Antileishmanial effects of Crotalaria spectabilis Roth aqueous extracts on Leishmania amazonensis.

Fifteen polar extracts from leaf, seed, pod, stem, flower and root of Crotalaria spectabilis were prepared using aqueous systems, based on the principles of green chemistry, and showed different protease inhibitor (PI) activities on trypsin, papain, pepsin and the extracellular L. amazonensis serine protease (LSPIII). The most pronounced inhibitory effect on LSPIII was observed in leaf (CS-P), root, stem, flower (CS-FPVPP) and pod (CS-VA) extracts. Crotalaria extracts exhibited low cytotoxicity on macrophages; however, they decreased the viability of L. amazonensis promastigotes and amastigotes, as observed in leaf (CS-AE, CS-P, CS-T and CS-PVPP), seed (CS-ST), flower and root (CS-RA) extracts. CS-P was chosen to study PI and secondary metabolites and a 10-12 kDa protein, analyzed by mass spectrometry, was identified as a serine PI homologous with papaya latex serine PI. Glycosylated flavonoids, such as quercetins, vitexin and tricin were the major secondary metabolites of CS-P. The presence of PIs in C. spectabilis is a new finding, especially in other organs than seeds since PIs have been reported only in seed legumes. Besides, this is the first report of antileishmanial activity of C. spectabilis extracts and the identification of serine polypeptide PI and glycosylated flavonoids from leaf.

Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis.

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.

Host cholesterol influences the activity of sterol biosynthesis inhibitors in Leishmania amazonensis.

A significant percentage of exogenous cholesterol was found in promastigotes and amastigotes of all studied species of Leishmania, suggesting a biological role for this molecule. Previous studies have shown that promastigotes of Leishmania uptake more low-density lipoprotein (LDL) particles under pharmacological pressure and are more susceptible to ergosterol inhibition in the absence of exogenous sources of cholesterol. This work shows that the host's LDL is available to intracellular amastigotes and that the absence of exogenous cholesterol enhances the potency of sterol biosynthesis inhibitors in infected macrophages. A complete understanding of cholesterol transport to the parasitophorous vacuole can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.

Past and future of trypanosomatids high-throughput phenotypic screening

Diseases caused by trypanosomatid parasites affect millions of people mainly living in developing countries. Novel drugs are highly needed since there are no vaccines and available treatment has several limitations, such as resistance, low efficacy, and high toxicity. The drug discovery process is often analogous to finding a needle in the haystack. In the last decades a so-called rational drug design paradigm, heavily dependent on computational approaches, has promised to deliver new drugs in a more cost-effective way. Paradoxically however, the mainstay of these computational methods is data-driven, meaning they need activity data for new compounds to be generated and available in databases. Therefore, high-throughput screening (HTS) of compounds still is a much-needed exercise in drug discovery to fuel other rational approaches. In trypanosomatids, due to the scarcity of validated molecular targets and biological complexity of these parasites, phenotypic screening has become an essential tool for the discovery of new bioactive compounds. In this article we discuss the perspectives of phenotypic HTS for trypanosomatid drug discovery with emphasis on the role of image-based, high-content methods. We also propose an ideal cascade of assays for the identification of new drug candidates for clinical development using leishmaniasis as an example.

Sertraline Delivered in Phosphatidylserine Liposomes Is Effective in an Experimental Model of Visceral Leishmaniasis.

Liposomes containing phosphatidylserine (PS) has been used for the delivery of drugs into the intramacrophage milieu. Leishmania (L.) infantum parasites live inside macrophages and cause a fatal and neglected viscerotropic disease, with a toxic treatment. Sertraline was studied as a free formulation (SERT) and also entrapped into phosphatidylserine liposomes (LP-SERT) against intracellular amastigotes and in a murine model of visceral leishmaniasis. LP-SERT showed a potent activity against intracellular amastigotes with an EC50 value of 2.5 µM. The in vivo efficacy of SERT demonstrated a therapeutic failure. However, when entrapped into negatively charged liposomes (-58 mV) of 125 nm, it significantly reduced the parasite burden in the mice liver by 89% at 1 mg/kg, reducing the serum levels of the cytokine IL-6 and upregulating the levels of the chemokine MCP-1. Histopathological studies demonstrated the presence of an inflammatory infiltrate with the development of granulomas in the liver, suggesting the resolution of the infection in the treated group. Delivery studies showed fluorescent-labeled LP-SERT in the liver and spleen of mice even after 48 h of administration. This study demonstrates the efficacy of PS liposomes containing sertraline in experimental VL. Considering the urgent need for VL treatments, the repurposing approach of SERT could be a promising alternative.

