Abstract
We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.
Highlights
The Leishmaniases, together with Human African Trypanosomiasis (HAT) and Chagas disease, are caused by kinetoplastid parasites of the TriTryp group
We have developed an assay for screening test compounds for their ability to kill intracellular amastigotes of Leishmania parasites, causative agents of human leishmaniasis
The assay is based on freeing amastigotes from infected macrophages by mild detergent lysis and measuring the number of parasites following their extracellular replication by a fluorescence-based method
Summary
The Leishmaniases, together with Human African Trypanosomiasis (HAT) and Chagas disease, are caused by kinetoplastid parasites of the TriTryp group All of these infections are diseases of poverty and cause severe impact, as measured in disability adjusted life years (DALY), in endemic countries (91 in total for the Leishmaniases, including countries in east and northern Africa, the Middle East, the Indian sub-continent and Central and South America [3]) They have received limited funding for research and development of new drugs [4] there are currently ‘‘repurposed’’ drugs in clinical or preclinical trials for all three disease groups e.g. fexinidazole for visceral leishmaniasis (VL) and HAT (acute and CNS stages) [5,6]; the antifungal lanosterol-14 alpha-demethylase inhibitors, posaconazole [7] and E1224 (a prodrug of ravuconazole [8,9]), for Chagas disease (see [4] and www.dndi.org/ for further details). Axenic amastigotes, adapted to replicate at acidic pH and elevated
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