both larval and adult stages of S. mansoni. Cytotoxicity was generally low, although several extracts lacked specific toxicity to test parasites. CONCLUSIONS: Our results indicated a number of plants and their parts to have antiparasitic activity not previously reported in the ethnopharmacological literature. Enhanced understanding of the primate diets, particularly during periods of intensified parasite infection risk may help to further narrow down plants of interest for lead compound development. The study of animal self-medication is a complementary approach, with precedence, to drug discovery of new lead drug compounds against human parasitic diseases.


Assuntos
Anti-Helmínticos/farmacologia , Antiprotozoários/farmacologia , Macaca fuscata/parasitologia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Infecções Protozoárias em Animais/tratamento farmacológico , Esquistossomose mansoni/tratamento farmacológico , Animais , Anti-Helmínticos/uso terapêutico , Antiprotozoários/uso terapêutico , Etnofarmacologia , Comportamento Alimentar , Feminino , Ilhas , Japão , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/isolamento & purificação , Masculino , Medicina Tradicional/métodos , Testes de Sensibilidade Parasitária , Extratos Vegetais/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Infecções Protozoárias em Animais/parasitologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/veterinária , Automedicação/veterinária , Testes de Toxicidade , Trypanosoma brucei rhodesiense/isolamento & purificação , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/isolamento & purificação
8.
Parasit Vectors ; 12(1): 600, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870416

RESUMO

BACKGROUND: The chemotherapeutic arsenal available to treat visceral leishmaniasis is currently limited, in view of many drawbacks such as high cost, toxicity or emerging resistance. New therapeutic strategies are particularly needed to improve the management and the outcome in immunosuppressed patients. The combination of an immunomodulatory drug to a conventional anti-Leishmania treatment is an emerging concept to reverse the immune bias from Th2 to Th1 response to boost healing and prevent relapses. METHODS: Here, immunostimulating and leishmanicidal properties of octyl-ß-D-galactofuranose (Galf) were assessed in human monocyte-derived macrophages (HM) and in a murine model, after challenge with Leishmania donovani promastigotes. We recorded parasite loads and expression of various cytokines and immune effectors in HM and mouse organs (liver, spleen, bone marrow), following treatment with free (Galf) and liposomal (L-Galf) formulations. RESULTS: Both treatments significantly reduced parasite proliferation in HM, as well as liver parasite burden in vivo (Galf, P < 0.05). Consistent with in vitro results, we showed that Galf- and L-Galf-treated mice displayed an enhanced Th1 immune response, particularly in the spleen where pro-inflammatory cytokines TNF-α, IL-1ß and IL-12 were significantly overexpressed compared to control group. The hepatic recruitment of myeloid cells was also favored by L-Galf treatment as evidenced by the five-fold increase of myeloperoxidase (MPO) induction, which was associated with a higher number of MPO-positive cells within granulomas. By contrast, the systemic level of various cytokines such as IL-1ß, IL-6, IL-17A or IL-27 was drastically reduced at the end of treatment. CONCLUSIONS: Overall, these results suggest that Galf could be tested as an adjuvant in combination with current anti-parasitic drugs, to restore an efficient immune response against infection in a model of immunosuppressed mice.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Dissacarídeos/administração & dosagem , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Feminino , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
9.
Int J Nanomedicine ; 14: 6073-6101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31686803

RESUMO

Background: Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. Methods: Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet-visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. Results: Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). Conclusion: GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.