Evaluation of Novel Chalcone-Thiosemicarbazones Derivatives as Potential Anti-Leishmania amazonensis Agents and Its HSA Binding Studies.

A series of seven chalcone-thiosemicarbazones (5a-5g) were synthesized and evaluated as potential new drugs (anti-leishmanial effect). Although four of the chalcone-thiosemicarbazones are already known, none of them or any compound in this class has been previously investigated for their effects on parasites of the Leishmania genus. The compounds were prepared in satisfactory yields (40-75%) and these compounds were evaluated against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis after 48 h of culture. The half maximal inhibitory concentration (IC50) values of the intracellular amastigotes were determined to be in the range of 3.40 to 5.95 µM for all compounds assayed. The selectivity index showed value of 15.05 for 5a, whereas pentamidine (reference drug) was more toxic in our model (SI = 2.32). Furthermore, to understand the preliminary relationship between the anti-leishmanial activity of the chalcone-thiosemicarbazones, their electronic (σ), steric (MR) and lipophilicity (π) properties were correlated, and the results indicated that moieties with electronic withdrawing effects increase the anti-leishmanial activity. The preliminary pharmacokinetic evaluation of one of the most active compound (5e) was studied via interaction to human serum albumin (HSA) using multiple spectroscopic techniques combined with molecular docking. The results of antiparasitic effects against L. amazonensis revealed the chalcone-thiosemicarbazone class to be novel prototypes for drug development against leishmaniasis.

Leishmanicidal Activity of Withanolides from Aureliana Fasciculata var. Fasciculata.

Leishmaniasis is the generic denomination to the neglected diseases caused by more than 20 species of protozoa belonging to the genus Leishmania. The toxic and parenteral-delivered pentavalent antimonials remain to be the first-line treatment. However, all the current used drugs have restrictions. The species Aureliana fasciculata (Vell.) Sendtner var. fasciculata is a native Brazilian species parsimoniously studied on a chemical point of view. In this study, the antileishmanial activity of A. fasciculata was evaluated. Among the evaluated samples of the leaves, the dichloromethane partition (AFfDi) showed the more pronounced activity, with IC50 1.85 µg/ml against promastigotes of L. amazonensis. From AFfDi, two active withanolides were isolated, the Aurelianolides A and B, with IC50 7.61 µM and 7.94 µM, respectively. The withanolides also proved to be active against the clinically important form, the intracellular amastigote, with IC50 2.25 µM and 6.43 µM for Aurelianolides A and B, respectively. Furthermore, withanolides showed results for in silico parameters of absorption, distribution, metabolism, excretion, and toxicity (ADMET) similar to miltefosine, the reference drug, and were predicted as good oral drugs, with the advantage of not being hepatotoxic. These results suggest that these compounds can be useful as scaffolds for planning drug design.

Anti-Mycobacterium tuberculosis activity of essential oil and 6,7-dehydroroyleanone isolated from leaves of Tetradenia riparia (Hochst.) Codd (Lamiaceae).

BACKGROUND: The global resurgence of tuberculosis (TB) and the development of drug resistance, as multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis isolates, are a threat to TB control and have created a need for new and more effective anti-TB drugs. AIM: The current study evaluated the in vitro cytotoxicity and activity of Tetradenia riparia essential oil (TrEO) and 6,7-dehydroroyleanone pure compound against M. tuberculosis H37Rv and susceptible and resistant clinical isolates. METHODS: The in vitro activities of TrEO and 6,7-dehydroroyleanone were determined by Resazurin Microtiter Assay Plate (REMA). The cytotoxicity was evaluated in murine peritoneal macrophages by Alamar Blue assay. The cytotoxic effects were expressed as median concentration cytotoxicity (CC50) and the selectivity index (SI) was calculated. RESULTS: TrEO and 6,7-dehydroroyleanone showed activity against M. tuberculosis H37Rv with minimum inhibitory concentration (MIC) 62.5 µg/ml and 31.2 µg/ml, respectively. Both of them exhibited activities against resistant and susceptible M. tuberculosis clinical isolates with MIC values between 31.2 and 62.5 µg/ml. Cytotoxicity assays showed SI 1.9 and 7.9 for TrEO and 6,7-dehydroroyleanone, respectively. CONCLUSION: These results revealed that TrEO isolated from leaves of T. riparia and the pure compound 6,7-dehydroroyleanone display good activity against M. tuberculosis clinical isolates, including MDR isolates, with low cytotoxicity to murine macrophages. The 6,7-dehydroroyleanone compound is a potential candidate for anti-TB drug.

