Synthesis of proteophosphoglycans of Leishmania major and Leishmania mexicana

A novel approach for the synthesis of various fragments of proteophosphoglycans from Leishmania major and Leishmania mexicana proteophosphoglycans has been developed. These compounds have been obtained by coupling alpha-mannosyl and alpha-N-acetyl-glucosamine phosphoramidite derivatives with the serine hydroxyl of various amino acids and peptides to give, after oxidation with tert-BuOOH, phosphotriesters exclusively as alpha-anomers in good yield. The resulting compounds could be deblocked using conventional methods. Glycophosphorylation of preassembled and properly protected peptides was found to be more efficient for the preparation of proteophosphoglycan fragments than a building block approach strategy using a phosphoglycosylserine derivative.     

A preliminary approach to the separation of Leishmania cell-surface antigens

The purpose of the current study was to characterize Leishmania cell-surface antigens by two different methods established for the purification of glycoproteins and proteins, and to point out a useful approach to define their size and mass heterogeneity. L. tropica parasites were initially isolated from patients with active cutaneous leishmaniasis and were then cultured in vitro. The parasite-cell layer was solubilised with 6 M guanidinium chloride (GuHCl) and subsequently prepared for the purification procedure. The methods used in this work were gel filtration chromatography and isopycnic density-gradient centrifugation. Because of the presence of a substantial amount of non-specific proteins in the culture medium, these methods were not effective alone in distinguishing these antigens. However, a good idea of their N-glycosylated structures could be obtained by using Periodic acid-Schiffs (PAS) and Con A lectin, and also size and mass heterogeneity. A combination of these methods effected a clear separation of the antigens. Amino acid analysis of the purified antigens was performed to positively identify them as well-known Leishmania cell-surface antigen gene products. The results confirmed the presence of more than one cell-surface antigen on the Leishmania parasite and the combination of gel chromatography and density-gradient centrifugation could be useful for their isolation.     

Microwave-assisted synthesis of 2-styrylquinoline-4-carboxylic acid derivatives to improve the toxic effect against Leishmania (Leishmania) amazonensis

The identification of new compounds is urgent to develop safe and efficacious candidates for leishmaniasis treatment, especially from natural products as a potential source of active molecules against neglected tropical parasite diseases. Inspired by the efficacious quinoline alkaloid microbial effects, we have previously reported the synthesis and biological activity of 2-phenylquinoline-4-carboxylic acids and poly-substituted quinolines against parasites. In this work, a series of eighteen 2-styryl-4-quinolinecarboxylic acids were synthesized under microwave irradiation settings obtaining from good to excellent yields (60%-90%), shorter reaction times (2 minutes), and eco-friendly experimental conditions. All these products were evaluated against infective forms of Leishmania (Leishmania) amazonensis, such as promastigotes and intracellular amastigotes, based on cytotoxicity assays, including host macrophage infection assays. Compounds 4 and 5 possessing a 2-chloro or 4-chlorostyryl moiety, respectively, were considered the most promising antileishmanial agents due to the parasite killing effect in intracellular forms inside infected macrophages. Thus, our results revealed that the 2-styryl-4-quinolinecarboxylic acid backbone structure was essential for the activity against intracellular pathogens like L. (L.) amazonensis.     

Efficient synthesis of the antigenic phosphoglycans of the Leishmania parasite

Antigenic phosphoglycan repeats of the Leishmania parasite can be assembled in a flexible and efficient manner without involving any glycosidation steps, and the chain can be extended either towards the non-reducing (6'-OH) or reducing (1-OH) end suitable for synthesis of lipophosphoglycan, proteophosphoglycan and analogues.     

Synthesis of Polycyclic Ether-Benzopyrans and In Vitro Inhibitory Activity against Leishmania tarentolae

Construction of a focused library of polycyclic ether-benzopyrans was undertaken in order to discover new therapeutic compounds that affect Leishmania growth and infectivity. This is especially of interest since there are few drug therapies for leishmaniasis that do not have serious drawbacks such high cost, side effects, and emerging drug resistance. The construction of these polycyclic ether-benzopyrans utilized an acetoxypyranone-alkene [5+2] cycloaddition and the Suzuki-Miyaura cross-coupling. The multi-gram quantity of the requisite aryl bromide was obtained followed by effective Pd-catalyzed coupling with boronic acid derivatives. Compounds were tested in vitro using the parasitic protozoan, Leishmania tarentolae. Effects of concentration, time, and exposure to light were evaluated. In addition, the effects on secreted acid phosphatase activity and nitric oxide production were investigated, since both have been implicated in parasite infectivity. The data presented herein are indicative of disruption of the Leishmania tarentolae and thus provide impetus for the development and testing of a more extensive library.     

