Low doses of 3-phenyl-lawsone or meglumine antimoniate delivery by tattooing route are successful in reducing parasite load in cutaneous lesions of Leishmania (Viannia) braziliensis-infected hamsters.

Current therapeutic ways adopted for the treatment of leishmaniasis are toxic and expensive including parasite resistance is a growing problem. Given this scenario, it is urgent to explore treatment alternatives for leishmaniasis. The aim of this study was to evaluate the effect of 3-phenyl-lawsone (3-PL) naphthoquinone on Leishmania (Viannia) braziliensis infection, both in vitro and in vivo, using two local routes of administration: subcutaneous (higher dose) and tattoo (lower dose). In vitro 3-PL showed low toxicity for macrophages (CC50 >3200 µM/48h) and activity against intracellular amastigotes (IC50 = 193 ± 19 µM/48h) and promastigotes (IC50 = 116 ± 26 µM/72h), in which induced increased ROS generation. Additionally, 3-PL up-regulated the production of cytokines such as tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6) and IL-10 in infected macrophages. However, the anti-amastigote action was independent of nitric oxide production. Treatment of hamsters infected with L. (V.) braziliensis from one week after infection with 3-PL by subcutaneous (25 µg/Kg) or tattooing (2.5 µg/Kg) route, during 3 weeks (3 times/week) or 2 weeks (2 times/week) significantly decreased the parasite load (p<0.001) in the lesion. The reduction of parasite load by 3-PL treatment was comparable to reference drug meglumine antimoniate administered by the same routes (subcutaneous 1mg/Kg and tattoo 0.1mg/Kg). In addition, treatment started from five weeks after infection with 3-PL per tattoo also decreased the parasite load. These results show the anti-leishmanial effect of 3-PL against L. (V.) braziliensis and its efficacy by subcutaneous (higher dose) and tattoo (lower dose) routes. In addition, this study shows that drug delivery by tattooing the lesion allows the use of lower doses than the conventional subcutaneous route, which may support the development of a new therapeutic strategy that can be adopted for leishmaniasis.

LQB-118 Suppresses Migration and Invasion of Prostate Cancer Cells by Modulating the Akt/GSK3ß Pathway and MMP-9/Reck Gene Expression.

BACKGROUND/AIM: Prostate cancer (PCa) is one of the most common malignancies in adult men. LQB-118 is a pterocarpanquinone with antitumor activity toward prostate cancer cells. It inhibits cell proliferation by down-regulating cyclins D1 and B1 and up-regulating p21. However, the effects of LQB-118 on PCa cell migration are still unclear. Herein, the LQB-118 effects on PCa metastatic cell migration/invasion and its mechanism of action were evaluated. MATERIALS AND METHODS: PC3 cells were treated with LQB-118 or Paclitaxel (PTX), and cell migration (wound healing and Boyden chamber assays) and invasion (matrigel assay) were determined. The LQB-118 mechanisms were evaluated by αVßIII protein expression (flow cytometry), protein phosphorylation (Western blot), and mRNA expression (qPCR). RESULTS: LQB-118 impaired PCa cell migration and invasion, down-regulated Akt phosphorylation, and also reduced GSK3ß phosphorylation, through a FAK-independent pathway. Also, it was observed that LQB-118 controlled the invasiveness behavior by reducing matrix metalloproteinase-9 (MMP-9) and up-regulating reversion-inducing cysteine rich protein with Kazal motifs (Reck) mRNA levels. Interestingly, LQB-118 increased integrin αvßIII expression, but this effect was not related to its activation, since the cell adhesion ability was reduced after LQB-118 treatment. CONCLUSION: These data highlight novel LQB-118 mechanisms in prostate cancer cells. LQB-118 acts as a negative regulator of the Akt/GSK3 signaling pathway and can modulate PCa cell proliferation, death, and migration/invasion. The results also support the use of LQB-118 for the treatment of metastatic PCa, alone or combined with another chemotherapeutic agent, due to its demonstrated pleiotropic activities.

Effects of anti-Leishmania compounds in the behavior of the sand fly vector Lutzomyia longipalpis.

BACKGROUND: Leishmaniasis is an infectious parasitic disease caused by pathogens of the genus Leishmania transmitted through the bite of adult female sand flies. To reduce case numbers, it is necessary to combine different control approaches, especially those aimed at the sand fly vectors. Innovative forms of control with the use of attractive sugar baits explored the fact that adult sand flies need to feed on sugars of plant origin. Leishmania parasites develop in the gut of sand flies, interacting with the sugars in the diet of adults. Recent studies have shown that sugar baits containing plant-derived compounds can reduce sand fly survival, the number of parasites per gut, and the percentage of infected sand flies. Several synthetic compounds produced from naphthoquinones and pterocarpans have anti-parasitic activity on Leishmania amazonensis and/or Leishmania infantum in cell culture. This work aimed to assess the inclusion of these compounds in sugar baits for blocking transmission, targeting the development of the Leishmania parasite inside the sand fly vector. RESULTS: We evaluated the attractant or repellent properties of these compounds, as well as of the reference compound N,N'-diethyl-m-toluamide (DEET), in sugar baits. We also observed changes in feeding preference caused by these compounds, looking for anti-feeding or stimulation of ingestion. Pterocarpanquinone L4 and pentamidine showed attractant and repellent properties, respectively. CONCLUSION: Based on the effects in feeding preference and intake volume, pterocarpanquinone L6, and the pyrazole-derived compound P8 were chosen as the most promising compounds for the future development of anti-Leishmania sugar baits. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi.

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 µM] and extracellular amastigotes [IC50/24 h = 3.3 µM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of "proton leak" (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and "ETS" (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration ("ROX") enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.

The pterocarpanquinone LQB 118 inhibits inflammation triggered by zymosan in vivo and in vitro.

LQB 118, a hydride molecule, has been described as an antineoplastic and antiparasitic drug. Recently, LQB118 was also shown to display anti-inflammatory properties using an LPS-induced lung inflammation model. However, LQB 118 effects on the inflammatory response induced by zymosan has not been demonstrated. In this study, swiss mice were LQB 118 intraperitoneally (i.p.) treated and zymosan was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for cell counting and proinflammatory cytokines quantification (IL-1ß, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). For in vitro studies, peritoneal macrophages zymosan-stimulated were used. Results demonstrated that LQB 118 treatment reduced polymorphonuclear cell migration and TNF-α, IL-1ß, and IL-6 levels in the peritoneal cavity. In macrophages, LQB 118 treatment display no cytotoxic effect and is also able to reduce cytokines levels. To investigate LQB 118 putative mechanism of action, TLR2, CD69, and P-p38 MAPK expression were evaluated. LQB 118 treatment reduced CD69 expression and p38 phosphorylation induced by zymosan. Furthermore, LQB 118 was able to negatively modulate TLR2 expression in the presence of inflammatory stimulus. Thus, our study provide new evidences for the mechanisms related to the anti-inflammatory effect of LQB 118 in vivo and in vitro.

The LQB-223 Compound Modulates Antiapoptotic Proteins and Impairs Breast Cancer Cell Growth and Migration.

Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.

Mutagenic and Cytotoxicity LQB 123 Profile: Safety and Tripanocidal Effect of a Phenyl-t-Butylnitrone Derivative.

The therapeutic options for Chagas disease are limited and its treatment presents a number of drawbacks including toxicity, drug resistance, and insufficient effectiveness against the chronic stage of the disease. Therefore, new therapeutical options are mandatory. In the present work, we evaluated the effect of a phenyl-tert-butylnitrone (PBN) derivate, LQB 123, against Trypanosoma cruzi forms. LQB 123 presented a trypanocidal effect against bloodstream trypomastigotes (IC50 = 259.4 ± 6.1 µM) and intracellular amastigotes infecting peritoneal macrophages (IC50 = 188.2 ± 47.5 µM), with no harmful effects upon the mammalian cells (CC50 values greater than 4 mM), resulting in a high selectivity index (CC50/IC50 > 20). Additionally, metacyclic trypomastigotes submitted to LQB 123 presented an IC50 of about 191.8 ± 10.5 µM and epimastigotes forms incubated with different concentrations of LQB 123 presented an inhibition of parasite growth with an IC50 of 255.1 ± 3.6 µM. Finally, we investigated the mutagenic potential of the nitrone by the Salmonella/microsome assay and observed no induction of mutagenicity even in concentrations as high as 33000 µM. Taken together, these results present a nonmutagenic compound, with trypanocidal activity against all relevant forms of T. cruzi, offering new insights into CD treatment suggesting additional in vivo tests.

Antileishmanial Activity of Ezetimibe: Inhibition of Sterol Biosynthesis, In Vitro Synergy with Azoles, and Efficacy in Experimental Cutaneous Leishmaniasis.

