Microtubule target for new antileishmanial drugs based on ethyl 3-haloacetamidobenzoates

A new family of antimicrotubule drugs named (3-haloacetamidobenzoyl) ureas and ethyl 3-haloacetamidobenzoates were found to be cytotoxic to the Leishmania parasite protozoa. While the benzoylureas were shown to strongly inhibit in vitro mammalian brain microtubule assembly, the ethyl ester derivatives were characterized as very poor inhibitors of this process. Ethyl 3-chloroacetamidobenzoate, MF29, was found to be the most efficient drug on the promastigote stage of three Leishmania species (IC₅₀: 0.3–1.8 μM). MF29 maintained its activity against the clinical relevant intracellular stage of L. mexicana with IC₅₀ value of 0.33 μM. It was the only compound that exhibits a high activity on all the Leishmania species tested. This compound appeared to alter parasite microtubule organisation as demonstrated by using antibodies directed against microtubule components and more precisely the class of microtubule decorated by the MAP2-like protein. It is interesting to notice that this MAP2-like protein was identified for the first time in a Leishmania parasite     

Inhibition of parasite protein kinase C by new antileishmanial imidazolidin-2-one compounds

The protein kinase C (PKC) family of isoenzymes mediate a wide range of signal transduction pathways in many different cells lines. Little is known regarding the presence and functional roles of PKC in Leishmania spp. Here we report the inhibition of parasite PKC by new imidazolidinone compounds. The most active derivative 7 showed an important activity (IC50 = 9.9 microM) against the clinical relevant stage of parasites in comparison with Glucantime (IC50 = 464.5 microM), without inducing toxicity on human fibroblast cells (IC50 = 102 microM). Pretreatment of intact parasites with 10 microM of compound 7 inhibited 80% of PKC activity. At the same concentration, this compound inhibited 70% of the parasite-host cell invasion process. An in vivo model showed that compound 7 reduced the liver parasite burden by 25% and spleen parasite burden by 44%. These results provide the first evidence that PKC plays a critical role in the invasion process. Thus Leishmania PKC activity could be a relevant therapeutic target and the imidazolidinones novel antileishmanial candidates.     

Inhibition of Parasite Protein Kinase C by New Antileishmanial Imidazolidin-2-one Compounds

The protein kinase C (PKC) family of isoenzymes mediate a wide range of signal transduction pathways in many different cells lines. Little is known regarding the presence and functional roles of PKC in Leishmania spp. Here we report the inhibition of parasite PKC by new imidazolidinone compounds. The most active derivative 7 showed an important activity (IC 50 =9.9 μM) against the clinical relevant stage of parasites in comparison with Glucantime ® (IC 50 =464.5 μM), without inducing toxicity on human fibroblast cells (IC 50 =102 μM). Pretreatment of intact parasites with 10 μM of compound 7 inhibited 80% of PKC activity. At the same concentration, this compound inhibited 70% of the parasite-host cell invasion process. An in vivo model showed that compound 7 reduced the liver parasite burden by 25% and spleen parasite burden by 44%. These results provide the first evidence that PKC plays a critical role in the invasion process. Thus Leishmania PKC activity could be a relevant therapeutic target and the imidazolidinones novel antileishmanial candidates.     

Antimicrobial and cytotoxicity activities of the medicinal plant Primula macrophylla

Primula macrophylla (Primulaceae) is reported as to be useful in asthma, restlessness, insomnia and fish poisoning. Antifungal and toxic activities of crude extract, fractions and a pure isolated compound exhibited statistically significant activities. Excellent antifungal activity was found in the crude extract, benzene and ethyl acetate fractions against T. longifusis and against M. canis with different MIC values. Antileishmanial activity (IC(50) = 50ug/mL) was observed as compared to standard drug Amphotericin B, and cytotoxic activity (LD(50) = 47.919microg/mL) was also found in the chloroform fraction. While pure compound 2-phenylchromone (Flavone) isolated from the chloroform fraction showed good activity (IC(50) = 25microg/mL) against Leishmania and cytotoxicity (LD(50) = 2.0116 microg/mL) in Brine Shrimp experiments. From antileishmanial and cytotoxic activity it can be concluded that 2-phenylchromone is the major compound responsible for these activities.     