New application for expanded porphyrins: sapphyrin and heterosapphyrins as inhibitors of Leishmania parasites

Sapphyrins and a series of related porphyrinoid macrocycles have been investigated as potential agents for the treatment of leishmaniasis. The effectiveness of the compounds was evaluated in vitro upon incubation with Leishmania tarentolae or L. panamensis amastigotes and promastigotes. Their effectiveness was also assessed against intracellular L. panamensis. The cytotoxicity of the compounds was evaluated in vitro using the U937 human promonocyte cell line. Effectiveness and cytotoxicity were assessed in the presence and absence of visible light to assess the photodynamic activity of the compounds. Sapphyrin and two related heterosapphyrins were shown to be particularly effective as inhibitors of Leishmania. A photodynamic effect was observed, which may be attributed to the formation of reactive oxygen species. Yields of singlet oxygen ((1)O(2)) produced were determined in ethanol solutions by direct measurement of (1)O(2) phosphorescence. Confocal microscopy demonstrated that sapphyrin and related macrocycles were taken up by the Leishmania cells and that their presence induces the formation of mitochondrial superoxide. Sapphyrins have been widely investigated as anticancer agents and we here show activity against the Leishmania parasites.     

Combinational sensitization of Leishmania with uroporphyrin and aluminum phthalocyanine synergistically enhances their photodynamic inactivation in vitro and in vivo

Leishmania were previously shown to undergo photolysis when their transgenic mutants were induced endogenously to accumulate cytoplasmic uroporphyrin or when loaded exogenously with aluminum phthalocyanine chloride. A combinational use of both is reported here, which renders Leishmania far more susceptible to photolysis. Fluorescence microscopy of cells loaded with the two photosensitizers localized them to different subcellular sites. Pre-exposure of Leishmania to both synergistically sensitized them for photolysis as extracellular promastigotes and intracellular amastigotes in infected macrophages in vitro when illuminated at specific wavelengths to excite the respective photosensitizers for production of reactive oxygen species. Both Leishmania stages lost their viability completely when doubly photosensitized optimally and illuminated at low intensity, the host cells being left unscathed. Inoculation of mice with photoinactivated Leishmania produced no lesions, which invariably developed in the control groups during a period of observations for 8 weeks. Pretreatment of Leishmania with both photosensitizers rendered these cells susceptible to clearance from the ear dermis by white light illumination. The results suggest that double photosensitization for synergistic activity enhances the efficacy and safety of photodynamic therapy in general and for Leishmania in particular.     

CE-ESI-MS metabolic fingerprinting of Leishmania resistance to antimony treatment

Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical-chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE-ESI-TOF-MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC(50) = 20.9 μM) and its variation when treated with 120 μM of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step-wise manner up to 180 μM. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol-dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.     

Synthesis and in vitro antiprotozoal activity of thiophene ring-containing quinones.

A series of quinones (3a-i, 4-9, 11) and aromatic compounds (2a, 2d, 2g) containing the thiophene ring were tested in vitro against the trypomastigote form of Trypanosoma cruzi and the promastigote forms of Leishmania. The quinones 3a-i, 4, 5a, b, 6 and 9 having the thiophene ring fused to a quinone nucleus were the most active members of the series. The electron affinities of the benzo[b]thiophene-4,7-quinones 3, evaluated by their LUMO energies and halfwave potentials, are reported.     

Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.     

Iterative synthesis of Leishmania phosphoglycans by solution,solid-phase,and polycondensation approaches without involving any glycosylation

A general strategy (solution, solid-phase, and polycondensation) for the synthesis of antigenic phosphoglycans (PG) of the protozoan parasite Leishmania is presented. Phosphoglycans constitute the variable structural and functional domain of major cell-surface lipophosphoglycan (LPG) and secreted proteophosphoglycan (PPG), the molecules involved in infectivity and survival of the Leishmania parasite inside human macrophages. We have shown that the chemically labile, anomerically phosphodiester-linked phosphoglycan repeats can be assembled in an iterative and efficient manner from a single key intermediate, without involving any glycosylation steps. Furthermore, the phosphoglycan chain can be extended toward either the nonreducing (6'-OH) or the reducing (1-OH) end. We also describe a new and efficient solid-phase methodology to construct phosphoglycans based on design and application of a novel cis-allylphosphoryl solid-phase linker that enabled the selective cleavage of the first anomeric-phosphodiester linkage without affecting any of the other internal anomeric-phosphodiester groups of the growing PG chain on the solid support. The strategy to construct larger phosphoglycans in a one-pot synthesis by polycondensation of a single key intermediate is also described, enabling CD spectrometric measurements to show the helical nature of phosphoglycans. Our versatile synthetic approach provides easy access to Leishmania phosphoglycans and the opportunity to address key immunological, biochemical, and biophysical questions pertaining to the phosphoglycan family (LPG and PPG) unique to the parasite.     

