Selenium-Containing (Hetero)Aryl Hybrids as Potential Antileishmanial Drug Candidates: In Vitro Screening against L. amazonensis.

Leishmaniasis remains a significant global health concern, with current treatments relying on outdated drugs associated with high toxicity, lengthy administration, elevated costs, and drug resistance. Consequently, the urgent need for safer and more effective therapeutic options in leishmaniasis treatment persists. Previous research has highlighted selenium compounds as promising candidates for innovative leishmaniasis therapy. In light of this, a library of 10 selenium-containing diverse compounds was designed and evaluated in this study. These compounds included selenium-substituted indole, coumarin, chromone, oxadiazole, imidazo[1,2-a]pyridine, Imidazo[2,1-b]thiazole, and oxazole, among others. These compounds were screened against Leishmania amazonensis promastigotes and intracellular amastigotes, and their cytotoxicity was assessed in peritoneal macrophages, NIH/3T3, and J774A.1 cells. Among the tested compounds, MRK-106 and MRK-108 displayed the highest potency against L. amazonensis promastigotes with reduced cytotoxicity. Notably, MRK-106 and MRK-108 exhibited IC50 values of 3.97 µM and 4.23 µM, respectively, and most of the tested compounds showed low cytotoxicity in host cells (CC50 > 200 µM). Also, compounds MRK-107 and MRK-113 showed activity against intracellular amastigotes (IC50 18.31 and 15.93 µM and SI 12.55 and 10.92, respectively). In conclusion, the identified selenium-containing compounds hold potential structures as antileishmanial drug candidates to be further explored in subsequent studies. These findings represent a significant step toward the development of safer and more effective therapies for leishmaniasis, addressing the pressing need for novel and improved treatments.

Flavonoid Derivatives as New Potent Inhibitors of Cysteine Proteases: An Important Step toward the Design of New Compounds for the Treatment of Leishmaniasis.

Leishmaniasis is a neglected tropical disease, affecting more than 350 million people globally. However, there is currently no vaccine available against human leishmaniasis, and current treatment is hampered by high cost, side-effects, and painful administration routes. It has become a United Nations goal to end leishmaniasis epidemics by 2030, and multitarget drug strategy emerges as a promising alternative. Among the multitarget compounds, flavonoids are a renowned class of natural products, and a structurally diverse library can be prepared through organic synthesis, which can be tested for biological effectiveness. In this study, we synthesised 17 flavonoid analogues using a scalable, easy-to-reproduce, and inexpensive method. All synthesised compounds presented an impressive inhibition capacity against rCPB2.8, rCPB3, and rH84Y enzymes, which are highly expressed in the amastigote stage, the target form of the parasite. Compounds 3c, f12a, and f12b were found to be effective against all isoforms. Furthermore, their intermolecular interactions were also investigated through a molecular modelling study. These compounds were highly potent against the parasite and demonstrated low cytotoxic action against mammalian cells. These results are pioneering, representing an advance in the investigation of the mechanisms behind the antileishmanial action of flavonoid derivatives. Moreover, compounds have been shown to be promising leads for the design of other cysteine protease inhibitors for the treatment of leishmaniasis diseases.

Antileishmanial Activity, Toxicity and Mechanism of Action of Complexes of Sodium Usnate with Lanthanide Ions: Eu(III), Sm(III), Gd(III), Nd(III), La(III) and Tb(III).

Leishmaniases are neglected diseases with limited therapeutic options. Diffuse cutaneous leishmaniasis can occur in Brazil due to Leishmania amazonensis. This study details the antileishmanial activity and cytotoxicity of complexes of sodium usnate (SAU) with lanthanide ions ([LnL3 (H2O)x] (Ln = La(III), Nd(III), Gd(III), Tb(III), Eu(III) and Sm(III); L = SAU). All lanthanide complexes were highly active and more potent than SAU against L. amazonensis promastigotes and intracellular amastigotes (Pro: IC50 < 1.50 µM; Ama: IC50 < 7.52 µM). EuL3·3H2O and NdL3·3H2O were the most selective and effective on intracellular amastigotes, with a selectivity index of approximately 7.0. In silico predictions showed no evidence of mutagenicity, tumorigenicity or irritation for all complexes. Treatment with EuL3·3H2O triggered NO release even at the lowest concentration, indicating NO production as a mechanism of action against the parasite. Incubating promastigotes with the lanthanide complexes, particularly with SmL3·4H2O and GdL3·3H2O, led to a change in the mitochondrial membrane potential, indicating the ability of these complexes to target this essential organelle. The same complexes caused cell death through cell membrane disruption, but their relationship with early or late apoptotic processes remains unclear. Thus, the inclusion of lanthanide ions in SAU improves selectivity with a promising mechanism of action targeting the mitochondria.