Original antileishmanial hits: Variations around amidoximes.

In continuation to our previous findings on amidoximes' antiparasitic activities, a new series of 23 original derivatives was designed and obtained by convergent synthesis. First, new terminal alkenes were synthesized by cross-coupling reaction. Then, cyclization was performed between terminal alkenes and ß-ketosulfones using manganese(III) acetate reactivity. Twenty-three amidoximes were tested for their in vitro activity against Leishmania amazonensis promastigotes and their toxicity on murine macrophages. Seven of the tested compounds exhibited an antileishmanial activity at lower than 10 µM with moderate to low toxicity. Six of these molecules showed activity at lower than 10 µM against promastigotes and toxicity at higher than 50 µM were selected and evaluated for their activity against intracellular Leishmania amazonensis amastigotes. Modulating chemical substituents in position 2 of dihydrofuran highly influenced their antileishmanial activities. The introduction of a methyl or trifluoromethyl group on the benzene ring of the benzyl group had a positive influence on activity without significantly increasing toxicity (52, 59, 60).

Second-generation pterocarpanquinones: synthesis and antileishmanial activity

Despite the development of new therapies for leishmaniasis, among the 200 countries or territories reporting to the WHO, 87 were identified as endemic for Tegumentary Leishmaniasis and 75 as endemic for Visceral Leishmaniasis. The identification of antileishmanial drug candidates is essential to fill the drug discovery pipeline for leishmaniasis. In the hit molecule LQB-118 selected, the first generation of pterocarpanquinones was effective and safe against experimental visceral and cutaneous leishmaniasis via oral delivery. In this paper, we report the synthesis and antileishmanial activity of the second generation of pterocarpanoquinones. Methods: The second generation of pterocarpanquinones 2a-f was prepared through a palladium-catalyzed oxyarylation of dihydronaphtalen and chromens with iodolawsone, easily prepared by iodination of lawsone. The spectrum of antileishmanial activity was evaluated in promastigotes and intracellular amastigotes of L. amazonensis, L. braziliensis, and L. infantum. Toxicity was assessed in peritoneal macrophages and selective index calculated by CC50/IC50. Oxidative stress was measured by intracellular ROS levels and mitochondrial membrane potential in treated cells. Results: In this work, we answered two pertinent questions about the structure of the first-generation pterocarpanquinones: the configuration and positions of rings B (pyran) and C (furan) and the presence of oxygen in the B ring. When rings B and C are exchanged, we noted an improvement of the activity against promastigotes and amastigotes of L. amazonensis and promastigotes of L. infantum. As to the oxygen in ring B of the new generation, we observed that the oxygenated compound 2b is approximately twice as active against L. braziliensis promastigotes than its deoxy derivative 2a. Another modification that improved the activity was the addition of the methylenedioxy group. A variation in the susceptibility among species was evident in the clinically relevant form of the parasite, the intracellular amastigote. L. amazonensis was the species most susceptible to novel derivatives, whilst L. infantum was resistant to most of them. The pterocarpanoquinones (2b and 2c) that possess the oxygen atom in ring B showed induction of increased ROS production. Conclusions: The data presented indicate that the pterocarpanoquinones are promising compounds for the development of new leishmanicidal agents.(AU)

Cyclobenzaprine Raises ROS Levels in Leishmania infantum and Reduces Parasite Burden in Infected Mice.