Evidence that the fully assembled capsid of Leishmania RNA virus 1-4 possesses catalytically active endoribonuclease activity

In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonuclease activity was found to be at 37(O)C to 42(O)C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.     

Novel multi-epitope protein containing conserved epitopes from different Leishmania species as potential vaccine candidate: Integrated immunoinformatics and molecular dynamics approach

Leishmaniosis, caused by intracellular parasites of the genus Leishmania, has become a serious public health problem around the world, and for which there are currently extensive limitations. In this work, a theoretical model was proposed for the development of a multi-epitope vaccine. The protein GP63 of the parasite was selected for epitopes prediction, due to its important biological role for the infection process and abundance. IEDB tools were used to determine epitopes B and T in Leishmania braziliensis; besides, other conserved epitopes in three species were selected. To improve immunogenicity, 50S ribosomal protein L7 / L12 (ID: P9WHE3) was used as a domain of adjuvant in the assembly process. The folding arrangement of the vaccine was obtained through homologous modeling multi-template with MODELLER v9.21, and a Ramachandran plot analysis was done. Furthermore, physicochemical properties were described with the ProtParam tool and secondary structure prediction combining GOR-IV and SOPMA tools. Finally, a molecular dynamics simulation (50 ns) was performed to establish flexibility and conformational changes. The analysis of the results indicates high conservancy in the epitopes predicted among the four species. Moreover, Ramachandran plot, physicochemical parameters, and secondary structure prediction suggest a stable conformation of the vaccine, after a minimum conformational change that was evaluated with the free energy landscape. The conformational change does not drive any substantial change for epitope exposition on the surface. The vaccine proposed could be tested experimentally to guide new approaches in the development of pan-vaccines; vaccines with regions conserved in multiple species.     

Cinnamides Target Leishmania amazonensis Arginase Selectively

Caffeic acid and related natural compounds were previously described as Leishmania amazonensis arginase (L-ARG) inhibitors, and against the whole parasite in vitro. In this study, we tested cinnamides that were previously synthesized to target human arginase. The compound caffeic acid phenethyl amide (CAPA), a weak inhibitor of human arginase (IC₅₀ = 60.3 ± 7.8 μM) was found to have 9-fold more potency against L-ARG (IC₅₀ = 6.9 ± 0.7 μM). The other compounds that did not inhibit human arginase were characterized as L-ARG, showing an IC₅₀ between 1.3–17.8 μM, and where the most active was compound 15 (IC₅₀ = 1.3 ± 0.1 μM). All compounds were also tested against L. amazonensis promastigotes, and only the compound CAPA showed an inhibitory activity (IC₅₀ = 80 μM). In addition, in an attempt to gain an insight into the mechanism of competitive L-ARG inhibitors, and their selectivity over mammalian enzymes, we performed an extensive computational investigation, to provide the basis for the selective inhibition of L-ARG for this series of compounds. In conclusion, our results indicated that the compounds based on cinnamoyl or 3,4-hydroxy cinnamoyl moiety could be a promising starting point for the design of potential antileishmanial drugs based on selective L-ARG inhibitors.     

Boswellic Acids Show In Vitro Activity against Leishmania donovani

In continuation of our search for leads from medicinal plants against protozoal pathogens, we detected antileishmanial activity in polar fractions of a dichloromethane extract from Boswellia serrata resin. 11-keto-β-boswellic acid (KBA) could be isolated from these fractions and was tested in vitro against Leishmania donovani axenic amastigotes along with five further boswellic acid derivatives. 3-O-acetyl-11-keto-β-boswellic acid (AKBA) showed the strongest activity with an IC₅₀ value of 0.88 µM against axenic amastigotes but was inactive against intracellular amastigotes in murine macrophages     

1-Oxabicyclic beta-lactams as new inhibitors of elongating MPT--a key enzyme responsible for assembly of cell-surface phosphoglycans of Leishmania parasite

New iminosugars (1-oxabicyclic beta-lactam disaccharides) have been synthesized as inhibitors of elongating alpha-D-mannosyl phosphate transferase (eMPT), a key enzyme involved in the iterative biosynthesis of cell-surface phosphoglycans of the Leishmania parasite. The design is based on a transition-state model for this remarkable enzyme that transfers intact alpha-D-mannosyl-phosphate from GDP-Man. Since these phosphoglycans are unique to Leishmania and are essential for its infectivity and survival, their biosynthetic pathway has emerged as a novel target for anti-leishmanial drug and vaccine design.     