The immunologically protective P-4 antigen of Leishmania amastigotes. A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum

The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum (20.8%) and the C-terminal domain of the 3' nucleotidase of Leishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP > CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.     

Antiprotozoal and antimicrobial activities of O-alkylated and formylated acylphloroglucinols

In the present article, we examined the antileishmanial, antimalarial, antibacterial, and antifungal activities of several newly synthesized O-alkylated phloroglucinol compounds (11-19) which are analogues of the naturally occurring antimalarial compound 1. Analogues 12 and 16 exhibited antileishmanial activity against, Leishmania donovani promastigotes with IC(50)s of 5.3 and 4.2microg/mL, respectively. Naturally occurring monomeric formylated acylphloroglucinol compounds, grandinol (2), jensenone (3), and their analogues (29-37), were also synthesized and evaluated for antileishmanial, antimalarial, antibacterial, and antifungal activities. Amongst these, both grandinol and jensenone showed mild to moderate antibacterial, antifungal, and antileishmanial activities. Jensenone (3) was effective against Candida albicans with an IC(50) of 5.5microg/mL but was ineffective against Cryptococcus neoformans and methicillin-resistant Staphylococcus aureus. Among the analogues, 34 was the most active against C. albicans and C. neoformans with IC(50)s of 2.0 and 2.5microg/mL, respectively, and was fungicidal toward Candida albicans.     

Synthesis and antimalarial properties of new chloro-9H-xanthones with an aminoalkyl side chain

The synthesis and antimalarial properties of twelve new chlorinated 9H-xanthones, carrying a [2-(diethylamino)ethyl]amino group in position 1, are reported. All compounds were found to be active towards the chloroquine-susceptible and chloroquine-resistant strains 3D7 and Dd2, resp., of the protozoa parasite Plasmodium falciparum. Especially one compound, 6-chloro-1-{[2-(diethylamino)ethyl]amino}-9H-xanthen-9-one (1k), was found to exhibit significant in vitro activity (IC50 = 3.9 microM) towards the resistant Dd2 strain.     

The in vitro leishmanicidal activity of hexadecylphosphocholine (miltefosine) against four medically relevant Leishmania species of Brazil

The in vitro leishmanicidal activity of miltefosine? (Zentaris GmbH) was assessed against four medically relevant Leishmania species of Brazil: Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) chagasi. The activity of miltefosine against these New World species was compared to its activity against the Old World strain, Leishmania (Leishmania) donovani, which is known to be sensitive to the effects of miltefosine. The IC50 and IC90 results suggested the New World species harboured similar in vitro susceptibilities to miltefosine; however, miltefosine was approximately 20 times more active against the Old World L. (L.) donovani than against the New World L. (L.) chagasi species. The selectivity index varied from 17.2-28.9 for the New World Leishmania species and up to 420.0 for L. (L.) donovani. The differences in susceptibility to miltefosine suggest that future clinical trials with this drug should include a laboratory pre-evaluation and a dose-defining step.     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.     

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.     

Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 microM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16-66 microM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.     

Trypanocidal activity of (-)-cubebin derivatives against free amastigote forms of Trypanosoma cruzi

Five (-)-cubebin derivative compounds, (-)-O-acetyl cubebin (3), (-)-O-benzyl cubebin (4), (-)-O-(N,N-dimethylaminoethyl)-cubebin (5), (-)-hinokinin (6) and (-)-6,6'-dinitrohinokinin (7), previously synthesised by our research group, were evaluated on in vitro assay against free amastigote forms of Trypanosoma cruzi, the asogic agent of Chagas' disease. It was observed that 6 was the most active compound (IC(50)=0.7 microM), and that 4 and 5 displayed moderate activity against the parasite, giving IC(50) values of 5.7 and 4.7 microM, respectively. In contrast, it was observed that compound 3 was inactive and that 7 displayed low activity with IC(50) values of congruent with 1.5 x 10(4) and 95.3 microM, respectively.     