Assuntos
Anfotericina B/síntese química , Antiprotozoários/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Anfotericina B/química , Anfotericina B/farmacologia , Animais , Antiprotozoários/química , Candida albicans/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Difusão Dinâmica da Luz , Ergosterol/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Nanopartículas Metálicas/ultraestrutura , Camundongos , Carbonilação Proteica/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Ácido Tióctico/química , Resultado do Tratamento
10.
SAR QSAR Environ Res ; 30(12): 919-933, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31702401

RESUMO

Folates are essential biomolecules required to carry out many crucial processes in leishmania parasite. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) involved in folate biosynthesis in leishmania have been established as suitable targets for development of chemotherapy against leishmaniasis. In the present study, various computational tools such as homology modelling, pharmacophore modelling, docking, molecular dynamics and molecular mechanics have been employed to design dual DHFR-TS and PTR1 inhibitors. Two designed molecules, i.e. 2-(4-((4-nitrobenzyl)oxy)phenyl)-1H-benzo[d]imidazole and 2-(4-((2,4-dichlorobenzyl)oxy)phenyl)-1H-benzo[d]oxazolemolecules were synthesized. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to evaluate in vitro activity of molecules against promastigote form of Leishmania donovani using Miltefosine as standard. 2-(4-((4-nitrobenzyl)oxy)phenyl)-1H-benzo[d]imidazole and 2-(4-((2,4-dichlorobenzyl)oxy)phenyl)-1H-benzo[d]oxazolemolecules were found to be moderately active with showed IC50 = 68 ± 2.8 µM and 57 ± 4.2 µM, respectively.


Assuntos
Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Complexos Multienzimáticos/química , Oxirredutases/química , Proteínas de Protozoários/química , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/química , Antiprotozoários/síntese química , Antiprotozoários/química , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzoxazóis/síntese química , Benzoxazóis/química , Benzoxazóis/farmacologia , Descoberta de Drogas , Concentração Inibidora 50 , Leishmania donovani/metabolismo , Modelos Moleculares , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Relação Estrutura-Atividade
11.
Int J Parasitol Drugs Drug Resist ; 10: 125-132, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31493763

RESUMO

Visceral leishmaniasis (VL) is associated with treatment complications due to the continued growth of resistant parasites toward currently available pathogen-directed therapeutics. To limit the emergence and combat resistant parasites there is a need to develop new anti-leishmanial drugs and alternative treatment approaches, such as host-directed therapeutics (HDTs). Discovery of new anti-leishmanial drugs including HDTs requires suitable in vitro assay systems. Herein, we modified and evaluated a series of resazurin assays against different life-stages of the VL causing parasite, Leishmania donovani to identify novel HDTs. We further analyzed the synergy of combinatorial interactions between traditionally used pathogen-directed drugs and HDTs for clearance of intracellular L. donovani. The inhibitory concentration at 50% (IC50) of the five evaluated therapies [amphotericin B (AMB), miltefosine, paromomycin, DNER-4, and AR-12 (OSU-03012)] was determined against promastigotes, extracellular amastigotes, and intracellular amastigotes of L. donovani via a resazurin-based assay and compared to image-based microscopy. Using the resazurin-based assay, all evaluated therapies showed reproducible anti-leishmanial activity against the parasite's different life-stages. These results were consistent to the traditional image-based technique. The gold standard of therapy, AMB, showed the highest potency against intracellular L. donovani, and was further evaluated for combinatorial effects with the HDTs. Among the combinations analyzed, pathogen-directed AMB and host-directed AR-12 showed a synergistic reduction of intracellular L. donovani compared to individual treatments. The modified resazurin assay used in this study demonstrated a useful technique to measure new anti-leishmanial drugs against both intracellular and extracellular parasites. The synergistic interactions between pathogen-directed AMB and host-directed AR-12 showed a great promise to combat VL, with the potential to reduce the emergence of drug-resistant strains.


Assuntos
Antiprotozoários/administração & dosagem , Quimioterapia Combinada/métodos , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Anfotericina B/administração & dosagem , Animais , Sinergismo Farmacológico , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/diagnóstico por imagem , Leishmaniose Visceral/parasitologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/administração & dosagem , Fosforilcolina/análogos & derivados , Pirazóis/administração & dosagem , Sulfonamidas/administração & dosagem
12.
Acta Trop ; 199: 105158, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31491399