BACKGROUND: The leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP) is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity. METHODOLOGY/PRINCIPAL FINDINGS: In vitro antileishmanial activity was determined in promastigotes and in L. infantum-infected macrophages. For in vivo studies, L. infantum-infected BALB/c mice were treated with CBP by oral gavage for five days and the parasite load was estimated. Trypanothione reductase activity was assessed in the soluble fraction of promastigotes of L. infantum. For evaluation of cytokines, L. infantum-infected macrophages were co-cultured with BALB/c splenocytes and treated with CBP for 48 h. The supernatant was analyzed for IL-6, IL-10, MCP-1, IFN-γ and TNF-α. CBP demonstrated an IC50 of 14.5±1.1µM and an IC90 of 74.5±1.2 µM in promastigotes and an IC50 of 12.6±1.05 µM and an IC90 of 28.7±1.3 µM in intracellular amastigotes. CBP also reduced the parasite load in L. infantum-infected mice by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg, after a short-term treatment. CBP inhibited the trypanothione reductase activity with a Ki of 86 ± 7.7 µM and increased the ROS production in promastigotes. CBP inhibited in 53% the production of IL-6 in infected macrophages co-culture. CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this study is the first report of the in vivo antileishmanial activity of the FDA-approved drug CBP. Modulation of immune response and induction of oxidative stress in parasite seem to contribute to this efficacy.

Antileishmanial activity of amides from Piper amalago and synthetic analogs

Two natural amides isolated from the chloroform extract of Piper amalago L., Piperaceae, leaves, a hydrogenated derivative and seven synthetic analogs were tested against the promastigote and intracellular amastigote forms of Leishmania amazonensis. The antileishmanial activity was evaluated in terms of growth inhibitory concentration for 50% of protozoa (IC50). The cytotoxicity toward the J774A1 macrophages was evaluated in terms of the cytotoxic concentrations for 50% of macrophages (CC50). The ability to induce nitric oxide production was also investigated for all compounds. The saturated amide 7-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-1-heptanone was obtained by hydrogenation of the natural compound N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl]pyrrolidine. Synthetic amides were prepared by addition of the appropriate amine to the corresponding acyl chloride. The natural compound, N-[7-(3',4'-methylenedioxyphenyl)-2(E),4(E)-heptadienoyl]pyrrolidine, was the most active of all tested compounds against the promastigote and intracellular amastigote forms with IC50 values of 15 µM and 14.5 µM, respectively. None of the compounds modulated the production of nitric oxide.

The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.

Pterocarpanquinones, aza-pterocarpanquinone and derivatives: synthesis, antineoplasic activity on human malignant cell lines and antileishmanial activity on Leishmania amazonensis.

Pterocarpanquinones (1a-e) and the aza-pterocarpanquinone (2) were synthesized through palladium catalyzed oxyarylation and azaarylation of conjugate olefins, and showed antineoplasic effect on leukemic cell lines (K562 and HL-60) as well as colon cancer (HCT-8), gliobastoma (SF-295) and melanoma (MDA-MB435) cell lines. Some derivatives were prepared (3-8) and evaluated, allowing establishing the structural requirements for the antineoplasic activity in each series. Compound 1a showed the best selectivity index in special for leukemic cells while 2 showed to be more bioselective for HCT-8, SF-295 and MDA-MB435 cells. Pterocarpanquinones 1a and 1c-e, as well as 8 were the most active on amastigote form of Leishmania amazonensis in culture. Compounds 1a, 1c and 8 showed the best selectivity index.

Modulation of P2X(7) purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination.

The purinergic P2X(7) receptor is a membrane protein of leucocytes involved in the clearance of intracellular bacteria such as Chlamydia and Mycobacterium. In this work, we investigated the role and modulation of macrophage P2X(7)R in intracellular infection with the protozoan parasite Leishmania amazonensis. Upon infection, isolated murine macrophages displayed enhanced expression of P2X(7)R and were significantly more responsive to extracellular ATP (ATPe)-induced pore opening, as demonstrated by the increased uptake of Lucifer Yellow. This was extended to the in vivo situation, where cells from established cutaneous lesions were more sensitive to ATPe than cells from uninfected mice. ATP treatment of infected macrophages inhibited parasite growth, and this was prevented by pre-treatment with oxidized ATP, a selective antagonist of P2X(7)R. Parasite killing was unlikely due to induction of nitric oxide production or cytolysis of infected macrophage, as those functions were unaltered with parasite-effective ATPe concentrations. A direct drug effect is also unlike, as ATPe enhanced axenic parasite growth. We found that leishmanial infection rendered wild-type but not P2X(7)R-deficient macrophages more prone to ATP-induced apoptosis. These results show that macrophage infection with L. amazonensis leads to enhanced expression of functional P2X(7)R, that upon ligation with ATPe helps in the elimination of the parasites by an as yet unclear mechanism possibly involving host cell apoptosis.