Synthesis and in vitro antileishmanial activity of 5-substituted-2'-deoxyuridine derivatives

We report herein the synthesis and the in vitro antileishmanial evaluation of 5-substituted-2'-deoxyuridine nucleosides. The most active compound against Leishmania donovani promastigotes was Thia-dU (3a) with an IC50 =3 microM. This compound exhibited the same activity as zidovudine (3'-azido-2'-deoxythymidine) used as nucleoside reference compound. Considering the cytotoxicity of synthetic compounds on peritoneal murine macrophages, the most toxic compound was MeThio-dU (3d) with a MTC at 10 microM. Only Methia-dU (3b) was active against intramacrophagic amastigotes with an IC50 =6.5 microM. This latter can now be evaluated in vivo, for further investigations through structure-based drug design.     

Abietane diterpenoids and triterpenoic acids from Salvia cilicica and their antileishmanial activities

Bioguided-fractionation of an acetone extract of the roots of Salvia cilicica (Lamiaceae) led to isolation of two new diterpenes, 7-hydroxy-12-methoxy-20-nor-abieta-1,5(10),7,9,12-pentaen-6,14-dione and abieta-8,12-dien-11,14-dione (12-deoxy-royleanone), together with oleanolic acid, ursolic acid, ferruginol, inuroyoleanol and cryptanol. Their structures were determined spectroscopically, which included HREIMS and 2D NMR spectroscopic analysis. The new abietane derivatives showed appreciable in vitro antileishmanial activity against intracellular amastigote forms of both Leishmania donovani (IC(50) values of 170 and 120 nM, respectively) and Leishmania major (IC(50) values of 290 and 180 nM, respectively). The triterpenoic acids were found to be potently active against amastigote (IC(50) values of 7-120 nM) and moderately active against promastigote stages (IC(50) values of 51-137 nM) of the two Leishmania species.     

Inhibition by Dications of in vitro growth of Leishmania major and Leishmania tropica: causative agents of old world cutaneous leishmaniasis

Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.     

Synthesis and preliminary evaluation of new ursolic and oleanolic acids derivatives as antileishmanial agents

A series of new ursolic and oleanolic acids derivatives was synthesized via ursolic or oleanolic acids, previously extracted from South American Ilex species. These new compounds were tested for in vitro antiparasitic activity on Leishmania amazonensis and Leishmania infantum strains. Some of these compounds showed activity against the promastigote forms of L. amazonensis or L. infantum, with IC(50) ranging from 5 to 12 microM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC(50) = 2-50 microM). From a structure-activity relationships point of view, these pharmacological results enlightened mainly the importance of an acetylation at position 3 of the oleanolic acid skeleton in the activity against the L. amazonensis strain, and of a bis-(3-aminopropyl)piperazine moiety on the carboxylic function of ursolic acid against the L. infantum strain.     

Evaluation of sage phenolics for their antileishmanial activity and modulatory effects on interleukin-6, interferon and tumour necrosis factor-alpha-release in RAW 264.7 cells

