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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., Author summary There are numerous drawbacks in the fight against leishmaniasis which includes difficulty in drug administration, lengthy time of treatment, high toxicity, adverse side effects, high cost of drugs and increasing parasite resistance to treatment. These have made the search for new antileishmanial chemotherapeutics very essential. The Medicine for Malaria Venture (MMV) with the aim of accelerating drug development for poverty-related diseases has assembled some 400 diverse, drug-like molecules active against neglected diseases called the Pathogen box compounds. Thus, in this study we explored the antileishmanial potency and elucidated some possible mechanisms of action of some of the compounds against the Leishmania donovani parasites. The six compounds studied caused a distortion in the mitochondrion morphology, loss of kinetoplastid DNA and eventual nuclear degeneration upon treatment for 72 hours. Parasites treated with two of the cytocidal compounds MMV676057 (E03C) and MMV688942 (D06A) showed no significant programmed cell death due to apoptosis when compared to the untreated parasites but rather showed a cell cycle growth arrest in the G0-G1 and S-phases.

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., Author summary The available drugs used in treatment of leishmaniasis, a disease caused by the Leishmania parasite, include pentavalent antimonials and amphotericin B. The cost and associated toxicity of the drugs, side effects and the emergence of drug resistance parasites, as well as the discomfort in drug administration suggests the need for new and better chemotherapeutic interventions. Iron is involved in several biological processes in the Leishmania parasite and it’s important for its growth and survival. The effects of using iron chelators to deprive parasite of iron was investigated in this study. Treatment of L. donovani with rosmarinic acid and apigenin inhibited the growth of promastigotes and intracellular amastigotes in a dose dependent manner. Morphologically, the compounds induced alterations in the parasites upon treatments. Rosmarinic acid was observed to cause the dysfunction of the mitochondria and alter the expression of iron dependent enzymes. Rosmarinic acid ability to chelate iron could be responsible for the changes in cell morphology and cell cycle observed.