Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

Embilica officinalis L. inhibits the growth and proliferation of Leishmania donovani through the induction of ultrastructural changes, mitochondrial dysfunction, oxidative stress and apoptosis-like cell death.

Visceral leishmaniasis (VL) is caused by a protozoan parasite, Leishmania donovani (L. donovani). It affects around 1-2 million people around the world annually. There is an urgent need to investigate new medicament of it due to difficult method of drug administration, long period of treatment, high cost of the drug, adverse side-effects, low efficacy and development of parasite resistance to the available drugs. Medicinal plants have also been used for the treatment of different diseases in traditional system of medicines due to their holistic effects. The Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland has already started the program for identification of potential medicinal plant and plant products having antileishmanial potential. Keeping all these in consideration, we planned to study the antileishmanial activity of one of the medicinal plant, Embilica officinalis L. (EO) fruit extract. EO fruit extract inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes in dose-dependent manner. EO fruit extract induced morphological and ultrastructural changes in parasites as observed under Electron Microscope. It also induced the oxidative stress, mitochondrial dysfunction, DNA laddering and apotosis-like cell death in parasites. Here, we for the first time reported such a detailed mechanism of action of antileishmanial activity of EO fruit extract. Our results suggested that EO fruit extract could be used for the development of new phytomedicine against leishmaniasis.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani.

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

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-ß-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-ß-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.

Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Bio-guided isolation of anti-leishmanial natural products from Diospyros gracilescens L. (Ebenaceae).

BACKGROUND: Plants represent an intricate and innovative source for the discovery of novel therapeutic remedies for the management of infectious diseases. The current study aimed at discovering new inhibitors of Leishmania spp., using anti-leishmanial activity-guided investigation approach of extracts from Diospyros gracilescens Gürke (1911) (Ebenaceae), targeting the extracellular (promastigotes) and intracellular (amastigotes) forms of Leishmania donovani. METHODS: The plant extracts were prepared by maceration using H20: EtOH (30:70, v/v) and further fractionated using a bio-guided approach. Different concentrations of D. gracilescens extracts, fractions and isolated compounds were tested in triplicate against L. donovani promastigotes and amastigotes in vitro. The antileishmanial potency and cytotoxicity on RAW 264.7 cells were determined using the resazurin colorimetric assay. The time kill kinetic profile of the most active sample was also investigated. The structures of all compounds were elucidated on the basis of extensive spectroscopic analyses, including 1D and 2D NMR, and HR-ESI-MS and by comparison of their data with those reported in the literature. RESULTS: The hydroethanolic crude extract of D. gracilescens trunk showed the most potent antileishmanial activity (IC50 = 5.84 µg/mL). Further fractionation of this extract led to four (4) fractions of which, the hexane fraction showed the most potent activity (IC50 = 0.79 µg/mL), and seven (07) compounds that exhibited moderate potency (IC50 = 13.69-241.71 µM) against L. donovani. Compound 1-deoxyinositol (7) inhibited the promastigote and amastigote forms of L. donovani with IC50 values of 241.71 µM and 120 µM respectively and also showed the highest selectivity against L. donovani promastigotes (SI > 5.04). To the best of our knowledge, the antileishmanial activity of this compound is being reported here for the first time. The promising hexane fraction showed significant inhibition of parasites growth at different concentrations, but with no evidence of cidal effect over an exposure period of 120 h. CONCLUSIONS: The results obtained indicated that the hydroethanolic extract from the D. gracilescens trunk and the derived hexane fraction have very potent inhibitory effect on cultivated promastigotes and amastigotes of L. donovani parasite. The isolated compounds showed a lesser extent of potency and selectivity. However, further structure-activity-relationship studies of 1-deoxyinositol could lead to more potent and selective hit derivatives of interest for detailed drug discovery program against visceral leishmaniasis.

Antiplasmodial and antileishmanial inhibitory activity of triterpenes and steroidal alkaloid from the leaves of Funtumia elastica (Preuss) Stapf (Apocynaceae).