A series of sage phenolics was tested for activity against a panel of Leishmania parasites and for immunomodulatory effects on macrophage functions including release of tumour necrosis factor (TNF), interleukin-6 (IL-6), and interferon (IFN)-like activities. For this, functional bioassays were employed including an in vitro model for leishmaniasis in which macrophage-like RAW 264.7 cells were infected with Leishmania parasites, an extracellular Leishmania growth-inhibition assay, a fibroblast-lysis assay for TNF-activity, a cell proliferation assay using IL-6 sensitive murine B9 hybridoma cells, and a virus protection assay for IFN-like activity. Whereas none of the test samples exhibited marked activities against extracellular Leishmania promastigotes (IC50 > 700 to > 2800 nM; > 500 microg/ml), caffeic acid, salvianolic acids K and L as well as the methyl ester of salvianolic acid I showed pronounced antileishmanial activities against intracellular amastigote stages within RAW cells (IC50 3-23 nM vs. 10-11 nM for the reference Pentostam). Noteworthy, the phenolic samples showed no cytotoxicity against the host cells (IC50 > 600 to > 2200 nM; > 400 microg/ml). Tested sage phenolics activated Leishmania-infected RAW 264.7 for release of TNF ranging 22-117 U/ml and IL-6 ranging 3-42 U/ml. In contrast, their TNF- or IL-6-inducing potential in experiments with non-infected host cells was negligible. Furthermore, caffeic acid and salvianolic acid K induced a modest release of IFN-like activity (5-9 and 2-4 U/ml, respectively) as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on L929 cells. The results support the emerging picture that plant polyphenols may be credited for the profound health-beneficial properties of various herbal medicines and agricultural products.     

Synthesis, antimalarial activity and cytotoxicity of 10-aminoethylether derivatives of artemisinin

In this study, a series of 11 10-aminoethylether derivatives of artemisinin were synthesised and their antimalarial activity against both the chloroquine sensitive (D10) and resistant (Dd2) strains of Plasmodium falciparum was determined. The compounds were prepared by introducing aliphatic, alicyclic and aromatic amine groups with linkers of various chain lengths through an ethyl ether bridge at C-10 of artemisinin using conventional and microwave assisted syntheses, and their structures were confirmed by NMR and HRMS. All derivatives proved to be active against both strains of the parasite. The highest overall activity was displayed by the short chain aromatic derivative 8 (IC(50)=1.44nM), containing only one nitrogen atom, while long chain polyamine derivatives were found to have the lowest activity against both strains. An interesting correlation between the IC(50), pK(a) values and resistance index (RI) was found.     

Rapid fluorescent assay for screening drugs on Leishmania amastigotes

A rapid fluorescent viability assay employing alamarBlue was optimized for use with Leishmania axenic amastigotes, the stage of the parasite responsible for disease pathology. The activity of two protein kinase inhibitors, Staurosporine and H-89, as well as Amphotericin B, on promastigotes and amastigotes of Leishmania donovani and Leishmania tropica was compared. Both protein kinase inhibitors inhibited promastigote growth at lower concentrations than amastigotes, while the GI(50) for Amphotericin B on both stages was similar. This assay only requires a limited number of axenic amastigotes (50,000 cells/well) and can be used to rapidly screen large chemical or natural product libraries for activity against amastigotes.     

Synthesis and antileishmanial activity of 3-imidazolylalkylindoles. Part I

The present study was designed to investigate conazoles as new antileishmanial agents. Several 3-imidazolylalkyl-indoles were prepared under mild reaction conditions and pharmacomodulation at N1 and C5 of the indole ring and at the level of the alkyl chain (R) was carried out starting from the corresponding 3-formylindoles 7-10. All target imidazolyl compounds 38-52 were evaluated in vitro against Leishmania mexicana promastigotes; ketoconazole, amphotericin B and meglumine antimoniate were used as references. Eight out of fifteen compounds (40,43,44,47,48, 50, 51 and 52) exerted similar activity to ketoconazole, with IC50 values in the range of 2.10-3.30 microg/mL. However the most potent compound, 1-(2-bromobenzyl)-3-(1H-imidazol-1-ylmethyl)-1H-indole (38), exhibited IC50 value (0.011+/-0.003 microg/mL) 270-fold lower than that of ketoconazole. Four compounds (38, 43, 50 and 52) were also tested against intracellular amastigotes of L. mexicana; compound 38 exhibited the highest activity with an IC50 value of 0.018+/-0.004 microg/mL.