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In the midst of numerous setbacks that beclouds the fight against leishmaniasis; a neglected tropical disease, the search for new chemotherapeutics against this disease is of utmost importance. Leishmaniasis is a disease closely associated with poverty and endemic in Africa, Asia, southern Europe and the Americas. It is caused by parasites of the genus Leishmania and transmitted by a sandfly vector. In this study, we evaluated the antileishmanial potency of eighteen pathogen box compounds and elucidated their biosafety and possible mechanisms of action against Leishmania donovani promastigotes and amastigotes in vitro. IC50s range of 0.12±0.15 to >6.25 μg/ml and 0.13±0.004 to >6.25μg/ml were observed for the promastigotes and amastigotes, respectively. We demonstrated the ability of some of the compounds to cause cytocidal effect on the parasites, induce increased production of reactive oxygen species (ROS), disrupt the normal parasite morphology and cause the accumulation of parasites at the DNA synthesis phase of the cell cycle. We recommend a further in vivo study on these compounds to validate the findings.

Leishmaniasis is one of the Neglected Tropical Diseases (NTDs) which is closely associated with poverty and has gained much relevance recently due to its opportunistic coinfection with HIV. It is a protozoan zoonotic disease transmitted by a dipteran Phlebotomus, Lutzomyia/ Sergentomyia sandfly; during blood meals on its vertebrate intermediate hosts. It is a four-faceted disease with its visceral form being more deadly if left untreated. It is endemic across the tropics and sub-tropical regions of the world. It can be considered the third most important NTD after malaria and lymphatic filariasis. Currently, there are numerous drawbacks on the fight against leishmaniasis which includes: non-availability of vaccines, limited availability of drugs, high cost of mainstay drugs and parasite resistance to current treatments. In this study, we screened the antileishmanial activity, selectivity, morphological alterations, cell cycle progression and apoptotic potentials of six Pathogen box compounds from Medicine for Malaria Venture (MMV) against Leishmania donovani promastigotes and amastigotes. From this study, five of the compounds showed great promise as lead chemotherapeutics based on their high selectivity against the Leishmania donovani parasite when tested against the murine mammalian macrophage RAW 264.7 cell line (with a therapeutic index ranging between 19-914 (promastigotes) and 1-453 (amastigotes)). The cell cycle progression showed growth arrest at the G0-G1 phase of mitotic division, with an indication of apoptosis induced by two (2) of the pathogen box compounds tested. Our findings present useful information on the therapeutic potential of these compounds in leishmaniasis. We recommend further in vivo studies on these compounds to substantiate observations made in the in vitro study.

BACKGROUND:Leishmaniasis is a disease caused by the protozoan parasite, Leishmania. The disease remains a global threat to public health requiring effective chemotherapy for control and treatment. In this study, the effect of some selected phenolic compounds on Leishmania donovani was investigated. The compounds were screened for their anti-leishmanial activities against promastigote and intracellular amastigote forms of Leishmania donovani. METHODOLOGY/PRINCIPAL FINDINGS:The dose dependent effect and cytotoxicity of the compounds were determined by the MTT assay. Flow cytometry was used to determine the effect of the compounds on the cell cycle. Parasite morphological analysis was done by microscopy and growth kinetic studies were conducted by culturing cells and counting at 24 hours intervals over 120 hours. The cellular levels of iron in promastigotes treated with compounds was determined by atomic absorption spectroscopy and the effect of compounds on the expression of iron dependent enzymes was investigated using RT-qPCR. The IC50 of the compounds ranged from 16.34 μM to 198 μM compared to amphotericin B and deferoxamine controls. Rosmarinic acid and apigenin were the most effective against the promastigote and the intracellular amastigote forms. Selectivity indexes (SI) of rosmarinic acid and apigenin were 15.03 and 10.45 respectively for promastigotes while the SI of 12.70 and 5.21 respectively was obtained for intracellular amastigotes. Morphologically, 70% of rosmarinic acid treated promastigotes showed rounded morphology similar to the deferoxamine control. About 30% of cells treated with apigenin showed distorted cell membrane. Rosmarinic acid and apigenin induced cell arrest in the G0/G1 phase in promastigotes. Elevated intracellular iron levels were observed in promastigotes when parasites were treated with rosmarinic acid and this correlated with the level of expression of iron dependent genes. CONCLUSIONS/SIGNIFICANCE:The data suggests that rosmarinic acid exerts its anti-leishmanial effect via iron chelation resulting in variable morphological changes and cell cycle arrest.

African trypanosomiasis remains a lethal disease to both humans and livestock. The disease persists due to limited drug availability, toxicity and drug resistance, hence the need for a better understanding of the parasite's biology and provision of alternative forms of therapy. In this study, the in vitro effects of phenolic acids were assessed for their trypanocidal activities against Trypanosoma brucei brucei. The effect of the phenolic acids on Trypanosoma brucei brucei was determined by the alamarBlue assay. The cell cycle effects were determined by flow cytometry and parasite morphological analysis was done by microscopy. Effect on cell proliferation was determined by growth kinetic analysis. Reverse Transcriptase quantitative Polymerase Chain Reaction was used to determine expression of iron dependent enzymes and iron distribution determined by atomic absorption spectroscopy. Gallic acid gave an IC50 of 14.2±1.5 μM. Deferoxamine, gallic acid and diminazene aceturate showed a dose dependent effect on the cell viability and the mitochondrion membrane integrity. Gallic acid, deferoxamine and diminazene aceturate caused loss of kinetoplast in 22%, 26% and 82% of trypanosomes respectively and less than 10% increase in the number of trypanosomes in S phase was observed. Gallic acid caused a 0.6 fold decrease, 50 fold increase and 7 fold increase in the expression levels of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes respectively while treatment with deferoxamine and diminazene aceturate also showed differential expressions of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes. The data suggests that gallic acid possibly exerts its effect on T. brucei via iron chelation leading to structural and morphological changes and arrest of the cell cycle. These together provide information on the cell biology of the parasite under iron starved conditions and provide leads into alternative therapeutic approaches in the treatment of African trypanosomiasis.