The phytochemical study of leaves of Funtumia elastica led to the isolation of three undescribed ursane derivatives, funtumic acids A, B and C (1-3), as well as one steroidal alkaloid, elasticine (4) and five other known compounds (5-9). Their structures were elucidated on the basis of NMR, MS, IR, UV spectroscopic data as well as by comparison with the literature. The compound 5-hydroxypyridine-3-carboxamide (9) was isolated for the first time from the Apocynaceae family. All the isolated compounds were evaluated for their antiparasitic effects against 3D7 and Dd2 strains of Plasmodium falciparum and promastigotes of Leishmania donovani (MHOM/SD/62/1S). Compounds 1-4 possessed good in vitro antimalarial activities against CQR Dd2 with IC50 values ranging from 4.68 to 5.36 µg/mL and moderate on CQS 3D7. Only compounds 1 and 2 showed leishmanicidal activities with IC50 values ranging between 10.49 and 13.21 µg/mL. In addition, crude extract exhibited potent antiplasmodial (IC50 0.91 and 3.12 µg/mL) and antileishmanial (IC50 3.32 µg/mL) activities, thus demonstrating their potential synergistic action.

Metabolomic approach of the antiprotozoal activity of medicinal Piper species used in Peruvian Amazon.

ETHNOPHARMACOLOGICAL RELEVANCE: In the Peruvian Amazon as in the tropical countries of South America, the use of medicinal Piper species (cordoncillos) is common practice, particularly against symptoms of infection by protozoal parasites. However, there is few documented information about the practical aspects of their use and few scientific validation. The starting point of this work was a set of interviews of people living in six rural communities from the Peruvian Amazon (Alto Amazonas Province) about their uses of plants from Piper genus: one community of Amerindian native people (Shawi community) and five communities of mestizos. Infections caused by parasitic protozoa take a huge toll on public health in the Amazonian communities, who partly fight it using traditional remedies. Validation of these traditional practices contributes to public health care efficiency and may help to identify new antiprotozoal compounds. AIMS OF STUDY: To record and validate the use of medicinal Piper species by rural people of Alto Amazonas Province (Peru) and annotate active compounds using a correlation study and a data mining approach. MATERIALS AND METHODS: Rural communities were interviewed about traditional medication against parasite infections with medicinal Piper species. Ethnopharmacological surveys were undertaken in five mestizo villages, namely: Nueva Arica, Shucushuyacu, Parinari, Lagunas and Esperanza, and one Shawi community (Balsapuerto village). All communities belong to the Alto Amazonas Province (Loreto region, Peru). Seventeen Piper species were collected according to their traditional use for the treatment of parasitic diseases, 35 extracts (leaves or leaves and stems) were tested in vitro on P. falciparum (3D7 chloroquine-sensitive strain and W2 chloroquine-resistant strain), Leishmania donovani LV9 strain and Trypanosoma brucei gambiense. Assessments were performed on HUVEC cells and RAW 264.7 macrophages. The annotation of active compounds was realized by metabolomic analysis and molecular networking approach. RESULTS: Nine extracts were active (IC50 ≤ 10 µg/mL) on 3D7 P. falciparum and only one on W2 P. falciparum, six on L. donovani (axenic and intramacrophagic amastigotes) and seven on Trypanosoma brucei gambiense. Only one extract was active on all three parasites (P. lineatum). After metabolomic analyses and annotation of compounds active on Leishmania, P. strigosum and P. pseudoarboreum were considered as potential sources of leishmanicidal compounds. CONCLUSIONS: This ethnopharmacological study and the associated in vitro bioassays corroborated the relevance of use of Piper species in the Amazonian traditional medicine, especially in Peru. A series of Piper species with few previously available phytochemical data have good antiprotozoal activity and could be a starting point for subsequent promising work. Metabolomic approach appears to be a smart, quick but still limited methodology to identify compounds with high probability of biological activity.

Bioassay-based Corchorus capsularis L. leaf-derived ß-sitosterol exerts antileishmanial effects against Leishmania donovani by targeting trypanothione reductase.

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived ß-sitosterol (ß-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC-MS). The inhibitory efficacy of this ß-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). ß-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of ß-sitosterolCCL was validated by enzyme inhibition and an in silico study in which ß-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, ß-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.

(±)-trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents: Synthesis, in vitro evaluation and SAR analysis.

Leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania, causes a serious burden of disease around the world, represents a threat to the life of millions of people, and therefore is a major public health problem. More effective and non-toxic new treatments are required, especially for visceral leishmaniasis, the most severe form of the disease. On the backdrop that dihydrobenzofurans have previously shown antileishmanial activity, we present here the synthesis of a set of seventy trans-2-phenyl-2,3-dihydrobenzofurans and evaluation of their in vitro activity against Leishmania donovani as well as a discussion of structure-activity relationships. Compounds 8m-o and 8r displayed the highest potency (IC50 < 2 µmol/L) and interesting selectivity of the antileishmanial activity over cytotoxicity against mammalian cells (SI > 4.6). Nonetheless, structural optimization as further requirement was inferred from the high clearance of the most potent compound (8m) observed during determination in vitro of its metabolic stability. On the other hand, chiral separation of 8m and subsequent biological evaluation of its enantiomers demonstrated no effect of chirality on activity and cytotoxicity. Holistic analysis of in silico ADME-like properties and ligand efficiency metrics by a simple scoring function estimating druglikeness highlighted compounds 16c, 18 and 23 as promising candidates for further development. Overall, the potential of trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents was confirmed.

The Effect of Iodium 30c on Experimental Visceral Leishmaniasis.

BACKGROUND: Leishmaniasis is one of several neglected tropical diseases that warrant serious attention. A disease of socio-economically poor people, it demands safer and cheaper drugs that help to overcome the limitations faced by the existing anti-leishmanials. Complementary or traditional medicines might be a good option, with an added advantage that resistance may not develop against these drugs. Thus, the present investigation was performed to evaluate the anti-leishmanial efficacy of an ultra-diluted homeopathic medicine (Iodium 30c) in experimental visceral leishmaniasis (VL). METHODS: Compliant with strict ethical standards in animal experimentation, the study was performed in-vivo in inbred BALB/c mice which were injected intravenously with 1 × 107 promastigotes of Leishmania donovani before (therapeutic) or after (prophylactic) treatment with Iodium 30c for 30 days. In other groups of mice (n = 6 per group), amphotericin B served as positive control, infected animals as the disease control, while the naïve controls included normal animals; animals receiving only Iodium 30c or Alcohol 30c served as sham controls. The anti-leishmanial efficacy was assessed by determining the hepatic parasite load and analysing percentages of CD4+ and CD8+ T cells. Biochemical analysis and histological studies were performed to check any toxicities. RESULTS: Iodium-treated animals showed a significantly reduced parasite load (to 1503 ± 39 Leishman Donovan Units, LDU) as compared with the infected controls (4489 ± 256 LDU) (p < 0.05): thus, the mean therapeutic efficacy of Iodium 30c was 66.5%. In addition, the population of CD4+ and CD8+ T cells was significantly increased (p < 0.05) after treatment. No toxicity was observed, as evidenced from biochemical and histopathological studies of the liver and kidneys. Efficacy of Iodium 30c prophylaxis was 58.3%, while the therapeutic efficacy of amphotericin B was 85.9%. CONCLUSION: This original study has shown that Iodium 30c had significant impact in controlling parasite replication in experimental VL, though the effect was less than that using standard pharmaceutical treatment.

Flavones reversibly inhibit Leishmania donovani tyrosine aminotransferase by binding to the catalytic pocket: An integrated in silico-in vitro approach.

The current drugs for treating Leishmaniasis are toxic, non-economical and with the emergence of drug resistance makes the need for novel therapeutics urgent and necessary. In the current study, we report the identification of compounds TI 1-5 against tyrosine aminotransferase of L. donovani from a curated ZINC15 database containing 183,659 compounds. These flavonoid compounds had binding energies < -8 kcal/mol and interacted with the active site residues S151, K286, C290, and P291. Assessment of physicochemical descriptors and ADMET properties established the drug likeliness of these compounds. The all-atom molecular dynamic simulations of the TAT-TI complexes exhibited stable geometrical properties and further trajectory analysis revealed the high-affinity interactions of TI 1, 3, 4, and 5 with the active site residues. DFT calculations reported the high electrophilic nature of TI 2 while other TI compounds demonstrated good kinetic stability and reactivity. From in vitro studies, TI 3 and TI 4 had the highest inhibition with Ki values of 0.9 ± 0.2 µM and 0.30 ± 0.1 µM, respectively. Taken together, the results from this study indicate the potentiality of TI 1, 3, 4, and 5 as anti-leishmanial leads, and these compounds can be exploited to manage the growing Leishmaniasis crisis in the world.

Curative efficacy of purified serine protease inhibitor PTF3 from potato tuber in experimental visceral leishmaniasis.