RESUMO

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan Leishmania donovani parasite which may be fatal if left untreated. While drug-sensitive parasites are able to live and multiply within the host macrophages, they develop resistance to drugs used against them for survival and multiplication in the infected patients undergoing routine treatment. Development of new agents devoid of such drug resistance potential is achievable by identifying new drug targets in the parasite. One such target is the key regulator of intracellular vesicular trafficking protein, RabGTPase which belongs to the Ras GTPase superfamily. We recently elucidated whole genome sequence (WGS) of L. donovani (clinical Indian isolate; BHU 1220, GenBank: AVPQ00000000.1) and identified Ldrab6 gene. We now provide experimental evidence for this gene's ability to impart drug-resistant phenotype to wild-type (sensitive) Leishmania upon transfection. trans-Dibenzalacetone (DBA), a synthetic analog of curcumin, was used to determine its antileishmanial activity in wild-type parasites and parasites transfected with Ldrab6 gene. Dose-response study showed that DBA had no effect on transfected parasites at 20 µg/mL dose, whereas wild-type promastigotes showed 50% inhibition (IC50) at the same dose. This indicates the development of resistant mechanism in the transfected parasites due to enhancement of the copy number of Ldrab6 gene in L. donovani parasites. Flow cytometric analysis revealed elevated level of thiols in transfectants when compared to wild-type parasites treated with DBA. To assess the functional activity of multidrug resistance-associated protein (MRP) pump in transfectants, the accumulation of calcein, a known MRP pump substrate and probenecid, a known MRP pump regulator, were analyzed. The results indicate that Ldrab6 gene in Leishmania conferred resistance by the well-established mechanism of drug-thiol conjugation and sequestration by ABC transporter multidrug resistance-protein A (MRPA). Accordingly, Leishmania parasites transfected with Ldrab6 gene can be used as an experimental cell line for the screening of new lead molecules for their propensity to develop drug resistance.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Antiprotozoários/farmacologia , Dosagem de Genes , Leishmania donovani/genética , Proteínas rab de Ligação ao GTP/genética , Animais , Linhagem Celular , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Leishmania donovani/efeitos dos fármacos , Pentanonas/farmacologia , Compostos de Sulfidrila/química
13.
Eur J Med Chem ; 182: 111632, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31499363

RESUMO

ß-amino acids and their analogues are gathering increased attention not only because of their antibacterial and antifungal activity, but also for their use in designing peptidomimetics with increased oral bioavailability and resistance to metabolic degradation. In this study, a series of α-phenyl substituted chalcones, α-phenyl, ß-amino substituted dihydrochalcones and ß-amino acid derivatives were synthesized and evaluated for their antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all synthesized derivatives, 10c showed promising antileishmanial efficacy against both extracellular promastigote and intracellular amastigote (IC50 8.2 µM and 20.5 µM respectively) of L. donovani with negligible cytotoxic effect towards J774 macrophages and Vero cells. 10c effectively reduced spleen and liver parasite burden (>90%) in both hamster and Balb/c model of VL without any hepatotoxicity. In vitro pharmacokinetic analysis showed that 10c was stable in gastric fluid and plasma of Balb/c mice at 10 µg/ml. Further analysis of the molecular mechanism revealed that 10c entered into the parasite by depolarizing the plasma membrane rather than forming nonspecific pores and induced molecular events like loss in mitochondrial membrane potential with a gradual decline in ATP production. This, in turn, did not induce programmed cell death of the parasite; rather 10c induced bioenergetic collapse of the parasite by decreasing ATP synthesis through specific inhibition of mitochondrial complex III activity. Altogether, our results allude to the therapeutic potential of ß-amino acid derivatives as novel antileishmanials, identifying them as lead compounds for further exploration in the design of potent candidates for the treatment of visceral leishmaniasis.