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50 = 28.5 ± 2.0 µM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.

Amentoflavone as an Ally in the Treatment of Cutaneous Leishmaniasis: Analysis of Its Antioxidant/Prooxidant Mechanisms.

Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoan Leishmania amazonensis. Although the antileishmanial activity of amentoflavone has already been reported in vitro, the mechanisms involved in the parasite death, as well as its action in vivo, remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC50 2.3 ± 0.93 µM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected with L. amazonensis we verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infected in vitro. The increase of ROS in vitro, associated with the reduction of NO and iNOS expression in vivo, points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion.

Spatio-temporal modeling of visceral leishmaniasis in Midwest Brazil: An ecological study of 18-years data (2001-2018).

Visceral leishmaniasis (VL) is a neglected vector-borne disease associated with socioeconomic and environmental issues. In Brazil, epidemics of VL have occurred in major cities since 1980. Applied models for medical and epidemiological research have been used to assess the distribution and characteristics of disease endpoints and identify and characterize potential risk factors. This study described the demographic features of VL and modeled the spatio-temporal distribution of human VL cases and their relationship with underlying predicitve factors using generalized additive models. We conducted an ecological study covering an 18-year period from the first report of an autochthonous case of VL in Campo Grande, state of Mato Grosso do Sul, in 2001 to 2018. The urban area of the city has 74 neighborhoods, and they were the units of analysis of our work. Socioeconomic and demographic data available from Brazilian public databases were considered as covariables. A total of 1,855 VL cases were reported during the study period, with an annual mean incidence rate of 13.23 cases per 100,000 population and a cumulative crude incidence of 235.77 per 100,000 population. The results showed the rapid transition from epidemic to endemic and the centrifugal dispersal pattern of the disease. Moreover, the model highlighted that the urban quality of life index, which is calculated based on income, education, housing conditions, and environmental sanitation data, plays a role in VL occurrence. Our findings highlighted the potential for improving spatio-temporal segmentation of control measures and the cost-effectiveness of integrated disease management programs as soon as VL is difficult to control and prevent and has rapid geographical dispersion and increased incidence rates.

Characterization of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) activity in mouse peritoneal cavity cells.

This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E-NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+ . A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E-NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii.

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Hairless mice as an experimental model of infection with Leishmania (Leishmania) amazonensis.

HRS/J Hairless mice have been investigated as an experimental model in cutaneous leishmaniasis induced by Leishmania (Leishmania) amazonensis. The animals were inoculated with 10(6) promastigotes into the right hind footpad and the course of infection was followed up for 30, 60 and 90 days. BALB/c mice were infected and used as control. Hairless mice were susceptible to L. (L.) amazonensis infection and a progressive increase in number of parasites and footpad thickness was detected over time. Signals of dissemination and visceralization were confirmed by the presence of parasite in the draining lymph node of lesion and spleen, at different times post infection. IL-10 gene expression evaluated by RT-PCR was significantly higher in Hairless mice at 60 days post infection, corroborating the pattern of susceptibility. These results point this inbred strain as a promising susceptible model for the study of experimental infection induced by L. (L.) amazonensis. This model would allow the use of other infection sites that minimize secondary interference and best monitoring the skin lesion, as in the case of in vivo assays of potential drugs for LT.

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis.

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 µg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 µg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

Epidemiological study on leishmaniasis in an area of environmental tourism and ecotourism, State of Mato Grosso do Sul, 2006-2007.

The aims of this study were to carry out a serological survey of canine leishmaniasis and identify the phlebotomine fauna in the urban area of Bonito, Mato Grosso do Sul. The serological survey was conducted on a sample of 303 dogs, by means of the indirect immunofluorescence test. Phlebotomines were captured using automated light traps. The serological survey found that 30% of the dogs were seropositive, both from the center and from all districts of the town. A total of 2,772 specimens of phlebotomines were caught and the species most found was Lutzomyia longipalpis (90.4%), which corroborated its role as the vector of for canine visceral leishmaniasis in the region. Phlebotomines of the species Bichromomyia flaviscutellata (the main vector for Leishmania (Leishmania) amazonensis) and Nyssomyia whitmani (the vector for Leishmania (Viannia) brasiliensis) were also caught. The findings indicate the need for continuous epidemiological surveillance, with attention towards diminishing the vector breeding sites and the transmission of these diseases in that region.