Leishmaniasis affects mainly low-income populations in tropical regions. Radical innovation in drug discovery is time-consuming and expensive, imposing severe restrictions on the ability to launch new chemical entities for the treatment of neglected diseases. Drug repositioning is an attractive strategy for addressing a specific demand more easily. In this project, we have evaluated the antileishmanial activities of 30 drugs currently in clinical use for various morbidities. Ezetimibe, clinically used to reduce intestinal cholesterol absorption in dyslipidemic patients, killed Leishmania amazonensis promastigotes with a 50% inhibitory concentration (IC50) of 30 µM. Morphological analysis revealed that ezetimibe caused the parasites to become rounded, with multiple nuclei and flagella. Analysis by gas chromatography (GC)-mass spectrometry (MS) showed that promastigotes treated with ezetimibe had smaller amounts of C-14-demethylated sterols, and accumulated more cholesterol and lanosterol, than untreated promastigotes. We then evaluated the combination of ezetimibe with well-known antileishmanial azoles. The fractional inhibitory concentration index (FICI) indicated synergy when ezetimibe was combined with ketoconazole or miconazole. The activity of ezetimibe against intracellular amastigotes was confirmed, with an IC50 of 20 µM, and ezetimibe reduced the IC90s of ketoconazole and miconazole from 11.3 and 11.5 µM to 4.14 and 8.25 µM, respectively. Subsequently, we confirmed the activity of ezetimibe in vivo, showing that it decreased lesion development and parasite loads in murine cutaneous leishmaniasis. We concluded that ezetimibe has promising antileishmanial activity and should be considered in combination with azoles in further preclinical and clinical studies.

Preclinical Studies Evaluating Subacute Toxicity and Therapeutic Efficacy of LQB-118 in Experimental Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is the second major cause of death by parasites, after malaria. The arsenal of drugs against leishmaniasis is small, and each has a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. Our group has focused on studying new drug candidates as alternatives to current treatments. The pterocarpanquinone LQB-118 was designed and synthesized based on molecular hybridization, and it exhibited antiprotozoal and anti-leukemic cell line activities. Our previous work demonstrated that LQB-118 was an effective treatment for experimental cutaneous leishmaniasis. In this study, we observed that treatment with 10 mg/kg of body weight/day LQB-118 orally inhibited the development of hepatosplenomegaly with a 99% reduction in parasite load. An in vivo toxicological analysis showed no change in the clinical, biochemical, or hematological parameters. Histologically, all of the analyzed organs were normal, with the exception of the liver, where focal points of necrosis with leukocytic infiltration were observed at treatment doses 5 times higher than the therapeutic dose; however, these changes were not accompanied by an increase in transaminases. Our findings indicate that LQB-118 is effective at treating different clinical forms of leishmaniasis and presents no relevant signs of toxicity at therapeutic doses; thus, this framework is demonstrated suitable for developing promising drug candidates for the oral treatment of leishmaniasis.

Further evidence that naphthoquinone inhibits Toxoplasma gondii growth in vitro.

Toxoplasmosis is a widely disseminated disease caused by Toxoplasma gondii, an intracellular protozoan parasite. Standard treatment causes many side effects, such as depletion of bone marrow cells, skin rashes and gastrointestinal implications. Therefore, it is necessary to find chemotherapeutic alternatives for the treatment of this disease. It was shown that a naphthoquinone derivative compound is active against T. gondii, RH strain, with an IC50 around 2.5 µM. Here, three different naphthoquinone derivative compounds with activity against leukemia cells and breast carcinoma cell were tested against T. gondii (RH strain) infected LLC-MK2 cell line. All the compounds were able to inhibit parasite growth in vitro, but one of them showed an IC50 activity below 1 µM after 48 h of treatment. The compounds showed low toxicity to the host cell. In addition, these compounds were able to induce tachyzoite-bradyzoite conversion confirmed by morphological changes, Dolichus biflorus lectin cyst wall labeling and characterization of amylopectin granules in the parasites by electron microscopy analysis using the Thierry technique. Furthermore, the compounds induced alterations on the ultrastructure of the parasite. Taken together, our results point to the naphthoquinone derivative (LQB 151) as a potential compound for the development of new drugs for the treatment of toxoplasmosis.

The pterocarpanquinone LQB-118 induces apoptosis in acute myeloid leukemia cells of distinct molecular subtypes and targets FoxO3a and FoxM1 transcription factors.