Assuntos
Aminoácidos/farmacologia , Antiprotozoários/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Aminoácidos/química , Animais , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Células Vero
15.
PLoS Negl Trop Dis ; 13(8): e0007643, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381572

RESUMO

Bacterial strains isolated from attine ants showed activity against the insect specialized fungal pathogen Escovopsis and also against the human protozoan parasite Leishmania donovani. The bioassay guided fractionation of extracts from cultures of Streptomyces sp. ICBG292, isolated from the exoskeleton of Cyphomyrmex workers, led to the isolation of Mer-A2026B (1), piericidin-A1 (2) and nigericin (3). Nigericin (3) presented high activity against intracellular amastigotes of L. donovani (IC50 0.129 ± 0.008 µM). Streptomyces puniceus ICBG378, isolated from workers of Acromyrmex rugosus rugosus, produced dinactin (4) with potent anti-L. donovani activity against intracellular amastigotes (IC50 0.018 ± 0.003 µM). Compounds 3 and 4 showed good selectivity indexes, 88.91 and 656.11 respectively, and were more active than positive control, miltefosine. Compounds 1-4 were also active against some Escovopsis strains. Compounds 1 and 2 were also produced by Streptomyces sp. ICBG233, isolated from workers of Atta sexdens, and detected in ants' extracts by mass spectrometry, suggesting they are produced in the natural environment as defensive compounds involved in the symbiotic interaction.


Assuntos
Anti-Infecciosos/metabolismo , Formigas/microbiologia , Hypocreales/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Streptomyces/isolamento & purificação , Streptomyces/metabolismo , Animais , Anti-Infecciosos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Streptomyces/classificação
16.
Eur J Med Chem ; 182: 111568, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31419778

RESUMO

The human protozoan parasites Leishmania donovani and L. infantum are the causative agents of visceral leishmaniasis, as such, responsible for approximately 30,000 deaths annually. The available chemotherapeutic treatments are reduced to a few drugs whose effectiveness is limited by rising drug resistance/therapeutic failure, and noxious side-effects. Therefore, new therapeutic hits are needed. Compounds displaying the imidazo[2,1-a]isoindole skeleton have shown antichagasic, anti-HIV, antimalarial and anorectic activities. Here, we report the leishmanicidal activity of thirty one imidazo[2,1-a]isoindol-5-ol derivatives on promastigotes and intracellular amastigotes of L. donovani. Eight out of thirty one assayed compounds showed EC50 values ranging between 1 and 2 µM with selectivity indexes from 29 to 69 on infected THP-1 cells. Six compounds were selected for further elucidation of their leishmanicidal mechanism. In this regard, compound 29, the imidazoisoindolol with the highest activity on intracellular amastigotes, induced an early decrease of intracellular ATP levels, as well as mitochondrial depolarization, together with a partial plasma membrane destructuration, as assessed by transmission electron microscopy. Consequently, the inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound, even when other additional targets cannot be ruled out. In all, the results supported the inclusion of the imidazoisoindole scaffold for the development of new leishmanicidal drugs.


Assuntos
Antiprotozoários/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Leishmania donovani/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Imidazóis/síntese química , Imidazóis/química , Indóis/síntese química , Indóis/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
17.
Int J Antimicrob Agents ; 54(4): 496-501, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31323307

RESUMO

Leishmania parasites are the causative agents of a wide spectrum of human diseases. The clinical manifestations of leishmaniasis range from self-healing skin lesions to fatality. The World Health Organization has classed leishmaniasis as a category 1 neglected tropical disease. Leishmaniasis represents a major international health challenge, affecting 12 million people per year and with nearly 310 million people at risk. The first-line chemotherapies used to treat leishmaniasis are intravenous pentavalent antimonials; however, these drugs are highly toxic. As the use of oral treatment options such as paromomycin and miltefosine has increased, the incidence of disease relapse has increased and drug resistance to antimonials has developed, emphasizing the importance of identifying new chemotherapies. A novel, target-free fluorometric high-throughput screen with an average Z-score of 0.73 +/- 0.13 has been developed to identify small molecules with antileishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVER-set™ library cassette #5 yielded 210 compounds that killed 80% of parasites, resulting in a hit rate of 2.1%. One hundred and nine molecular scaffolds were represented within the hit compounds, and one scaffold that exhibited potent antileishmanial activity was 2,4-diaminoquinazoline. Host cell toxicity was determined prior to in-vitro infection of human THP-1 macrophages with Leishmania donovani mCherry expressing promastigotes; successful drug treatment was considered when the half maximal inhibitory concentration was <10 µM. BALB/c mice were infected with Leishmania major mCherry promastigotes and treated with small molecules that were successful during in-vitro infections. Several small molecules tested were as efficacious at resolving cutaneous leishmaniasis lesions in mice as known antimonial treatments.