To overcome the drug toxicity and frequent resistance of parasites against the conventional drugs for the healing of human visceral leishmaniasis, innovative plant derived antileishmanial components are very imperative. Fuelled by the complications of clinically available antileishmanial drugs, a novel potato serine protease inhibitor was identified with its efficacy on experimental visceral leishmaniasis (VL). The serine protease inhibitors from potato tuber extract (PTEx) bearing molecular mass of 39 kDa (PTF1), 23 kDa (PTF2) and 17 kDa (PTF3) were purified and identified. Among them, PTF3 was selected as the most active inhibitor (IC50 143.5 ± 2.4 µg/ml) regarding its antileishmanial property. Again, intracellular amastigote load was reduced upto 83.1 ± 1.7% in pre-treated parasite and 88.5 ± 0.5% in in vivo model with effective dose of PTF3. Protective immune response by PTF3 was noted with increased production of antimicrobial substances and up-regulation of pro-inflammatory cytokines. Therapeutic potency of PTF3 is also followed by 80% survival in infected hamster. The peptide mass fingerprint (MALDI-TOF) results showed similarity of PTF3 with serine protease inhibitors database. Altogether, these results strongly propose the effectiveness of PTF3 as potent immunomodulatory therapeutics for controlling VL.

In Vitro and in Vivo Activity of mTOR Kinase and PI3K Inhibitors Against Leishmania donovani and Trypanosoma brucei.

Kinetoplastid parasites, including Leishmania and Trypanosoma spp., are life threatening pathogens with a worldwide distribution. Next-generation therapeutics for treatment are needed as current treatments have limitations, such as toxicity and drug resistance. In this study, we examined the activities of established mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitors against these tropical diseases. High-throughput screening of a library of 1742 bioactive compounds against intracellular L. donovani was performed, and seven mTOR/PI3K inhibitors were identified. Dose-dilution assays revealed that these inhibitors had half maximal effective concentration (EC50) values ranging from 0.14 to 13.44 µM for L. donovani amastigotes and from 0.00005 to 8.16 µM for T. brucei. The results of a visceral leishmaniasis mouse model indicated that treatment with Torin2, dactolisib, or NVP-BGT226 resulted in reductions of 35%, 53%, and 54%, respectively, in the numbers of liver parasites. In an acute T. brucei mouse model using NVP-BGT226 parasite numbers were reduced to under the limits of detection by five consecutive days of treatment. Multiple sequence and structural alignment results indicated high similarities between mTOR and kinetoplastid TORs; the inhibitors are predicted to bind in a similar manner. Taken together, these results indicated that the TOR pathways of parasites have potential for the discovery of novel targets and new potent inhibitors.

Leishmania donovani Growth Inhibitors from Pathogen Box Compounds of Medicine for Malaria Venture.

INTRODUCTION: Leishmaniasis is a collective term used to describe various pathological conditions caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the neglected diseases and has been given minimal attention by drug discovery and development stakeholders to narrow the safety and efficacy gaps of the drugs currently used to treat leishmaniasis. The challenge is further exacerbated by the emergence of drug resistance by the parasites. METHODS: Aiming to look for potential anti-leishmanial hits and leads, we screened Medicines for Malaria Venture (MMV) Pathogen Box compounds against clinically isolated Leishmania donovani strain. In this medium-throughput primary screening assay, the compounds were screened against promastigotes, and then against amastigote stages. RESULTS: From the total 400 compounds screened, 35 compounds showed >50% inhibitory activity on promastigotes in the initial screen (1 µM). Out of these compounds, nine showed >70% inhibition, with median inhibitory concentration (IC50) ranging from 12 to 491 nM using the anti-promastigote assay, and from 53 to 704 nM using the intracellular amastigote assay. Identified compounds demonstrated acceptable safety profiles on THP-1 cell lines and sheep red blood cells, and had appropriate physicochemical properties suitable for further drug development. Two compounds (MMV690102 and MMV688262) were identified as leads. The anti-tubercular agent MMV688262 (delamanid) showed a synergistic effect with amphotericin B, indicating the prospect of using this compound for combination therapy. CONCLUSION: The current study indicates the presence of additional hits which may hold promise as starting points for anti-leishmanial drug discovery and in-depth structure-activity relationship studies.

Evaluation of in vitro antileishmanial efficacy of cyclosporin A and its non-immunosuppressive derivative, dihydrocyclosporin A.

BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.

Phosphonamidates are the first phosphorus-based zinc binding motif to show inhibition of ß-class carbonic anhydrases from bacteria, fungi, and protozoa.