Epidemiological study on leishmaniasis in an area of environmental tourism and ecotourism, State of Mato Grosso do Sul, 2006-2007 / Estudo epidemiológico das leishmanioses em área de turismo ambiental e ecoturismo, Estado de Mato Grosso do Sul, 2006-2007

The aims of this study were to carry out a serological survey of canine leishmaniasis and identify the phlebotomine fauna in the urban area of Bonito, Mato Grosso do Sul. The serological survey was conducted on a sample of 303 dogs, by means of the indirect immunofluorescence test. Phlebotomines were captured using automated light traps. The serological survey found that 30 percent of the dogs were seropositive, both from the center and from all districts of the town. A total of 2,772 specimens of phlebotomines were caught and the species most found was Lutzomyia longipalpis (90.4 percent), which corroborated its role as the vector of for canine visceral leishmaniasis in the region. Phlebotomines of the species Bichromomyia flaviscutellata (the main vector for Leishmania (Leishmania) amazonensis) and Nyssomyia whitmani (the vector for Leishmania (Viannia) brasiliensis) were also caught. The findings indicate the need for continuous epidemiological surveillance, with attention towards diminishing the vector breeding sites and the transmission of these diseases in that region.

O presente trabalho teve por objetivo proceder ao levantamento sorológico para leishmanioses em cães e identificar a fauna flebotomínea da zona urbana de Bonito, Mato Grosso do Sul. O inquérito sorológico foi realizado em amostras de 303 cães com a utilização da reação de imunofluorescência indireta. As capturas de flebotomíneos realizaram-se com armadilhas automáticas luminosas. O inquérito sorológico identificou 30 por cento cães reagentes procedentes do centro e de todos os bairros da cidade. Foram capturados 2,772 exemplares de flebotomineos, sendo a espécie mais freqüente foi Lutzomyia longipalpis (90.4 por cento), o que corrobora o seu papel de vetora de leishmaniose visceral canina na região. Foram capturados, também, flebotomíneos da espécie Bichromomyia flaviscutellata, principal vetora da Leishmania (Leishmania) amazonensis, e Nyssomyia whitmani, vetora da Leishmania (Viannia) braziliensis. Os achados indicam a necessidade de uma contínua vigilância epidemiológica, atentando para a diminuição dos criadouros dos vetores e da transmissão desses agravos naquela região.

Indução de resistência à infecção por Leishmania amazonensis (Kinetoplastida: Trypanosomatidae) no modelo murino por formas atenuadas do parasito / Induction of resistance against Leishmania amazonensis (Kinetoplastida: Trypanosomatidae) infection in murine model by attenuated forms of the parasite

As leishmanioses tegumentares compreendem importantes doenças em termos de morbidade, principalmene em áreas tropicais do mundo. No Brasil, a Leishmaniose Tegumentar Americana encontra-se em expansão em todas as regiões, sendo assinalada em praticamente todos os Estados. O controle da leishmaniose torna-se difícil pela grande variedade de espécies distintas de Leishmania e seus respectivos perfis epidemiológicos. Uma solução seria o desenvolvimento de vacinas contra a doença, o que se torna extremamente difícil devido ao polimorfismo genético e à diversidade biológica dos parasitos. No presente trabalho, os processos de fotoferese (associação do 8-metoxipsoraleno com a radiação UVA) e exposição à radiação-g foram utilizados como mecanismos de atenuação das formas promastigotas de L. amazonensis, visando à indução de resistência contra a infecção com as formas virulentas do parasito no modelo murino. Foi observado que, apesar das formas promastigotas tratadas pela dose de 50 Krad de radiação-g não provocarem lesões cutâneas características de L. amazonensis, as mesmas não foram capazes de induzir imunidade protetora em camundongos suíços, fato verificado através do desafio com formas virulentas do parasitos, onde foi observada uma alta carga parasitária no local da inoculação. A imunomodulação da vacinação com promastigotas g-irradiadas pelo BCG conferiu proteção aos camundongos desafiados, evidenciada pelo estudo histopatológico, que demonstrou ausência de parasitos e presença de reação inflamatória local. A vacinação com promastigotas tratadas pelo 8-MOP associado à radiação UVA induziu resistência dos camundongos ao desafio com formas virulentas, não sendo observados parasitos através de estudo histopatológico até 90 dias após o desafio. A vacinação com formas tratadas somente pela radiação UVA ou pelo 8-MOP, no entanto, não foi capaz de induzir tal imunidade, verificando-se lesões semelhantes às da infecção pelas formas virulentas do parasito. A reinfecção com formas promastigotas de L. amazonensis realizada seis dias após a primeira inoculação também foi indutora de resistência, uma vez que não foram encontrados parasitos nos camundongos reinfectados até 90 dias após a dose reinfectiva através de estudo histopatológico.