Assuntos
Antiprotozoários/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Leishmania donovani/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Modelos Animais de Doenças , Feminino , Fluorometria/métodos , Humanos , Camundongos Endogâmicos BALB C , Recidiva , Células THP-1/parasitologia , Resultado do Tratamento
18.
Parasitol Res ; 118(9): 2705-2713, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31359134

RESUMO

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.


Assuntos
Antiprotozoários/administração & dosagem , Artemisininas/administração & dosagem , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Extratos Vegetais/administração & dosagem , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Artemisia annua/química , Artesunato/administração & dosagem , Feminino , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/imunologia , Interações Hospedeiro-Parasita , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/fisiopatologia , Macrófagos/imunologia , Camundongos Endogâmicos BALB C
19.
Artigo em Inglês | MEDLINE | ID: mdl-31158574

RESUMO

Visceral leishmaniasis (VL) and Chagas disease (CD) are caused by kinetoplastid parasites that affect millions of people worldwide and impart a heavy burden against human health. Due to the partial efficacy and toxicity-related limitations of the existing treatments, there is an urgent need to develop novel therapies with superior efficacy and safety profiles to successfully treat these diseases. Herein we report the application of whole-cell phenotypic assays to screen a set of 150,000 compounds against Leishmania donovani, a causative agent of VL, and Trypanosoma cruzi, the causative agent of CD, with the objective of finding new starting points to develop novel drugs to effectively treat and control these diseases. The screening campaign, conducted with the purpose of global open access, identified twelve novel chemotypes with low to sub-micromolar activity against T. cruzi and/or L. donovani. We disclose these hit structures and associated activity with the goal to contribute to the drug discovery community by providing unique chemical tools to probe kinetoplastid biology and as hit-to-lead candidates for drug discovery.


Assuntos
Antiprotozoários/farmacologia , Doença de Chagas/parasitologia , Descoberta de Drogas/métodos , Leishmaniose/parasitologia , Animais , Antiprotozoários/química , Linhagem Celular , Doença de Chagas/tratamento farmacológico , Descoberta de Drogas/instrumentação , Avaliação Pré-Clínica de Medicamentos , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose/tratamento farmacológico , Testes de Sensibilidade Parasitária , Ratos , Relação Estrutura-Atividade , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento
20.
J Enzyme Inhib Med Chem ; 34(1): 1164-1171, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31219348

RESUMO

Chagas disease and leishmaniasis are neglected tropical disorders caused by the protozoans Trypanosoma cruzi and Leishmania spp. Carbonic anhydrases (CAs, EC 4.2.1.1) from these protozoans (α-TcCA and ß-LdcCA) have been validated as promising targets for chemotherapic interventions. Many anti-protozoan agents, such as nitroimidazoles, nifurtimox, and benznidazole possess a nitro aromatic group in their structure which is crucial for their activity. As a continuation of our previous work on N-nitrosulfonamides as anti-protozoan agents, we investigated benzenesulfonamides bearing a nitro aromatic moiety against TcCA and LdcCA, observing selective inhibitions over human off-target CAs. Selected derivatives were assessed in vitro in different developmental stages of T. cruzi and Leishmania spp. A lack of significant growth inhibition has been found, which has been connected to the low permeability of this class of derivatives through cell membranes. Further strategies necessarily need to be designed for targeting Chagas disease and leishmaniasis with nitro-containing CA inhibitors.


Assuntos
Antiprotozoários/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Leishmania donovani/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Leishmania donovani/enzimologia , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Trypanosoma cruzi/enzimologia
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