A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from pathogens (bacteria, fungi, and protozoa) was shown to produce an impairment of the microorganism growth and virulence. As phosphonamidates have been recently validated as human α-CA inhibitors (CAIs) and no phosphorus-based zinc-binding group have been assessed to date against ß-class CAs, herein we report an inhibition study with this class of compounds against ß-CAs from pathogenic bacteria, fungi, and protozoa. Our data suggest that phosphonamidates are among the CAIs with the best selectivity for ß-class over human isozymes, making them interesting leads for the development of new anti-infectives.

Antiprotozoal and antihelminthic properties of plants ingested by wild Japanese macaques (Macaca fuscata yakui) in Yakushima Island.

ETHNOPHARMACOLOGICAL RELEVANCE: Primates forage on a variety of plant parts to balance their dietary intake to meet requirements of energy, nutrition and maintenance, however the reason(s) leading them to ingest some plants which have no nutritional value and/or contain bioactive or even toxic secondary metabolites is recently gaining closer attention. The growing literature suggests that primates consume plants for medicinal purposes (self-medication) as well, particularly when infected with parasites and pathogens (bacteria, viruses, microbes). Interestingly, some of the plants they consume are also used by humans for similar purposes or may have potential uses for humans. MATERIALS AND METHODS: As part of a 16-month study of the parasite ecology of a sub-species of Japanese macaques (Macaca fuscata yakui) on the island of Yakushima, we surveyed their feeding habits and collected a subset of plants and plant parts observed being ingested by macaques. The ethnomedicinal value of these plants was surveyed and methanolic extracts of 45 plant parts were tested in vitro against important parasites of humans, including four protozoan parasites Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, and the trematode flatworm Schistosoma mansoni. Potential toxicity of the extracts was also assessed on mammalian cells. RESULTS: A wide range of ethnomedicinal uses in Asia for these plants is noted, with 37% associated with the treatment of parasites, pathogens and related symptoms. Additionally, the 45 extracts tested showed broad and significant activity against our test organisms. All extracts were active against T. b. rhodesiense. The majority (over 80%) inhibited the growth of P. falciparum and L. donovani. Half of the extracts also displayed antiprotozoal potential against T. cruzi while only several extracts were active against both larval and adult stages of S. mansoni. Cytotoxicity was generally low, although several extracts lacked specific toxicity to test parasites. CONCLUSIONS: Our results indicated a number of plants and their parts to have antiparasitic activity not previously reported in the ethnopharmacological literature. Enhanced understanding of the primate diets, particularly during periods of intensified parasite infection risk may help to further narrow down plants of interest for lead compound development. The study of animal self-medication is a complementary approach, with precedence, to drug discovery of new lead drug compounds against human parasitic diseases.

2-furyl(phenyl)methanol isolated from Atractilis gummifera rhizome exhibits anti-leishmanial activity.

We report for the first time the isolation of 2-furyl(phenyl)methanol (5) from the chloroform extracts of the Atractylis gummifera roots. A. gummifera is a thistle belonging to the Asteraceae family that produces the ent-kaurane diterpenoid glycoside atractyloside (ATR). ATR (1) was isolated and chemically modified to obtain its aglycone atractyligenin (2) and the methylated derivatives ATR-OMe (3) and genine-OMe (4). The compounds 1-5 were structurally characterised and evaluated against the intracellular amastigote, cultured within macrophages, and the extracellular promastigote of Leishmania donovani, the protozoan parasite responsible for the highly infective disease visceral leishmaniasis, which is fatal if untreated. The 2-furyl(phenyl)methanol 5 exhibited notable activity against the promastigote.

Tropical and Subtropical Parasitic Diseases: Targets for a New Approach to Virtual Screening.

Computational techniques are widely used to reduce costs associated with new drug development with the ability to bind a specific molecular target. These studies need a Brookhaven protein data bank structure sample of the enzyme interaction with an inhibitor of adequate size. In this context, a new computational methodology is postulated to be used when there are no published samples fulfilling this requirements. In this study, 7 compounds, which showed anti-T. cruzi, L. donovani and L. infantum properties, and proved to be inhibitors of their Fe-SOD enzymes, have been theoretically evaluated against related parasites Fe-SOD enzymes, which have been proposed as targets for antiparasitic drugs. This methodology may be applied to similar cases and also to generate starting structures to be used with different CADD methods.