Acute myeloid leukemia (AML) patients' outcome is usually poor, mainly because of drug resistance phenotype. The identification of new drugs able to overcome mechanisms of chemoresistance is essential. The pterocarpanquinone LQB-118 compound has been shown to have a potent cytotoxic activity in myeloid leukemia cell lines and patient cells. Our aim was to investigate if LQB-118 is able to target FoxO3a and FoxM1 signaling pathways while sensitizing AML cell lines. LQB-118 induced apoptosis in both AML cell lines HL60 (M3 FAB subtype) and U937 (M4/M5 FAB subtype). Cell death occurred independently of alterations in cell cycle distribution. In vivo administration revealed that LQB-118 was not cytotoxic to normal bone marrow-derived cells isolated from mice. LQB-118 induced FoxO3a nuclear translocation and upregulation of its direct transcriptional target Bim, in HL60 cells. However, LQB-118 induced FoxO3a nuclear exclusion, followed by Bim downregulation, in U937 cells. Concomitantly, LQB-118 exposure reduced FoxM1 and Survivin expression in U937 cells, but this effect was more subtle in HL60 cells. Taken together, our data suggest that LQB-118 has a selective and potent antitumor activity against AML cells with distinct molecular subtypes, and it involves differential modulation of the signaling pathways associated with FoxO3a and FoxM1 transcription factors.

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro.

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC(50) of 2.5 µM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.

Effectiveness of the local or oral delivery of the novel naphthopterocarpanquinone LQB-118 against cutaneous leishmaniasis.

OBJECTIVES: This paper describes the antileishmanial properties of LQB-118, a new compound designed by molecular hybridization, orally active in Leishmania amazonensis-infected BALB/c mice. METHODS: In vitro antileishmanial activity was determined in L. amazonensis-infected macrophages. For in vivo studies, LQB-118 was administered intralesionally (15 µg/kg/day, five times a week), intraperitoneally (4.5 mg/kg/day, five times a week) or orally (4.5 mg/kg/day, five times a week) to L. amazonensis-infected BALB/c mice throughout experiments lasting 85 or 105 days. At the end of the experiments, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine were measured as toxicological parameters. RESULTS: LQB-118 was active against intracellular amastigotes of L. amazonensis [50% inhibitory concentration (IC(50)) 1.4 µM] and significantly less so against macrophages (IC(50) 18.5 µM). LQB-118 administered intralesionally, intraperitoneally or orally was found to control both lesion and parasite growth in L. amazonensis-infected BALB/c mice, without altering serological markers of toxicity. CONCLUSIONS: These results demonstrate that the molecular hybridization of a naphthoquinone core to pterocarpan yielded a novel antileishmanial compound that was locally and orally active in an experimental cutaneous leishmaniasis model.

2-Methoxy-3,8,9-trihydroxy coumestan: a new synthetic inhibitor of Na+,K+-ATPase with an original mechanism of action.

The aim of the present work was to analyse the interaction between Na(+),K(+)-ATPase and one of our recent synthesized coumestans, namely the original molecule 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). Rat brain (mainly alpha 2 and alpha 3 Na(+),K(+)-ATPase isoforms) and kidney (alpha 1 isoform) fractions enriched with Na(+),K(+)-ATPase were utilized to compare the inhibition promoted by PCALC36 with that of classical inhibitors like ouabain and vanadate. Analysis of inhibition curves revealed that unlike ouabain, which was about a thousand times more potent to inhibit brain isoforms than kidney isoform, PCALC36 had a similar affinity for brain (IC(50)=4.33+/-0.90 microM) and kidney (IC(50)=11.04+/-0.86 microM) isoforms. The inhibitory effect of PCALC36 was not antagonized by 1-10 mM K(+), as observed with ouabain. Whereas vanadate was more potent in ionic conditions promoting the E2 conformation of the enzyme, the inhibitory effect of PCALC36 was equal in ionic conditions favouring either the E1 or E2 conformations. Equilibrium binding assays with [3H]ouabain revealed that the addition of 2-10 microM PCALC36 did not change the K(d) of ouabain but decreased its maximal binding (B(max)) in a concentration-dependent manner (from 76.6 to 44.0 pmol/mg protein). This inhibitory effect of PCALC36 was not reverted after an extensive washing procedure indicating that it forms a very stable complex with Na(+),K(+)-ATPase. We conclude that PCALC36, a new molecule with a non-steroidal skeleton, inhibits the Na(+),K(+)-ATPase by a mechanism of action different from the cardiac glycosides and could thus serve as a structural paradigm to develop new inotropic drugs.