Vaccination of mice against Leishmania mexicana amazonensis with microsomal fraction associated with BCG

Attempts to develop a satisfactory vaccine against New World cutaneous leishmaniasis have been made with varying success. We found that in mice, pretreated subcutaneously with 2×10⁶ BCG organisms 2 weeks prior to immunization at the same site with as low as 10 μg of a microsomal preparation (Pol-F) of Leishmania mexicana amazonensis, a profound alteration was observed in the course of infection produced by inoculation of virulent amastigotes of the same strain. The BCG-Pol-F vaccine preparation was consistently shown to have a protective capacity associated with larger classical cellular immune response (skin test) and higher specific antibody titres in response to leishmanial challenge dose (1 × 10⁶ amastigotes/mouse).     

seco-limonoids and quinoline alkaloids from Raputia heptaphylla and their antileishmanial activity

A novel seco-limonoid, rel-(1S,5R,9S,7R,8S,9R,10S,11R,13S,14R,15R,17R)-11,19-dihydroxy-7-acetoxy-7-deoxoichangin (raputiolide) (1), and two novel quinolone alkaloids N-methyl-2-phenoxyquinolin-4(1H)-one (heptaphyllone A) (2) and 6-methylbenzofuro[2,3-b]quinolin-4(1H)-one (heptaphyllone B) (3), along with the known seco-limonoid ichangin (4), were isolated from Raputia heptaphylla PITTIER (Rutaceae) stem bark. Five known alkaloids, N-methyl-8-methoxyflindersine (5), skimmianine (6), kokusaginine (7), dictamnine (8) and flindersiamine (9), were also isolated from R. heptaphylla leaves. Their structures were established on the basis of full spectroscopic data interpretation supported by data from the pertinent literature. seco-Limonoid 1 configuration was determined by enhanced nuclear Overhauser effect spectroscopy (NOESY) experiments and density functional theory (DFT) molecular modeling. The antileishmanial effect of the isolated compounds was evaluated on Leishmania Viannia panamensis (promastigotes and amastigotes). Whereas alkaloids 2-3, 6-8 and limonoid 4 exhibited no significant parasitocide activity against internalized L. (V.) panamensis amastigotes, limonoid 1 and alkaloid 5 had leishmanicidal activity on intracellular amastigotes (EC??: 8.7 μg/ml) and promastigotes (EC(50): 14.3 μg/ml), respectively.     

Bioflavonoid-Induced Apoptosis and DNA Damage in Amastigotes and Promastigotes of Leishmania donovani: Deciphering the Mode of Action

Natural products from plants contain many interesting biomolecules. Among them, quercetin (Q), gallic acid (GA), and rutin (R) all have well-reported antileishmanial activity; however, their exact mechanisms of action are still not known. The current study is a step forward towards unveil the possible modes of action of these compounds against Leishmania donovani (the causative agent of visceral leishmaniasis). The selected compounds were checked for their mechanisms of action against L. donovani using different biological assays including apoptosis and necrosis evaluation, effects on genetic material (DNA), quantitative testing of nitric oxide production, ultrastructural modification via transmission electron microscopy, and real-time PCR analysis. The results confirmed that these compounds are active against L. donovani, with IC₅₀ values of 84.65 µg/mL, 86 µg/mL, and 98 µg/mL for Q, GA, and R, respectively. These compounds increased nitric oxide production and caused apoptosis and DNA damage, which led to changes in the treated cells’ ultrastructural behavior and finally to the death of L. donovani. These compounds also suppressed essential enzymes like trypanothione reductase and trypanothione synthetase, which are critical for leishmanial survival. The selected compounds have high antileishmanial potentials, and thus in-vivo testing and further screening are highly recommended.     

Activity of Compounds from Temperate Propolis against Trypanosoma brucei and Leishmania mexicana

Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three Trypanosoma brucei strains and displayed EC₅₀ values < 20 µg/mL. The extracts were fractionated, from which 12 compounds and one two-component mixture were isolated, and characterized by NMR and high-resolution mass spectrometry, as 3-acetoxypinobanksin, tectochrysin, kaempferol, pinocembrin, 4′-methoxykaempferol, galangin, chrysin, apigenin, pinostrobin, cinnamic acid, coumaric acid, cinnamyl ester/coumaric acid benzyl ester (mixture), 4′,7-dimethoxykaempferol, and naringenin 4′,7-dimethyl ether. The isolated compounds were tested against drug-sensitive and drug-resistant strains of T. brucei and Leishmania mexicana, with the highest activities ≤ 15 µM. The most active compounds against T. brucei were naringenin 4′,7 dimethyl ether and 4′methoxy kaempferol with activity of 15–20 µM against the three T. brucei strains. The most active compounds against L. mexicana were 4′,7-dimethoxykaempferol and the coumaric acid ester mixture, with EC₅₀ values of 12.9 ± 3.7 µM and 13.1 ± 1.0 µM. No loss of activity was found with the diamidine- and arsenical-resistant or phenanthridine-resistant T. brucei strains, or the miltefosine-resistant L. mexicana strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity.